BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON … · MB MS MCh FNCCP FIACS Professor of Cardiothoracic...
Transcript of BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON … · MB MS MCh FNCCP FIACS Professor of Cardiothoracic...
BIOCHEMICAL AND IMMUNOLOGICAL STUDIES ON CIRCULATING AUTOANTIBODIES IN
DILATED CARDIOMYOPATHY
^ i - ' j » - * '
THESIS SUBMITTED FOR THE DEGREE OF
Bottor of $i)tlo£(opI)p
BIOCHEMISTRY
\ BY
NABIHA YUSUF ^
"'•"' • %W-y'. Approved: - ^ - * ^ ^•^*
£)r. Najmul Islam (Supervisor) ''vuy-
DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE
JAWAHARLAL NEHRU MEDICAL COLLEGE ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
1998
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m W: 1999
T5110
(Htvtiiicnit
I certify that the work presented in the following pages has been
carried out by Ms. Nabiha Yusuf and is suitable for the award of Ph.D.
degree in Biochemistry of the Aligarh Muslim University, Aligarh.
•i^'d^/'
(Najmul Islam) Lecturer Department of Biochemistry Faculty of Medicine J.N. Medical College Aligarh Muslim University Aligarh-202002
D R A M I T BANERJEE MB MS MCh FNCCP FIACS
Professor of Cardiothoracic Surgery MAULANA AZAD MEDICAL COLLEGE AND ASSOCIATED G B PANT HOSPITAL Ex-Professor. JIPMER, Pondicherry Ex-Medical Officer. Intelligence Bureau and Police Training School, New Delhi Edifor, Indian Journal of
THORAaC AND CARDIOVASCULAR SURGERY
ROOM NO 102 ACADEMIC BLOCK
DEPARTMENT OF CARDIOTHORACIC SURGERY GoviND BALLABH PANT HOSPITAL
NEW DELHI 110 002 INDIA
Telephones: Office: 3234242/5102, 3234726
Residence: 6446522 Fax: 3239442
August 9,1996
TO W H O M S O E V E R !T MAY r O N C F R N
This is to certif\'that we have provided a human hear! from an autopsy rase to Ms Nabiha Ynsnfforher
research work She is involved in cardiovascular research in the Department of Biochemistry' in Medical
College, AMU, Aligarh Farlier she was a research associate in our institution
/ , 'L
7'.-Dr. Am if Baneriee
Dr. AN^T FAVERJEE
G I. : . ' • 'c-f i tal , .S. O b i
^n ianh vexmmhxnnce of muDAD
^ 0 my 'role mohel' my MOTHER
Sc my 'ancijor'
my BROTHER for tijetr lobe, encouragement an^ support
mo my SIR fottii
profounh gra t i tuhe
mijxs piece of foork is hehicateb to tl]e cause of tlte mi l l i ons suffer ing from carhiac
ailments (norlbioihe
ACKNOWLEDGEMENT
'He who goes in search of knowledge walks in the path of GOD'.
Perhaps, this is the most appropriate quote to sum up this quest. I take this
opportunity to thank the Almighty for His benevolent presence and invisible
prowess to guide me in His path It was my immense faith in Him which kept
me going undeterred and reach this destination. I pray to Him to keep my
faith strong and guide me with His guiding light After all 'Light dawns
after every darkness, believe in the divine'.
In this search for knowledge, my mind automatically goes to my
supervisor, Dr. Najmul Islam, who worked hand in glove with me to achieve
this mission. We were on no ordinary mission, it was a 'mission impossible'
and for us where the odds weighed heavily against the evens, mutual
understanding was the key words. He M'as willing to discuss my problems at
all levels, convince me and console me in moments of despair. I was allowed
to voice my opinions too and together we could knit this piece of work in its
present shape. I can never thank him enough for being a friend, philosopher
and guide to me Ms. Zeba Islam was also always around to put in a word
of encouragement.
I am grateful to Prof. Rashid AH for allow ing me to work m his
laboratory which was a great source of help in executing my experiments.
Without this, I could have never accomplished this task I am also thankful
for his constructive criticism often at the cost of his great discomfort and
inconvenience.
/ owe a deep sense of gratitude to Dr. Asif All, Chairman, who has
been a great source of help and encouragement during some of the trying
moments. Whenever, the going was tough, he did his best to remove every
hurdle that came my way. I am particularly grateful to him for his
affectionate ways and encouragement for this humble effort of mine.
Thanks are also due to Drs. Khushtar Salman, A.F. Faizy and
Moinuddin for their help from time to time. A special note of thanks to Dr.
M.U. Siddiqui, who always shielded me and gave a lot of moral support
whenever I needed it.
There have been noteworthy contributions from persons outside this
department who have always encouraged me to go ahead when the pace
slowed down and did their best whenever I approach them for help. This
includes Prof. W.A. Nizami, Deptt. of Zoology, A.M.U. and Dr. Saad Tayyab,
Reader, Interdisciplinary Biotechnology Unit.
This work would have remains a distant dream but for the love and
encouragement from my uncle. Dr. M. Khalilullah, Consultant in
Cardiology, Inderprastha Apollo Hospital, New Delhi. He has been a father
figure and source of inspiration to me. He was the one who was really keen
that I should undertake a study in the field of cardiology. He helped me
during the course of this study in every possible way. In his absence. Dr.
M. Ahmad, Cardiologist, J.N.M.C., Aligarh ably supported me with the
clinical details and also encouraged me to forge ahead. Thanks are also
due to Prof. Amit Bannerjee, Deptt. of Cardiothoracic Surgery, G.B. Pant
Hospital, New Delhi for providing a human heart which formed the pivotal
basis of this study.
My thanks are also due to my departmental colleagues, Drs. Aslam
and Sajid, Mr. Rizwan Ahmad, Waris, Deepak, Haseeb, Sharique, Humra,
Talat and Subia for their support at times. Here, I would like to mention a
special note of thanks to Dr. Rizwan Manzer whose incorrigible support
helped me to overcome the last and most crucial phase of this crusade.
My friends Drs. Shagufta and Farah, Rana, Shahroz, Farah, Lubna,
Minhaj, Salman and especially Saba and Jehangir, who stood with me
through thick and thin and gave me comfort whenever I needed it. I also
extend my thanks to my close friends and relatives who may be far but yet
near to encourage me.
I would also like to mention the relentless effort of Mr. Wajid and Mr.
Mansoor of Central Photographs. My genuine thanks are also due to Mr.
Saleemuddin, cartographer for his fine sketches.
The non-teaching staff of the department mainly Mr. Aleem, Mr.
Dabeer and Jumman who were everready to help me. The office staff mainly
Mr. Gautam and the staff of clinical lab^ Mr. Aslahuddin. The animal house
attendants also need a special mention for taking care of my animals.
Financial assistance from the U.G.C. Government of India in the form ofS.R.F. is
gratefully acknowledged.
Last but not the least, I would like to express my gratitude to Mr
Alaishya Hood for taking pains to print this manuscript in its present form.
Nabiha Yusuf
CONTENTS
PAGE NO
ABSTRACT
LIST OF FIGURES
LIST OF TABLES
ABBREVIATIONS
INTRODUCTION
EXPERIMENTAL
RESULTS
DISCUSSION
REFERENCES
1
vi
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Xi
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41
75
149
161
^bstvnti
p i l a t e d c a r d i o m y o p a t h y ( D C M ) is a c h r o n i c hear t
muscle disease of unknown etiology characterized by dilatation and
contracti le dysfunction of left and/or the right ventricle. In man,
myocarditis is known to be the precursor of DCM in many cases, al
though clinical and histological diagnoses remain obscure. Since in
DCM, heart is the only affected organ, it fits into the criteria of organ
specific autoimmune disease. Autoimmune features in patients with
DCM include familial aggregation, a weak association with HLA-DR4
and with immunoglobulin genes, abnormal expression of HLA class II
on cardiac endothelium and increased levels of circulating cytokines.
Cardiac antibodies have been found against various antigens in
DCM patients. Multiplicity of parallel autoimmune reactions directed
towards a single organ may be due to spread sensitizatio n. Among
the various cardiac antigens, 150 kD C-protein has been found to in
duce myocarditis leading to DCM in experimental animals. C-protein
plays an important role in contractile activity and is one of the major
constituent proteins which are essential to compete with natural anti
gens at MHC antigen binding site.
In the present study, 150 kD human cardiac C-protein was
electroeluted and further purified by HPLC. C-protein was character
ized by ultraviolet and circular dichroic spectroscopy andSDS-PAGE.
Due to identical molecular weights, IgG and C-protein gave a similar
pattern on 7.5% SDS-PAGE. C-protein is an intracellular member of
immunoglobulin superfamily and it shares some of its domains with
1 1
IgG. In order to further substantiate the similarity pattern between
the two, the above were subjected to urea denaturation. Both IgG and
C-protein were denatured to give two prominent bands on urea SDS-
PAGf using an acrylamide (5-20%) and a urea (1-lOM) gradient.
The thermodynamical data speculate the tremendous structural
stability exhibited by C-protein which is a highly stable dimeric pro
tein molecule as evident from the observation that only 4.4 percent
had undergone thermal unfolding till TS^C. Fifty percent denaturation
was observed at around TQ.S^C. The negative AGj values above 79°C sug
gests the transition of completely folded C-protein to the unfolding
state. In comparision to native DNA, our results are indicative for C-
protein to be topologically less constrained. The 150 kD C-protein
which is acting as an autoantigen in DCM was found to be equally
reactive with naturally occuring SLE autoantibodies in which DNA is
acting as an autoantigen. The antigenicity of electroeluted HPLC puri
fied 150 kD cardiac C-protein was assessed by inducing the produc
tion of polyclonal antibodies in rabbit and rats. The repertoire of
specificities of induced antibodies was ascertained by direct binding
and competition ELISA.
DNA-protein crosslinks have been produced by typical photo
chemical reactions occuring in biological systems. The crosslinks have
been found to be covalent in nature and have been responsible for many
deleterious pathological processes. In this case, the HPLC purified C-
protein was photoadducted to commercially available calf thymus na-
I l l
tive DNA purified of protein and single stranded regions. Time course
kinetics revealed that the formation of photoadduct increased with
increase irradiation time. This was further reiterated by the amount
of bound lysine residues (from C-protein) to native DNA. Time course
kinetic study indicates that the formation of photoadduct obeys ap
parent first order reaction.
The prima donna in the category of oxygen ^ e e radicals (OFR's)
are oxygen itself, superoxide, hydrogen peroxide, transition metal ions
and hydroxyl (OH) radicals, the first four of which interact to gener
ate the last. These reactive oxygen species also produce DNA-protein
crosslinks in biological systems. In this study we have induced DNA-
C-protein crosslink by the action of H2O2 and OH. Among the two,
OH appears to form a better DNA-C-protein adduct in terms of recog
nition with DCM, SLE and immune sera. The adducts/photoadducts
were characterized by nuclease SI sensitivity assay.
Autoantibodies found in DCM patients showed remarkable bind
ing with HPLC purified C-protein. Competition ELISA results showed
inhibition in antibody activity ranging from 64.7% to 82.1%. More
over, inhibition of antibody activity was achieved at a very low mag-
nitude of inhibitor concentration. The induced anti C-protein antibod
ies exhibited much higher magnitude of binding. These results indi
cate the major involvement of 150 kD C-protein as an autoantigen in
DCM.
Native DNA showed poor reactivity with sera of DCM patients.
On the contrary, mitochondrial DNA (mt DNA) showed appreciably
I V
higher binding with DCM autoant ibodies . On ROS modif ica t ion , the
magnitude of binding decreased in case of mt DNA whi le it increased
in case of ds DNA but a relat ively low magni tude of binding was
observed when nDNA was photoadducted with C-pro te in . Conversely ,
the photoadduct ion of nDNA-C-protein in p resence of OH exhibi ted a
high degree of b inding. This could be due to cumula t ive binding efect
of immunogenic epi topes on C-protein as well as somewhat s imilar
epi topes to the immunogen on ROS modified DNA and hence when
both ROS-DNA and C-protein were photoadduc ted , a h igher degree of
binding was observed. This finding suggests that p robab ly apart from
150kD C-protein, the photoadducted ROS-DNA-C-pro te in complex
could also act as an al ternate autoantigen for myoca rd i t i s /DCM.
The binding of induced anti C-protein an t ibod ie s with h is tones ,
ROS his tones , po ly-D- lys ine , poly-L-glutamate and poly (L+G) com
plex was of low magni tude , thereby, suggest ing for the presence of a
minor subpopulation of epitopes responding to the anti-C-protein antibod
ies. Similar binding patterns with DCM autoantibodies further substanti
ates the finding.
In the present study, attempts were also made to probe the possible
involvement of 150kD cardiac C-protein which is an autoant igen in
myocarditis/DCM, in the induction of autoantibodies in SLE patients. As
says of anti-DNA and anti-human heart antigens in the sera of DCM and
SLE patients were carried out by employing direct binding ELISA where as
specificity determination was accomplished by competition ELISA. Inter-
estingly, as evident from the binding results, human heart extract proteinic
antigen (s) reactive with DCM autoantibodies showed strong recognition of
SLE anti-DNA antibodies. This is suggestive for the possible involvement
of a common or somewhat similar trigger/epitope (s) for immune response
in autoimmune myocarditis/DCM and autoimmune SLE, although such in
ferences could not lead to confirmity at such a preliminary level of investi
gation. The mechanism or the role of myocarditis inducing 150kD C-pro-
tein in stimulation of immune system for production of antibodies in SLE
remains to be ascertained.
In conclusion, 150kD human cardiac C-protein has been found to be a
potent immunogen capable of inducing polyclonal antibodies in experimen
tal animals. It is a thermodynamically stable molecule and shares some of
its domains with IgG as revealed by thermal denaturation and urea denatur-
ation studies. C-protein showed strong recognition with DCM sera as well
as immunoaffinity purified SLE anti-DNA autoantibodies. Out of the ad-
ducts/photoadducts formed by crosslinking C-protein with native DNA, the
one formed in presence of OH showed strongest recognition towards DCM
and anti C-protein autoantibodies, thereby indicating the possibility of ROS-
DNA-C-protein photoadduct as an alternate autoantigen in myocarditis/
DCM.
V I
LIST OF FIGURES
Figure 1
Figure 2
Figure 3
Piguie 4
Figuie 5
Figuie 6
Figuie 7
Figuie 8
Figuie 9
F-iguie 10
Figui e ] 1
Figuie 12
Fmuie 13
Eiec t rophore t ic pat tern of total human heart extract 76
(THHt ) in 5-20% gradient SDS-PAGE
Elution profile of purification of electroeluted 150 kD 77
human cardiac C-protein by HPLC
Agarose gel electrophoresis to show the absence of any 73
trace of DNA in THHE
Ciicular dichroic spectia of 150 kD human cardiac C- 80
protein
Thermal denaturation profile of HPLC purified 150 kD 81
human cardiac C-piotein
Electiophoretic pattein of C-piotein and IgG on urea SDS- 87
PAGE
Binding of anti C-piotein antibody in rabbit with C- 89
piotein
Binding of anti C-piotein antibodv in rat with C-piotein 89
Inhibition of anti C-piotein antibodv in rabbit with C- 90
piotein
Inhibition of anti C~piotein antibodv in lat with C-protein 90
Elution piofile of IgG on protein-A.-sepharose CL - 48 92
column
Binding of DC\1 and immune IgG with C-piotein 93
Inhibition of immune IgG activitv bv native and ROS C- 95
protein
V l l
Figure-14 Inhibition of immune IgG activity by nDNA C-protein 96
adducts/photoadducts.
Figure 15 Inhibition of immune IgG activity by native and ROS 97
histone.
Figure 16 Inhibition of immune IgG activity by Poly-L-lysine, Poly- 9 8
L-glutamate and Poly (L+G) complex.
Figure 17 Inhibition of immune IgG activity by ROS DNA and RNA. 99
Figure 18 Direct Binding ELISA of THHE with DCM autoantibodies. 102
Figure 19 Inhibition ELISA of DCM autoantibodies with C-protein lOa
as inhibitor.
Figure 20 Direct binding ELISA of DCM autoantibodies with 1 50 kD 105
C-protein.
Figure 21 Direct binding ELIS.A of DCM autoantibodies with native 106
DNA.
Figure 22 Inhibition of DCM IgG activity by native and ROS C- 108
protein.
Figure 23 : Inhibit ion of DCM IgG activity by nDNA C-protein 109
adducts/photoadducts.
Figure 24 Inhibition of DCM IgG activity by native and ROS llO
modified histone.
Figure 25 Inhibition of DCM IgG activity by Poly-D-lysine. Poly- 111
L-Glutamate and Poly (L+G) complex.
Figure 26 Inhibition of DCM IgG activity by ROS DNA and RNA. 112
Figure 27 Time course pattern of nDN.\-C-protein photoadducts us
formed by UV-B irradiation.
V l l l
Figure 28 UV absorption charactei is t ics of i rradiated nDNA-C- 116
protein
Figure 29 I'V absorption chractenstics of nDNA-C-protein and their ng
adducts after ROS modification
Figure 30 t lec t iophore t ic mobility of nDNA-C-protein and their ^21
adducts after ROS modification
Figure 31 Nuclease SI sensitivity assay of nDNA-C-protein adducts/ 122
photoadducts
Figure 32 Time course kinetic piofile of photoaddition of C-protein 124
to na tne D N ^
Figure 33 Standaid plot foi estimation of 1\ sine by T \ B S method 125
Figuie 34 kinetic profile of photobound 1\ sine residues (piesent in 126
C-piotein) to n D \ \
Figuie 35 'Xppaient first ordei kinetic profile of nD\A-C-protein 127
photoadducts formation at vanous inadiation times
Figure 36 Immunoaffinitv puiification of SLE IgG on nDNA poly 129
1\ s\ 1 sephaiose 4B column
Figure 37 Binding of n D \ ^ C-piotein photoadduct with DCM. SLE 131
and immune IgG
Figure 38 Binding of ROS D \ A C-piotein adduct with DCM. SLE 132
and immune IgG
Figuie 39 Binding of ROS D \ \ C-piotein photoadduct with DCM. 133
SI E and immune IgG
Figuie 40 Binding of SEE autoantibodies with ds DNA. ssDNA RNA134
and IHHE
IX
Figure 41 Inhibition ELISA of SLE autoantibodies with DNA, ssDNA 135
and RNA as inhibitors on plates coated with ds DNA.
Figure 42 Inhibition ELISA of SLE autoantibodies with THHE as i36
inhibitor on plates coated with ds DNA.
Figure 43 Inhibition ELISA of protein-A-sepharose isolated SLE IgG 137
with 150 kD C-protein on plates coated with ds DNA.
Figure 44 Inhibition ELISA of immunoaffinity purified SLE IgG with 138
150 kD C-protein on plates coated with ds DNA.
Figure 45 Direct binding ELISA of DCM autoantibodies with nativei40
and ROS mt DNA.
Figure 46 Inhibition ELISA of DCM autoantibodies with native ni t i4i
DNA as competitor.
Figure 47 Inhibition ELISA of DCM autoantibodies with ROS mti42
DNA as competitor.
Figure 48 Glucose le \e ls in sera of patients \\ith DCM. 143
Figure 49 Total serum cholesterol levels in patients with DCM. 144
Figure 50 Levels of uric acid is sera of patients with DCM. 145
Figure 51 C-reactive protein (CRP) level in sera of patients withl46
DCM.
LIST OF TABLES
Table 1 : Thermal denaturation of 150 kD human cardiac C-protein. 84
Table 2 : Thermal denaturation of IgG. 85
Table 3 : Antigenic specificity of anti C-protein antibodies. 100
Table 4 : Competition ELISA of 150 kD cardiac C-protein with DCM 104
autoantibodies.
Table 5 : Antigenic specificity of DCM autoantibodies. 113
Table 6 ; UV absorption characteristics of formation of nDNA C-protein 117
photoadduct.
Table 7 : UV absorption characteristics of formation of ROS DNA C- 120
protein adduct/photadduct.
Table 8 : Competition ELISA of mt DNA with DCM autoantibodies. 143
X I
ABBREVIATIONS
BSA : Bovine serum albumin
CD : Circular Dichroism
DCM : Dilated cardiomyopathy
DNA : Deoxyribonucleic acid
ds DNA : Double standard DNA
EDTA : Ethylene diamine tetra acetic acid
ELISA ; Enzyme linked immunosorbent assay
HPLC : High performance liquid chromatography
IgG : Imunoglobulin G
kD : Kilo Dalton
MHC : Major Histocompatibility complex
mt CM : Mitochondrial cardiomyopathy
mt DNA : Mitochondrial DNA
Oj" : Superoxide anion radical
°OH : Hydroxyl radical
RNS : Reactive Nitrogen species
ROS : Reactive Oxygen species
ss DNA : Single stranded DNA
SDS - PAGE : Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SLE : Systemic lupus erythematosus
THHE : Total human heart extract
TNBS : Trinitrobenzene sulphonic acid
Tris : Tris (hydroxymethyl) aminomethane
TEMED : N, N, N , N- Tetramethylethylene diamine
UV : Ultraviolet
^1 : Microlitre
Jig : Microgram
(3lntr0buctt0n
^ i l a t e d Cardiomyopathy (DCM) is a chronic heart muscle
disease characterized by impaired systolic function of left or/and right
ventricle (Richardson et al., 1996). It is a major cause of severe
congestive heart failure in young people and the commonest cause of
transplantat ion worldwide. The heart is suscept ible to immune
mediated injury. The myocytes may be injured during or after immune
response to endogenous or exogenous cardiac antigens. Immune
mediated myocyte injury may be reversible and manifested as a
t rans ien t depression of ven t r i cu la r function and/or e lec t r i ca l
instability, or it may be irreversible, ultimately leading to myocyte
necrosis.
Mechanisms of immune mediated injury may be classified into
humoral and cellular types. Humoral immunity requires recognition
of an epitope by antibody molecules expressed by B-lymphocytes and
depends on circulating antibodies to initiate injury. Several cardiac
disorders like post myocardial infarction syndrome, idiopathic dilated
cardiomyopathy, viral myocarditis and doxorubicin cardiotoxicity are
accompanied by deposition of antibodies within the myocardium,
particularly on the sarcolemma (Rose et al., 1988). Moreover, 13-95%
patients with various myocardial diseases such as idiopathic dilated
cardiomyopathy, myocarditis and hypertrophic cardiomyopathy have
shown the presence of heart react ive antibodies in their serum
(Neumann et al., 1990). The major mechanism of cell injury mediated
by humoral immunity may be the activation of complement via the
classic pathway.
Cellular immunity also plays a role in the mechanism underlying
cardiac dysfunction. It is initiated by recognition of an immunogenic
epitope by antigen specific T-lymphocytes. This initial recognition
and activation are effected by helper T-cells and require presence of
histocompatible antigen presenting cells. Presentation of myocardial
antigen to T-lymphocytes by antigen presenting cells can result in the
clonal expansion and differentiation of cytotoxic T-cells, stimulated
by the release of immune cytokines. As a consequence, myocyte
necrosis occurs, resulting in fibrosis (Sanderson et al., 1985).
Extensive studies have provided appreciable evidences for the
presence of a wide spectrum of autoantibodies against cardiac tissue
in myocardial diseases (Maisch et al., 1983; Caforio et al., 1992). In
patients with DCM, circulating autoantibodies to distinct cardiac
autoantigens have been described providing evidences for autoimmune
involvement. Thus, autoimmune disorders result due to immunologic
reactions, humoral and/or cellular, directed against the individuals own
tissue components. The precise mechanisms underlying the trigger of
autoimmune reaction are not yet completely understood, however
certain factors are said to be involved.
Shoenfeld and Isenberg (1989) described the wide spectrum of
autoimmune diseases as a mosaic of autoimmunity with many factors
leading to diverse diseases.
T-helper/T-suppresser cell imbalance:
The T-cell sub-sets control / regulate the immune response. In
autoimmune diseases, the ratio of T-helper (Th) to T-suppresser (Ts)
cells in peripheral blood tends to be nearly 10-15 : 1, particularly
during active or acute phase of the disease compared to 2 : 1 in normal
individuals (Deodhar, 1992).
Polyclonal B-cell activation:
Polyclonal B- and/or T-cell activation has been an initiating
mechanism. The proposition that polyclonal B-cell activators can
induce autoantibodies is predicted on the existence of non deleted
self reactive B-cells and/or developmentally arrested anergised B-cells
that might become active upon appropriate stimulation. A large number
of molecules, particularly of microbial origin have been found to act
as polyclonal B-cell activators and in the induction of autoantibodies.
Polyclonal stimulation of a large set of T-cells by bacterial/viral
surface antigens (SAgs) is another suggested scenario. T-cells that
react with MHC class II bound SAg on B-cells may mutually stimulate
the SAg displaying B-cells, thereby leading to production of polyclonal
immunoglobulins and in some instances, autoantibodies. Alternatively,
the activated T-cells themselves may induce tissue damage, through
cross reactions with self molecules (Theofilopoulos, 1995).
Genetic factors:
Gene t i c t ransmiss ion in Id iopa th ic DCM (IDC) has been
occasionally described in the past while importance of genetic factors
is a recent advance (Mestroni et al., 1990). In control studies.
familial IDC is detectable in over 20-25% of patients with a diagnosis
of I D C , with a prevalent autosomal dominant trait. A single dominant
locus was proposed and for the putative disease gene, a gene frequency
in the order of 10"* was estimated in overall population.
The candidate genes for familial IDC can be divided into two
main groups, genes that are responsible for normal heart function like
the ones encoding contractile proteins such as myosin light and heavy
chain, tropomyosin, troponins and actin. Other candidate genes are
genes coding for proteins involved in metabolic pathways: genes
encoding for atrial natriuretic factor and its receptor, phospho lamban,
G-proteins , p - adrenoceptors and Ca^' channels appear to be of
particular interest. According to autoimmune pathogenetic hypothesis
of IDC, the second group of genes are involved in immune function.
Immune dysfunctions in IDC involves cellular and humoral immunity
such as organ specif ic au toan t ibod ie s (Caforio et al . , 1994).
Association studies indicate a strong correlation between disease and
human leukocyte antigens (HLA). Associat ion between immune
response (Ir or MHC) and T-cell receptor (TCR) genes and the
development of immune mediated cardiac injury are not unexpected
since most autoimmune diseases are T-cell dependent and all T-cell
mediated responses are major histocompatibili ty complex (MHC)
restricted (Bach, 1995). MHC may alter predisposition by shaping of
TCR repertoire, antigenic peptide selection and presentation and
peptide transport. HLA-DR associations play a major role in patients
with DCM. HLA-DR positive patients were six times more likely to
have antibodies compared to those who did npt be long to this
phenotype. Antibodies against different cellular constituents may have
distinct immunogenetic associations. On the other hand, HLA-DR4
and HLA-DRl share common epitopes which may confer susceptibility
to autoimmunity in DCM.
In addi t ion , the e x i s t e n c e of an a s s o c i a t i o n b e t w e e n
autoantibodies, clinically overt disease and certain restriction fragment
length polymorphisms (RFLPs) of HLA-DRp and -DQa genes suggests
that subtypes of these molecules may have enhanced pathogenetic
significance (Limas, 1996).
Immune deficiency states:
The availability of severe combined immunodeficient (SCID)
mouse provides a potentially unique opportunity to study contributions
made by the immune system in induction of coxsackie virus B3 (CVB3)
induced myocarditis. Unlike congenitally athymic (nu/nu) mice, the
SCID mutation results in an absence of both B- and T- cell function,
due to an aberrant VDJ recombinase mechanism. While no functional
T- or B-cells are present in the SCID mouse, ant igen presenting
capabilities and natural killer cell activity are intact (Bosma and
Caroll, 1991).
In 1986, Cohen et al. described the first cases of rapidly fatal
DCM in three AIDS patients. Since then a number of prospective
clinical and echocardiography studies have shown that a subgroup of
HIV infected patients may be predisposed to the development of
clinically significant and progressive heart disease (Herskowitz et al.,
1992; Herskowitz & Baughman, 1994). This subgroup of patients
frequently present with class HI or IV congestive heart failure and
demonstrate rapid clinical deterioration. One emerging hypothesis to
explain the high frequency of dilated heart muscle disease in HIV
seropositive patients is the association between HIV related ventricular
dysfunction and myocarditis. The increased CD8+ T-lymphocytes and
sole induction of MHC class I in the HIV related myocard i t i s
popula t ion are l ikely due to the marked systemic decrease in
circulating CD4+ T-lymphocytes in AIDS patients (Levy et al., 1985).
Studies by Matsumori et al. (1994) have suggested alterations
in cytokine production in patients with dilated heart muscle disease.
Patients with HIV related myocarditis have elevated levels of TNF-a
and IL-6 compared to patients without myocarditis suggesting that
specific cytokines may play a role in the pathogenesis of HIV related
myocarditis (Herskowitz et al., 1995).
Molecular mimicry:
Most s tudies have addressed molecular mimicry as cross
reactions of antibodies with linear peptides shared by self and foreign
molecules (Theofilopoulos, 1995). The term 'molecular mimicry' was
i n i t i a l l y used in 1968 to exp la in pe r s i s t en t v i ra l i n f ec t i on .
Autoimmunity provided by molecular mimicry should occur only when
the microbial and host determinants are similar enough to cross react.
yet different enough to break immunologic tolerance. The induction
and breaking of tolerance at both the B- and T-cell levels have been
established in heterologous serum protein models , and the same
kinetics probably govern the establishment an4 breaking of tolerance
to microbial agents cross reacting to host proteins.
It is to be pointed out that an antibody reacts with the three
dimensional configuration of a molecule, the antibody could bind but
with a different affinity, to similar but not necessari ly identical
configurations. The multiple organ reactive antibodies recognized
either the same molecule present in more than one organ or different
molecules in multiple organs (Haspel et al., 1983). Molecular mimicry
is a common phenomenon. The role cross reacting antibodies play in
pathogenesis will depend on the nature and location of the autoantigens
as well as on the properties of the antibodies (Srinivasappa et al.,
1986). Homology by itself may not lead to a cross reacting immune
r e s p o n s e . Howeve r , unless the homology and subsequen t
immunological cross reactivity involve a host protein that can
precipitate disease, the autoimmune response is unlikely to lead to
autoimmune disease.
Infectious agents may participate in the induction of autoimmune
disease not only through the process of mimicry, but also through other
effects, including tissue damage and release of sequestered antigens,
availability of cryptic self determinants through increased MHC
expression, ec topic expression of molecules , redis t r ibut ion of
intracellular molecules to the cell surface, upregulation or shift in
the spectrum of cytokine production, immunological exhaustion and
bystander activation of T-cells (Theofilopoulos, 1995).
In humans, myocarditis is known to be the precursor of DCM in
some cases, although clinical and histological diagnosis remains
obscure (Aretz et al., 1985). The commonest cause of myocarditis is
said to be a virus infection, but majority of cases are of unknown
etiology (Woodruff, 1980). Pedigrees where myocarditis and DCM
were present in different family members have been repor ted
(O'Connell et al., 1983).
Since in DCM. the only affected organ is the heart muscle, to
prove autoimmune involvement, it is necessary that the criteria of an
organ specific autoimmune disease are fulfilled (Rose and Bona, 1991).
Autoimmune features in DCM include familial aggregation, a weak
association with HLA-DR4 and immunoglobulin genes, abnormal
expression of HLA class II on cardiac endothelium and increased levels
of circulating cytokines. The organ-specific antibodies produced a
diffuse cytoplasmic staining of myocytes but did not stain skeletal
muscle. Antibodies were classified as cross reactive 1, which exhibit
only partial heart specificity, gave a fine striational staining on cardiac
tissue and stained weakly skeletal muscle fibres. The entirely cross
reactive antibodies, classified as cross reactive 2. stained with a broad
striational pat tern both in heart and skele ta l muscle sec t ions .
Absorption studies with relevant tissues had confirmed the organ
specificity and cross reactivity of the antibody types (Caforio et al.,
1990).
Like other autoimmune conditions, DCM autoantibodies seem
to be produced against multiple antigen specificities. The multiplicity
of parallel autoimmune reactions directed towards a single organ could
be due to spread sensitization. Thus, a single, initial autoantigen would
give rise to the first attack of the target organ, which would result in
T-cell mediated inflammation or the release of degradation products.
These mechanisms would enhance autoantigen presentation or release
immunogenic cell components that would give rise to a secondary
autoimmune response that would be difficult to differentiate from the
primary one. Conversely, as suggested, the initial event is a target
cell abnormality or lesion that renders a set of immunogenic peptides
: only the peptide with a sufficient affinity for MHC molecules would
be recognized by the T-cell repertoire, leading to an autoimmune
response (Bach, 1995).
Several autoantigens which react with sera from patients with
DCM have been identif ied. Studies on t issue from pat ien ts of
myocardi t i s and DCM have identif ied increased express ion of
intracellular antigens such as adenine nucleotide translocator (ANT)
and branched chain ex-ketoacid dehydrogenase (BCKD) complex.
Antibodies against the intracellular mitochondrial ANT protein cross
react with myocyte sarcolemmal calcium ion (Ca^ ) channel proteins.
Binding of Ca' channel proteins by such cross reactive antibodies can
10
thus physiologically alter the metabolism of normal myocyte, possibly
leading to myocyte injury (Ansari et al., 1991). Since SR-Ca^* ATPase
constitutes the major calcium regulatory protein in the sarcoplamic
reticulum regulating myocardial contractility, autoimmune inhibition
of this enzyme may affect the functional outcome of the myocardium.
The time dependent association between SR-Ca^^ ATPase immunization
and development of severity of myocarditic lesions supports the
immunopathogenetic role of SR-Ca^' ATPase. The involvement of T-
cell immunity may contribute more to the generation of myonecrosis,
whereas the antibody mediated immunity may contribute more to
functional impairment and activation of complement (Sharaf et al.,
1994). The ADP/ATP carrier has organ specific antigenic determinants
although there is a partial identity among the carrier proteins from
heart, kidney and liver. The organ specificity of ADP/ATP carrier was
also conf i rmed by the organ specif ic r eac t ion of d i l a t ed
cardiomyopathy sera with this protein. The autoantibodies reacted with
antigenic determinants at the cardiac myocyte plasma membrane and
interacted with atleast one subunit of calcium channel. Therefore, the
autoantibody was also called the Ca-channel reactive antibody. A cross
reaction between antigenic determinants could be of pathogenic
significance because antibodies against the homologies were present
in pa t i en t s with m y o c a r d i t i s and DCM (Ansar i et a l . , 1 9 8 8 ;
Schwimmbeck et al., 1993).
After an au to immune response is in i t i a t ed , c i r c u l a t i n g
autoantibodies against the ADP/ATP carrier disturb myocardial energy
11
metabolism and cross react with myocyte sarcolemmal Ca^' channel
proteins. The antibodies were able to bind to the Ca^^ channel and
enhance calcium influx and calcium overload in cardiac myocytes,
finally resulting in cytotoxic damage (Liao, 1996).
The most important intracellular microorganisms associated with
viral disease include a variety of viruses (Woodruff, 1980), two
m e m b e r s of ch lamydiae (Chlamydia psittaci and Chlamydia
pneumoniae) (Kuo et al., 1993), several rickettsiae. Toxoplasma gondi
and Toxoplasma cruzi (Speirs et al., 1988). Trichinella spiralis is the
most prone worm to cause myocarditis. Myocarditis pathogenesis may
be complex and involve various immune mechanisms. In addition,
many gram-posi t ive and gram-negat ive bacter ia may persis t and
multiply intracellularly or extracellularly or both and cause damage
to the myocardium by various pathways . Some bacteria produce
exotoxins that may cause damage to the myocardium, whereas others
release endotoxins that may be harmful. The bacteria commonly
encountered in myocarditis are Beta-hemolytic streptococci (Putterman
et al., 1991), Corynebacterium diptheriac (Ua\a\daiT, 1992), Neisseria
meningitidis (Hardman and Earle. , 1969), Yersinia enierocolitica
(Zollner et al., 1992), Salmonella typhi or paratyphi and Borrelia
burgdorferi (Klein et al., 1991). Mycoplasma pneumoniae was
incriminated in 6% of consecutive military conscripts suffering from
unequivocal myocarditis, and in 1-2% of conscripts hospitalized with
mycoplasmal infections (Karjalainen, 1990). Trypanosoma cruzi
(Chagas ' disease) is a well recognized cause of myocarditis and
12
cardiomyopathy in both urban and rural areas of South America. The
African trypanosomes, T. gambiense and T. rhodesiense occasionally
cause myocarditis and cardiomyopathy (Tsala Mbala, 1988).
Coxsackie virion and other viral proteins share epitopes with
internal or plasma membrane proteins of normal cells (molecular
mimicry) and s t imulate immune responses which par t ic ipa te in
autoimmune reactions. These hypotheses may not be exclusive and
all may be operative in a single model. CVB3 or CVB4 particles share
epitopes with human cardiac myocyte sarcolemmal proteins, human
and mouse cardiac myos ins , s t reptococcal M-prote in , adenine-
translocator protein and an unidentified protein(s) on the plasma
membrane on no rma l mouse ca rd iac myocytes or f ib rob la s t s
(Srinivasappa et al., 1986).
This underlines the clearence of virus via rapid and other
immune responses significantly affects the outcome (acute resolved
versus acu te / ch ron ic m y o c a r d i t i s ) . Antecedent i n f e c t i o n s by
heterologous CVB3 serotypes can exacerbate or aneliorate in CVB3
induced myocarditic challenge mechanism by which shared epitopes
between viral cel lular prote ins can provide either protect ion or
increase severity of CVB3 induced myocarditis. Studies by Gauntt et
al. (1995) suggest that antibodies to only a limited numbers of epitopes
on the light meromyosin (LMM) rod portion of human cardiac myosin
(^M) are shared with CVB3m and participated in protection/enhanced
disease and antibodies to the majority of shared epitopes in LMM are
13
without consequence in CVBSm induced heart disease. Resistance to
this enterovirus and other intracellular infections partly depends on
natural kil ler cells (NK) which are vulnerable to the effects of
environmental pol lutants such as mercury, nickel, 8-tetrachloro-
dibenzo-p-dioxin and methyl mercury. The development of disease as
seen in the heart of CBS virus infected mice, can be severely affected
in different ways by different heavy metals . Furthermore, during
infection, the organ distribution of heavy metals may be changed in a
specific way for each compound. This may subsequently result in
altered infectious disease pathogenesis (Ilback et al., 1995).
The G-protein coupled membrane receptors are all members of
a superfamily of membrane proteins which structurally share the
overall bacteriorhodopsin architecture, seven a -helices, spanning
the membrane lipid bilayer and forming a hydrophobic pocket that
can serve as a pharmacophore. The seven helices are linked together
by three extracellular and three intracellular loops. The N-terminus
of the polypeptide is located in the extracellular space while the C-
terminus is located in the intracellular space. In order to induce an
immune r e s p o n s e aga ins t G-pro te in c o u p l e d r ecep to r s , two
requirements are necessary : firstly the receptor must be degraded by
proteolysis to small fragments and the fragments generated must be
able to form a complex with one of the MHC or HLA class II molecules
of the host.
The functionally important G-protein coupled receptors are (3 -
adrenoceptor and the M2 muscarinic receptor . Anti-p receptor
14
antibodies were present in 30-40% patients of dilated cardiomyopathy.
These antibodies were produced against P, peptide corresponding to
the second extracellular loop with little reactivity towards a P^ peptide
in patients with DCM. P,-adrenergic receptor has two antigenic
regions. Some of the sera recognize the first extracellular loop, other
sera the second extracellular loop of the P,-adrenoceptor. The
dominant epitope in the second loop is the cysteine-containing amino
acid sequence A-R-R-C-Y-N-D which forms a disulphide bridge with
the cysteine of first extracellular loop (Wallukat et al., 1995). The
immunoglobulin fraction isolated from serum samples of myocarditic
and cardiomyopathic patients contains stimulatory autoantibodies,
directed against P,-adrenoceptor which increase dose dependently,
the beating rate of the cultured neonatal heart myocytes. Recent
studies have pointed out the presence of anti-receptor autoantibodies
in DCM (Fu et al., 1994). Immunization of genetically predisposed
mice with cardiac myosin causes cardiac enlargement, the production
of heart-specific antibodies and a heart condition that resembles
human DCM. the autoantibodies in patients against human ventricular
myosin are directed against myosin heavy chain. The a - and P-myosin
heavy chains are relevant autoantigens by the heart specific antibodies
detected by immunofluorescence in DCM. In humans, the a -myosin
heavy chain isoform is expressed exclusively in atrial myocytes,
whereas the P -isoform is present both in ventricular myocytes and
slow skeletal muscle fibers (Caforio et al., 1992). Cardiac myosin
induced myocarditis is T-cell mediated and class II MHC restricted.
15
Myosin-reactive T-cells from normal myosin immunized donors
induced severe myocarditis in severe combined immunodeficiency
(SCID) mice. Potential myosin reactive T-cells, though lacking CD4
or CDS, still remain functional and hence CD4 or CDS molecules are
not essential for induction of autoimmune myocarditis (Pummerer et
al., 1995).
Myosin is an intracellular molecule. Viral infection or other
causes of tissue necrosis might lead to release or exposure of myosin
and trigger autoimmunity in individuals with a predisposing genetic
background. Virus may promote auto-sensi t izat ion to myosin by
molecular mimicry (Oldstone, 19S7; Srinivasappa, 19S6). Monoclonal
antibody to coxsackie VP-1 capsid protein has been demonstrated
which reacts with cardiac myosin heavy chain.
The mechanism by which myosin as well as other autoantigens
previously identified may trigger and perpetuate, although not directly
cause autoimmune disease is unknown. It also remains to be established
whether the antibodies found in DCM sera have a direct pathogenic
role or, like the anti-myosin antibodies found in murine autoimmune
myocarditis, are only markers of immune damage.
Role of 150 kD cardiac C-protein:
Cardiac C-protein is found to be a dominant antigen among many
types of cardiac pro te ins and is known to induce autoimmune
myocarditis as judged on the basis of experimental studies indicating
16
production of autoantibody by mouse specifically against 150 kD C-
protein on induction of myocarditis. This specific antibody is produced
inspite of immunization with whole cardiac tissue homogenate .
Purified C-protein also produced myocarditis and recombinant cardiac
C-protein (amino acid res idue 205-916) effect ively p roduced
myocarditis in some mice strains including SMA, DBA/IJ, SJL, and
02°/A, but not in A/J, AKR/J, or DBA/2J mice strains (Kasahara et
al., 1994). C-protein is a component of thick filament of skeletal and
cardiac muscles (Yamamoto and Moos, 1983). The function of C-
protein remains unknown, but it has been suggested that C-protein
may regulate thick filament assembly and length (Squire, 1981),
participate in thick filament structural support (Pepe et al., 1975),
maintain the structure and contribute to the radial elasticity of the
sarcomere, or regulate cross-bridge movement during contraction
(Magid et al., 1984).
C-protein has a number of properties that make particularly
attractive the suggestion that it plays a role in regulation of contractile
activity. For example, C-protein binds to both purified actin (Moos et
al., 1978) and myosin (Moos et al., 1975) and can alter the ability of
actin to stimulate myosin ATPase (Moos and Feng, 1980). The effects
of C-protein on ATPase activity are, however, complex . C-protein
inhibits skeletal muscle actomyosin ATPase, but stimulate cardiac
muscle actomyosin ATPase (Yamamoto and Moos, 1983). It has been
suggested that the physiological role of C-protein may involve a
calcium-regulated binding of C-protein to thin filaments, because C-
17
protein binding to native thin filaments occurs only in the presence
of micromolar concentrations of calcium (Moos, 1981). It has been
shown that C-protein in cardiac muscle becomes phosphorylated in
response to P-adrenergic agonists and dephosphorylated in response
to cholinergic agonists (Hartzell and Titus, 1982). The level of protein
phosphorylation correlates with the rate of relaxation of cardiac
contraction and it has been suggested that C-protein regulates twitch
relaxation in cardiac muscle. Electron microscopy shows C-protein
to be V-shaped. The other par t ic les could be seen as distorted,
stretched or collapsed V-shaped particles.
C-protein is one of the major constituent protein necessary to
compete with natural antigens such as invariant chain and MHC to
obtain the position at the MHC-antigen binding site (Demotz et al.,
1990; Harding and Unanue , 1990). Moreover, C-prote in is an
intracellular member of the immunoglobulin superfamily (Einheber
and Fischman, 1990). IgG like domains are conserved in most cell
surface immune recognition molecules and also TCR and MHC class
II have this domain (Williams and Barclay, 1988). The peptides of
recycled membrane proteins, class II HLA and invariant chain, have
been shown to be major self-antigens which are presented by class II
molecules. However, the HLA binding motifs are located in the
variable domain of this structure, not in this constant IgG like domain
(Rudensky et al., 1992; Baum et al., 1993). The question remains
whether or not these IgG like conformations work with each other at
cell surface similar to the directly bound MHC-TCR like superantigen
(Woodland and Blackman, 1993).
18
The findings of Kasahara et al. (1994) conflict with an earlier
report that myosin is the major antigen producing au to immune
myocarditis under H-2 genetic restriction (Neu et al., 1987). In SMA
mice, repeated injections of myosin-enriched heart extract with K 0 3
LPS still preferentially produced the autoantibody to C-protein. In
addition, myosin contamination was completely avoided by using the
fusion protein expressed in E. coli PI6-4, which is encoded by part of
the cDNA of C-protein. CB3 induced myocarditis which might further
trigger autoimmune reactions against myosin by a molecular mimicry
mechan ism, has been sugges ted to be a p o s s i b l e u n d e r l y i n g
pathogenesis in DCM (Noel et al., 1991). Kasahara et al. (1994)
suggested that the autoimmune triggering mechanism in C-protein
induced myocarditis may differ from a CB3-triggered or myosin-
triggered mechanism because cross-reactive epitopes between CBS and
C-protein have not been reported; contrary to the previous results that
A/J, C3H/He, Balb/c, DBA/2 strains have a high susceptibility to CB3
(Lawrence et al., 1991), the C-protein residue 205-916 hardly induced
myocarditis in these strains.
Fur ther assessment about the i nvo lvemen t of C-pro te in
t r iggered autoimmune myocardi t i s in DCM, immunoblot t ing was
employed with sera of 16 DCM patients against whole cardiac
proteins. Out of 16 DCM patients sera of 2 patients preferentially
recognized the C-proiein. On the contrary, none of the control sera
reacted specifically to C-protein (Kasahara et al., 1994).
20
crossreactivity. The maximum score possible for each peptide is
variable and is determined by its length and by the amino acids present,
rarer amino acids having a higher value. The scoring system used,
attaches the highest values to exact matching of rare amino acids. This
level of matching would be expected to lead to cross reactivity of
epitopes since far weaker matching occurs within conserved motifs of
peptide^ bound to class II major histocompatibility complex peptides.
Differences between individuals in MHC class II may influence the
selection of particular hsp epitope and the corresponding target antigen
that gives rise to an autoimmune disease (Jones et al., 1991).
The amino acid sequences/peptides of autoantigen region 384-
396 and 451-470 of hsp share homology with the amino acid sequences/
peptides of myosin heavy chain 136-148 and 253-272 with a similarity
score of 65 and 64, respectively, as seen in coxsackie myocarditis
(Jones et al., 1991). Latif et al. (1993) have reported antibodies against
a 60 kD band in 85% DCM patients by Western blotting. Two
dimensional gel analysis and protein sequencing revealed 100%
homology of this peptide with hsp 60. The presence of circulating
antibodies against hsp 60 in 85% of DCM patients is clear evidence
of a damaged myocardium. This suggests that myocyte injury induces
the expression of hsp60 or other stress proteins that may have immuno
modulatory activities and could influence the development and course
of autoimmune recognition of the myocardium. The exact pattern and
distribution of hsp60 in normal and diseased myocardium remains to
be established.
21
DNA-protein photoadducts/photointeractions:
A variety of chemicals and ionizing and ul traviolet (UV)
radiations are known to produce DNA-protein crosslinks (Oleinick et
al., 1986). Several reports indicate that the bonds formed between DNA
and proteins are of covalent nature (Oleinick et al., 1986; Cress and
Bowden, 1983). Proteins and nucleic acids are the two most important
classes of functional biomolecules in cel ls . These "co lour less"
macromolecules absorb in the UV region of spectrum. Typical
photochemical reactions have been identified that are relevant to
biological damage induced by exposure of organisms to radiation.
The crosslinking of nucleic acids to protein is one of the lesions
produced in biological systems by UV light, which has been observed
in bacteria (Smith, 1962; Alexander and Moroson, 1962; Bridges et
al., 1967) and in mammalian cells (Alexander and Moroson, 1962;
Habazin and Han, 1970; Han et al., 1975; Zimmernan et al., 1972).
The photobinding of serum albumin to DNA (Maukovitz, 1972) and
gene-5-protein to bacteriophage IdDNA (Anderson et al., 1975)
provides direct proof at a molecular level for the crossl inking
p h e n o m e n o n . The role of DNA-pro t e in c ros s l inks in ag ing ,
carcinogenesis and radiation biology has been well documented
(Smith, 1975).
The recognit ion between nucleic acids and proteins is of
fundamental importance since it triggers most steps of nucleic acid
metabolism. This recognition, presumably, first involves electrostatic
22
interactions of phosphate groups with positively charged regions in
proteins; then, more specific interactions take place which involve
possible interactions between nucleic acids bases (or other structural
elements) and amino acids side chains in proteins. A more extended
scope of examination reveals the possible recognition of secondary
or tertiary structures of nucleic acids by proteins. In either cases,
recognition may be dependent of nucleotide sequence (Duguet, 1981).
The initial isolation of a mixed photoproduct of thymine and
cysteine (5-S-cysteine-6-hydrothymine) from in vitro UV irradiation
of a solution of thymine and cysteine has served as a model for
crosslinking phenomenon. Fifteen amino acids have been found to
react photochemically with DNA. Cysteine, lysine, phenylalanine,
tryptophan and tyrosine were the most reactive; alanine, aspartic acid,
glutamic acid, serine and threonine are unreactive (Shetlar et al.,
1985). It has been suggested that the resistance of Micrococcus
radiodurans, one of the most radiation resistant organisms known, is
due to its extraordinary ability to repair pyrimidine dimers, but what
ultimately kills the organism is damage that involves both DNA and
protein. The crosslinking of DNA and protein may constitute one type
of such damage (Shetlar, 1980).
A different type of photochemisty arises when protein and DNA
are irradiated together as compared to when they are irradiated
separately. Since, DNA and proteins do not exist in cells as pure
solutions of the separate molecules but are in intimate contact with
23
each other, it is suggested that photochemical interaction of DNA and
protein would play a significant role in the inactivation of UV
irradiated cells under certain conditions.
Hydroxyl (OH) radicals produced from water by ionizing
radiation appeared to be responsible for the formation of ionizing
radiation induced DNA-protein crosslinks iil isolated chromatin and
in intact cells (Oleinick et al., 1986; Mee and Adelstein, 1981). The
nucleosomal core of the histones as well as non-histone proteins has
been found to be involved in the formation of DNA-protein crosslinks
in isolated chromatin exposed to ionizing radiation (Mee and
Adelstein, 1981). The hydroxyl radical induced formation of DNA-
protein crosslink between methyl group of a Thy moiety and the C-3
position of a Tyr moiety in calf thymus nucleohistone in aqueous
solution has been described (Dizdaroglu et al., 1989).
DNA-protein crosslinking in normal and solar UV sensitive ICR
2A frog cell lines exposed to solar UV radiation has been observed
(Rosenstein et al., 1989). Induction of DNA-protein crosslinks in these
mutant cell lines may help in examining the role played by this type
of DNA damage in the hypersensitivity to solar UV radiation which
are exhibited by these mutant cells. These crosslinks might be lethal
to the cell if they interfere with normal gene functions.
Role of free radicals:
A free radical can be defined as a chemical species possessing
an unpaired electron and is capable of independent existence. Free
24
radicals can be formed by:
i) Hemolytic cleavage of a covalent bond of a normal
molecule, with each fragment containing one of the
unpaired electrons
ii) By the loss of a single electron from a normal molecule
iii) By the addition of a single electron to a normal
molecule.
The prima donna in the biochemistry of oxygen free radicals
are oxygen itself, superoxide, hydrogen peroxide, transition metal ions
and the hydroxyl radical, the first four of which conspire by a variety
of interactions to generate the last (Halliwell and Gutteridge, 1990).
Under normal circumstances, the major source of free radicals in cells
is electron 'leakage' from electron transport chains. These can be
mediated by the action of enzymes or non-enzymatically, often through
the red-ox chemistry of transition metal ions (Cheeseman and Slater,
1993). Free radical production can also be increased by exogenous
sources like toxic foreign compounds and ionizing radiations.
Although there are several sites of ROS generation in cells, the main
source of O^'and its stoichiometric product, H^O^ is the auto oxidation
of the reduced components of the mitochondrial electron transport
chain (Haiku et al, 1993). In the presence of trace amounts of transition
metal (M) ions (i.e. Fe^^ Cu" ') found in biological systems (Halliwell
and Gutteridge, 1985), H^O^ can participate readily in Fenton-like
reactions (Fenton, 1894; Cohen, 1985) resulting in the production of
OH as shown.
25
Mn+ + H ^ O ^ ^ M(" ')+ OH + OH
Hydroxyl radicals have been implicated in the dele ter ious
processes such as gene mutation (Okada, 1970), cell transformation
(Borek, 1985) and cell death (Painter, 1980). Nitric oxide (NO') is an
important physiological free radical produced by phagocytes in which
the unpaired electron is delocalized between two atoms. Nitric oxide
or its derivative acts upon smooth muscle cells in vessel walls to
produce relaxation, besides acting as a neurotransmitter (Hirono et
al., 1997).
DNA damage by ROS:
DNA is an important target for free radicals since it is clearly
of major significance for cell function as well as its susceptibility of
being damaged by oxidizing radicals (Wiseman and Halliwell, 1996).
The most important reac t ions seems to be based on hydrogen
abstraction to form carbon-centered radicals followed by oxygen
addition to form peroxyl radicals that subsequently decay. Addition
of OH to double bonds is also an important mechanism. Both the
nucleobases and the sugar phosphates offer potent ia l targets for
hydroxyl radicals (*0H). The consequences of oxidat ive radical
damage to DNA are highly serious which inc ludes cell death,
reproductive cell death, mutation and transformation into cancer cells
(Feig et al., 1994). Oxidative damage to DNA includes a range of
specifically oxidized purines and pyrimidines as well as alkali labile
sites and strand breaks formed directly or by repair processes
26
.(Dizdaroglu, 1991; Dizdaroglu, 1994; Breen, 1995). The apparently
most abundant and certainly the most studied oxidative modification
of DNA bases involves the C-8 hydroxylation of guanine, frequently
est imated as the oxidized deoxynucleos ide , 8-oxo-7,8-dihydro-
2'deoxyguanosine (8-oxodG). Oxidation of guanine can also lead to
the ring opened product of 2,6-diamino-4-hydroxy-5-formamido
pyrimidine (Dizdaroglu, 1991). Other abundant oxidatively modified
purines and pyrimidines include 8-oxoadenine, 2-hydroxyadenine,
Fapy adenine, 5-hydroxy methyluracil, 5-hydroxycytosine, cytosine
glycol and thymine glycol (Tg). In addition, a large number of other
modifications of bases and sugars have been identified (Dizdaroglu,
1991; Dizdaroglu, 1994). All these modifications are present in DNA,
and the level can be increased in vivo or in vitro by systems generating
ROS. The yie ld of the individual DNA modificat ions is highly
dependent on which ROS are involved. Thus, whereas, singlet oxygen
induces preferentially 8-oxodG (Epe, 1991), superoxide has low
reactivity to induce this modification at all. Apriori to the above,
hydroxyl radicals can cause almost any modification (Dizdaroglu,
1991). In addition, the target molecule conditions, such as oxygen
tension, chelation of transition metals and the presence of reductants
may influence the yield and nature of oxidative DNA modifications
(Dizdaroglu, 1994).
Oxidative modifications of DNA bases can lead to mutations if
left unpaired or repaired with errors before replication. From studies
on bacteriophage and plasmid DNA, it appears that although radicals
21
generated by iojiizing radiations damage all four bases, mutations are
usually related to modifications of GC base pairs in contrast to AT
base pairs. Thus, AT base pair perturbations/modifications rarely lead
to mutations . The mutations are clustered in hotspots and are mainly
base pair substitutions, whereas, base deletions, large deletions, and
insertions are less frequent. Hydroxyl radicals generated from ionizing
radiation induce mainly GC to CG or AT base pair substi tutions,
depending on DNA expression system, whereas, hydrogen radicals
preferentially induce GC to AT substitutions (Retil et al., 1993).
Singlet oxygen and 1,2-dioxetanes preferentially induce 8-oxodG and
GC to AT base pair substitutions (Epe, 1991; Retel et al., 1993; Emmert
et al., 1995). In agreement, modified guanine such as 8-oxoguanine
and apurinic sites are mispaired mainly with adenine, at a frequency
dependent on the adjacent base sequences and the involved polymerase
(Kamiya et al., 1995). Majority of ROS induced mutations appear to
involve modification of guanine, in particular 8-oxoguanine, causing
G to T transversions. In addition to the mutations related to mispairing
to oxidised bases, ROS may also cause reduced fidelity of DNA J3
polymerase (Feig and Loeb, 1993) and alter the methylation of
cytosine and thus gene control (Weitzman et al., 1994; Loft and
Poulsen, 1996).
Protein damage by ROS:
Most proteins are susceptible to modification by OH or by OH
+ O " (+ Oj). The modification includes altered molecular weight
28
(aggregation or fragmentation), altered net electrical charge (+ or - ) ,
loss of tryptophan, and production of bityrosine. Since Oj 'alone has
no measurable effect on any of these parameters, it has been suggested
that OH is the initiating species. Oxygen and/or O2" appear to modify
significantly the damage induced by OH. Protein fragmentation has
been reported following exposure to OH + O^-*- Oj (Garrison et al.,
1962; Schuessler and Schilling, 1984). This process involves hydrogen
abstraction by OH from amino acid a-carbon atoms, followed by
reaction with O^to produce peroxyl species. Decomposition of carbon
peroxides was proposed as the mechanism for protein fragmentation
(Garrison et al., 1962).
All amino acids in the protein are susceptible to modification
by OH or by OH + 0 , ( + O^). In contrast, O^"alone appeared only to
reduce cysteine residues. In agreement with known rate constants for
reaction of free amino acids with H, tryptophan, histidine and cysteine
are more vulnerable than most other residues. Tyrosine is formed as a
simple addition product of OH + phenylalanine. The selectivity of
OH for tryptophan, tyrosine, histidine and cysteine is less than might
expected from reactions with free amino acids (Dorfman and Adams,
1973). The p rocess of aggregat ion by OH appears to involve
intermolecular bityrosine formation. It is highly unlikely, however,
that bityrosine is the only (non-disulfide) covalent modification.
Essentially any amino acid radical formed within a peptide chain could
crosslink with an amino acid radical in another protein. Alternatively,
some radicals may crosslink with amino acid molecules. Protein
29
denaturation also preceded increases in proteolytic suscept ibi l i ty
following exposure to OH + O^. Thus d e n a t u r a t i o n / i n c r e a s e d
hydrophobic i ty is a pos s ib l e cause for enhanced p r o t e o l y s i s .
Oxidatively denatured proteins are degraded by novel ATP and calcium
independent, soluble proteolytic systems (Davies et al., 1987).
ROS in SLE:
Antibodies to DNA in SLE have been extensively studied and
may be specific for ssDNA, reactive with both ssDNA and dsDNA or
specific for dsDNA (Worrall et al., 1990; Stollar, 1979). However, it
is ant ibodies to nat ive dsDNA that r epresen t a c h a r a c t e r i s t i c
serological finding in these patients and are of important diagnostic
and clinical value. It has been shown that modification of DNA can
render it more immunogenic (Ali et al., 1985). Native dsDNA has been
reported to be a weak immunogen (Tan and Stoughton, 1969) and yet
in certain autoimmune conditions like SLE, autoantibodies to multiple
nuclear antigens, including DNA and histones are formed. Blount et
al, (1991) postulated that DNA damaged by ROS may affect the
development of autoimmune diseases, like SLE. The process by which
DNA is damaged in such cases may involve the release of ROS
inteiinediates during the characteristic respiratory burst of activated
neutrophils, denature DNA in such a way that SLE serum anti-DNA
antibodies bind better to it (Blount et al., 1989). ROS denaturation
exposes base residues in the DNA backbone and minor regions of
ssDNA. Lymphocytes i so la ted from pat ien ts with SLE contain
30
increased levels of 8-oxodG in the DNA. In urine, the SLE patients
did not secrete 8-oxodG. A study on cultivated blood monocytes
isolated from SLE patients showed reduced capacity for repair of 8-
oxodG induced by incubation with hydrogen peroxide. In DNA isolated
from immune complexes precipitated from plasma, 8-oxodg was a 100
fold more abundant in SLE patients compared to usual values of
nuclear DNA. This oxidatively modified DNA may be a source of DNA
antibodies characteristic for SLE. The SLE patients suffer from an
increased rate of oxidative DNA damage and also from deficient repair.
This may contribute to the pathogenesis and even to increased risk of
malignant diseases in these patients (Lunec et al., 1994; Bashir et al.,
1993). Hydroxyl free-radical mediated in vitro modification of calf
thymus DNA showed single strand breaks, decrease in melt ing
temperature and structural alteration of purine and pyrimidine bases
leading to polyspecificity of induced antibodies (Alam et al., 1993,
Ara and Ali, 1993). Antibodies against free radical modified DNA
recognize B-conformation (Ara et al., 1992). ROS modified DNA
promises to be a more discriminating antigen than native DNA for
binding of SLE autoantibodies (Ara and Ali, 1993) and the binding
increased considerably with increase in DNA fragment size (Ara and
Ali, 1992).
ROS/RNS in autoimmune myocarditis/DCM:
Oxygen derived free radicals and their metabolites have been
implicated to participate in various types of myocardial injury. These
31
radicals and metabolites are thought to mainly consist of superoxide
anion (O^'" ) hydrogen peroxide (H^O^) and hydroxyl radical (OH),
however, the culprit playing the most important role is still
undetermined. The involvement of HjO^ generated through dismutation
of 02'caused severe myocardial damage (Kloner et al., 1989). Oxygen
derived free radicals and their metabolites can cause enzyme
inactivation either directly or indirectly (Miki et al., 1988). Thus,
functional deterioration may be linked to ATP reduction by inactivation
of enzymes necessary for glycosylation and oxidative phosphorylation
(Goldhaker et al., 1989). On the other hand, calcium transport in the
sarcoplasmic reticulum has been demonstrated to be decreased by
exposure to oxygen radicals (Hess et al., 1984). *0H, on the other
hand, is produced by H^Oj through iron catalysed Haber Weiss reaction
(Haber and Weiss, 1934) and is highly reactive. It causes lipid
peroxidation of the biological membranes (Meerson et al., 1982)
resulting in changes in membrane permeability and fluidity, and finally
results in loss of membrane integrity. The plasma membrane and
mitochondria are more damaged in the heart exposed to higher OH
levels. The combination of these changes may have resulted in
decreased contractility in hearts exposed to OH (Takemura et al.,
1994). Experimental studies by Fukuchi et al. (1991) have indicated
the possible involvement of free radicals and antioxidants in the early
stages of the development of cardiomyopathy in BIO 14.6 Syrian
hamster.
32
Recent studies on the molecular genetics of the myocardium have
revealed that a certain percentage of patients with DCM possess
mutation in the mitochondrial (mt) DNA of the myocardium. These
patients can be regarded as microchondrial cardiomyopathy (mtCM),
a new clinical entity. The cumulative increase in oxygen free radical
damage in human heart mt DNA closely correlates with deletions
associated with age and with mtCM. This led us to the ' redox
mechanism of ageing and degenerative disease ' in which the oxygen
free radical damage in mt DNA results in a cumulative increase in
somatic mutations, leading to bioenergetic deficit, cell death, ageing
and degenerative diseases. The accumulation of hydroxyl radical
adducts in mt DNA hydrolysate, such as 8-hydroxydeoxyguanosine
(8-OH-dG) in place of deoxyguanosine (dG), could satisfy this
prerequisite to the deletion (Hayakawa et al., 1992). The mt DNA,
having no protective protein, is located inside the inner mt membrane.
During the evolution from yeast to mammals, mt DNA reduced to one
fifth of its size by loss of introns. Thus, human mt DNA more easily
acquires oxygen free radical damage than single celled organisms. The
revelation that human mt DNA is highly susceptible to hydroxyl attack
resulting in breakdown into hundreds of mini circles suggests that the
major target of the active cell death machinery would be mt DNA.
The cumulative and synergistic increase in oxygen damage and
deletions in mt DNA associated with age implies that normal ageing
and development seem to be the two sides of a coin. The cell death
machinery could be easily imbalanced by the point mutat ions.
33
especially (syn) , in the mt DNA of patients with mtCM, leading to
their premature ageing (Ozawa, 1995).
In recent years, nitric oxide (NO) has been implicated in a wide
variety of immunologically mediated disorders. Pathologic release of
NO is considered to be one of the probable cause of diminished
myocardial contractility such as septic shock (Brady et al., 1992),
cardiac allograft rejection (Yang et al., 1994) and some form of
idiopathic dilated cardiomyopathy (Winlaw et al., 1994). Human
myocarditis varies greatly in origin and is generally a self limiting
process; acute congestive heart failure may develop during the active
phase, but cardiac function improves significantly in most cases as
myocarditis resolves histologically (Mason et al., 1995). It is possible
that the excessive production of NO by inducible nitric oxide synthase
(iNOS) is responsible for the development of myocardial organic
lesions as well as for functional changes in myocardial contractility
during the active phase of the disease.
Physiological actions of NO are mediated through the activation
of soluble guanyla te cyclase and the c o n s e q u e n t e levat ion of
intracellular cGMP level (Moncada and Higg, 1993). Studies suggest
that NO modulates myocardial contractility through an increase in
myocardial cGMP. It is reported that cGMP produces inhibition of
Ca^* influx into myocytes (Levi et al., 1989) or attenuation of the
positive inotropic effect of p-adrenergic stimulation (Watanabe and
Besch Jr., 1975). It is also possible that excessive and prolonged
34
production of NO by iNOS in infiltrating inflammatory cells modulates
the mitochondrial respiration in surrounding viable myocytes, thus
NO synthase inhibition in the early phase of viral myocarditis may
alter the adequate immune response to invading cardiotropic virus,
thus preventing the elimination of virus from cardiac tissue (Hirono
et al., 1997).
Systemic lupus erythematosus:
Systemic lupus erythematosus (SLE) is a multisystem prototype
auto immune disease (Isenberg and Shoenfeld , 1987) which is
characterized by the appearance of circulating of autoantibodies that
bind to numerous cellular and macromolecular antigens of self and
exogenous origin. Although there is a distinct correlation between SLE
and immune system, SLE continues to remain as a disease of unknown
etiology and origin. It has been postulated that immune responses
against host antigens could resul t from genetic p red ispos i t ion ,
exaggerated B-cell activity, cross reactivity between foreign and host
antigens or modification of host antigen as a consequence of infection,
inflammation, drug administration etc.
A lot of literature is available on the production and assay of
antibodies to DNA, structural analysis of specific antibody-nucleic
acid interaction and their application in biomedical research (Stollar,
1992). A central paradox of the pathogenesis of SLE is the origin of
anti-DNA antibodies. DNA to which most of the antibodies are
detected in SLE and other autoimmune disorders, is no longer regarded
35
as the inciting antigen because immunization with native DNA does
not induce SLE like disease. Animals immunized with covalently/non
covalently modified DNA induced high titre antibodies, which are
almost exclusively directed towards modified structures (Stollar,
1989). Genes encoding anti-DNA antibodies, however, are not
restricted to SLE and are part of normal B-cell repertoire (Cairns et
al., 1989; Stewart et al., 1993). The possible role of immunogenic
DNA, DNA-psoralen crosslink, hydroxyl radical, positively charged
amino acids and P-estradiol in the pathogenesis of SLE has also been
reported (Pisetsky et al., 1990; Hasan et al., 1991; Moinuddin and
Ali, 1994; Alam et al., 1993; Ara and Ali, 1993; Ara and Ali, 1992;
Alam et al., 1992; Ara et al., 1992).
Anti-DNA antibodies have been shown to cross react with
polynucleotides (Lafer et al., 1981), polynegatively charged structures
such as phospholipids (Smeenk et al., 1987), proteoglycans (Faaber
et al., 1986), cardiolipin (Eilat et al., 1986), dextran sulphate (Wollina,
1985), membrane associated proteins (Jacob et al., 1984), intermediate
filament of cytoskeleton (Andre - Schwartz et al., 1984), histones
(Gohill and Fritzler, 1987), bacterial products (Carroll et al., 1985),
platelets (Zouali et al., 1988), hyaluronic acid and chondroitin sulphate
(Faaber et al., 1984). Monoclonal anti-DNA antibodies of both human
and murine origin showed cross reactivity with antigens other than
DNA (Shoenfeld et al., 1983). There are many compounds in the
vicinity of DNA which may react upon irradiation. These compounds
include polyamines, histones and nuclear matrix elements. Polybasic
36
molecules found in cells have great affinity for acidic-constituents
such as DNA (Held and Awad, 1991) and their interaction may play a
role in the pathogenes is of SLE. Thus , the po lyspec i f i c i ty of
monoclonal anti-DNA antibodies and synthetic polypeptide antigens
suggests that antigens other than normal self ant igens serve as
immunogenic triggers.
SLE is a non organ specific autoimmune disease and as seen
earlier, a large panoply of autoantibodies have been identified in the
serum of lupus patients in varying frequency. The clinical picture of
SLE shows malar and discoid rashes, photosensitivity, oral ulcers, non-
eros ive a r t h r i t i s , p l e u r i t i s , p e r i c a r d i t i s , h e m o l y t i c anaemia ,
lymphopenia and thrombocytopenia.
Anti-DNA antibodies play a major role in the development of
lupus nephritis in MRL/lpr mice. Although anti - dsDNA antibodies
are considered to be the hallmarks of human lupus nephritis, most of
the anti-DNA antibodies in the murine lesions also bind to ssDNA
(Gavalchin et al., 1987). Before the onset of disease, the production
of anti-DNA antibodies shifts markedly from IgM to IgG isotype
(Papoian et al., 1974). This sw^itch is also associated with an increased
cationic charge of the anti-DNA antibody production (Batta et al.,
1987). The majority of anti - DNA antibodies in the renal lesions are
of lgG2a and IgG2b subclasses (Gavalchin et al., 1987).
Purified forms of nucleic acids are poor immunogens (StoUar,
1973). Modification of DNA, for example by ultraviolet light exposure
37
(Davis et al., 1976) or the presentation to the immune system of DNA
fragments (Lafer et al., 1981) has induced antibodies which bind to
immunogen but not to unmodified B-DNA.
The anti-DNA antibody response of lupus mice resembles an
antigen driven response that is composed of antibodies that are
heterogeneous in their DNA binding specificity. More compelling
evidence for the role of DNA as a selecting antigen has been obtained
from the sequencing of panels of anti-DNA antibodies isolated from
individual lupus mice (Schlomchik et al., 1990). Since the antibodies
in these panels all bind to DNA and show clonal restriction, it may be
concluded that DNA functions in vivo as a selecting antigen. A microbe
possessing both foreign epitopes and epitopes that mimic autoantigens
has the potential to elicit an autoimmune response (Rekvig, 1995).
Nucleic acids complexed with proteins have also been considered as
potential immunogens. Many human monoclonal anti-DNA antibodies
of both isotypes use VH genes which are members of fetally expressed
repertoire. It is possible that these genes contain CDR sequences which
lend themselves to formation of a DNA binding site. Comparison of
sequences of large numbers of anti-DNA antibodies enables us to
identify motifs which might contribute to such binding properties.
Comparison of the sequence of antibody cDNA with that of the
germline gene from which it is derived enables us to identify the
positions and nature of somatic mutations which may likewise
contribute to antigen binding (Isenberg et al, 1997).
38
Cardiovascular manifestations of SLE:
The cardiovascular manifestations of SLE have become more
apparent with prolonged survival and improvement in diagnost ic
modalities. While most patients still die of infection or renal failure,
the incidence of cardiovascular morbidity and mortality in SLE is
rising, and is now the third leading cause of death in SLE (Urowitz et
al., 1976). Whether there is a cardiomyopathy directly attributable to
lupus is unresolved. Most series cite a high incidence of heart failure
but attribute it to multiple problems, including; fever, infections,
anaemia , u raemia , h y p e r t e n s i o n , s t e ro ids and a c c e l e r a t e d
atherosclerosis. Studies on systolic time interval in SLE patients
indicate a shorter left ventricular ejection time and a longer pre-
ejection period than control subjects, suggesting left ventricular
systolic dysfunction, an indicator for primary lupus cardiomyopathy
(delRio et al., 1978). Haemodynamic studies have revealed that lupus
cardiomyopathy may exist even without clinical signs or may represent
abnormalities of the intrinsic contractile and relaxation properties of
the myocardium (Strauer et al., 1976). Various etiologies have been
proposed for lupus cardiomyopathy. Das and Cassidy (1973) detected
anti heart antibodies in 20 of 32 patients with SLE and proposed that
these antibodies might be involved in the genesis of myocardial
dysfunction in lupus. Bidani et al (1980) demonstrated immune
complexes in the myocardium in 9 of 10 necropsies. They proposed
that immune complex deposi t ion led to complement act ivation,
inflammation and myocardial damage. Either mechanism (anti heart
39
antibodies or immune complex deposition) may provoke the small
vessel vasculitis, focal myocarditis, fibrosis and myocardial necrosis
that is seen at autopsy in many SLE hearts. Further evidence is needed,
however, to establish the link between these pathologic findings,
immunologic abnormalities and cardiac dysfunction in v/vo (Doherty
and Siegel, 1985). Frustaci et al. (1996) have recently reported a case
of lupus myoca rd i t i s caus ing left v e n t r i c u l a r a n e u r y s m . An
immunologic mechanism of myocardial damage in this patient is
suggested by the results of serologic studies (increased anti-nuclear
antibodies, increased anti-DNA, decreased C3-4, increased ant i -
sarcolemmal antibodies) by the normal coronary arteriogram and by
the cardiac biopsy which showed active myocarditis and fibrinous
e n d o c a r d i t i s . Pos t -mor tem s t u d i e s have r evea led m y o c a r d i a l
inflammatory lesions in upto 70% of victims of SLE. It remains to be
seen if improved diagnosis and t reatment of the cardiovascular
manifestations of SLE can enhance survival.
Objectives of the present s tudy:
Organ specific au to immune d isease are charac ter ized by
production of autoantibodies which react with autoantigens unique to
that organ. Since in DCM, the only affected organ is the heart muscle,
it is necessary that the criteria of an organ specific autoimmune disease
is fulfil led. This includes gene t ic p red i spos i t ion , presence of
inflammation and abnormal HLA molecule expression in target organ,
reproduction of human disease in susceptible animals with relevant
40
autoantigens and detection of organ and disease specific antibodies
in affected patients and symptom free relatives. Although there is
evidence that autoantibodies of DCM patients react to several different
proteins, the epitopes for experimental autoimmune myocarditis and
DCM still remain limited. Therefore, the cardiac muscle protein
responsible for autoimmune myocarditis causing DCM must be
identified.
In the present investigation, a systematic study was carried out
to characterize the 150 kD C-protein autoantigen which is known to
induce autoimmune myocarditis leading to DCM. The autoantigen, 150
kD human cardiac C-protein was characterized by ultraviolet
spectroscopy, circular dichroism, thermal denaturation, SDS-PAGE and
urea denaturation studies. The immunogenicity of C-protein was as
certained by inducing polyclonal antibodies in experimental animals.
The repertoire of specificities of induced antibodies was assessed by
direct binding and competition ELISA. Commercially available calf
thymus native DNA was purified free of protein and single standed
regions and adducted to C-protein is presence of ROS and/or UV-B
irradiation. The DNA C-protein adduct/photoadduct was characterized
by nuclease SI sensitivity assay. Kinetic behaviour of addition of
lysine residues (present in C-protein) at various irradiation time was
studied in case of DNA C-protein photoadduct. The recognition of C-
protein and DNA C-protein adducts/photoadducts by DCM, SLE and
induced anti C-protein antibodies was evaluated by direct binding and
competition ELISA and band shift assay.
"^xptvxxntninl
41
Materials:
Calf Thymus DNA, Histone, Poly - D - lysine. Poly - L -
glutamate, Picryl sulfonic acid, bovine serum albumin, agarose NA,
anti - human / anti - rabbit IgG alkaline phosphatase, anti - rat IgG
horseradish peroxidase conjugate, ethidium bromide, coomassie
brilliant blue G - 250 and R - 250, standard protein markers, sodium
dodecyl sulphate, freund's complete and incomplete adjuvant and
Tween - 20 were purchased from Sigma Chemical Company, U.S.A.
Protein - A - Sepharose CL - 4B and Ficoll 400 were obtained
from Pharmacia Fine Chemica l , Sweden. Tris (hydroxymethyl )
methylamine, EDTA (disodium salt), chloroform, isoamyl alcohol and
h y d r o g e n p e r o x i d e were p u r c h a s e d from Q u a l i g e n s , India .
Immunod iagnos t i c ki ts were purchased from St ' n g e n , India.
Biochemical assay kits were purchased from Bayer i: gnistics.
Polystyrene microtitre flat bottomed plates were purchased from
Nunc, Denmark, p-nitrophenyl phosphate and Folin - Ciocalteau
reagent were obtained from Centre for Biochemical Technology, New
Delhi. Acrylamide, ammonium persulphate, bisacrylamide, N, N, N,'
N - tetra ethyl methylene diamine (TEMED) were from Bio - Rad
Laboratories, U.S.A. Diphenylamine and ethanol were chemically pure.
All other chemicals were of highest analytical grade available.
Equipment:
Shimadzu UV - 240 s p e c t r o p h o t o m e t e r e q u i p p e d with
thermoprogrammer and controller unit, ELISA microplate reader MR
42
- 600 (Dynatech, U.S.A..), Beckman DU - 640 spectrophotometer with
densitomt'ter, Avanti 30 table top high speed refrigerated centrifuge
(Beckman, U.S.A.), Ultraviolet lamp (Vilber Lourmat, France), agarose
gel electrophoresis assembly (GNA - 100), Po lyacry lamide gel
electrophoresis assembly (Bio - Rad, U.S.A.), gradient former model
385 (Bio - Rad, U.S.A.), electroeluter (Bio - Rad, U.S.A.) HPLC
(Beckman System Gold) and ELICO pH meter LI - 120 were the major
equipments used in this study.
Collection of sera:
Normal human sera were collected from healthy subjects without
evidence of DCM / SLE or other autoimmune disease. Sera from
patients with dilated cardiomyopathy (DCM) and systemic lupus
erythematosus (SLE) were obtained from the patients admitted to the
Medical College Hospital of A.M.U. and from outdoor Cardiology and
Medicine c l i n i c s , GB Pan t Hosp i t a l and AIIMS, New D e l h i ,
respectively. DCM patients were clinically diagnosed based on the
definition and classification of cardiomyopathies (Richardson et al.,
1996). The SLE s^ra were obtained from the patients who had elevated
levels of anti-DNA antibodies and met the revised criteria for the
American Rheumatism Association (ARA) for the diagnosis of the
d isease (Arnett et al . , 1988) . All serum samples were hea t
decomplemented at 56 °C for 30 min, and were subsequently stored at
-20 °C with 0.02% sodium azide as preservative.
43
DNA estimation:
Colorimetric estimation of DNA was carried out according to
Burton (1956) using diphenylamine reagent.
a) Diphenylamine reagent:
The reagent was prepared immediately before use by dissolving
750 mg of recrystallized diphenylamine in 50 ml of glacial acetic
acid containing 0.75 ml of concentrated sulphuric acid.
b) Assay procedure:
Varying amount of DNA adjusted to 1.0 ml with Phosphate
Buffer Saline (PBS), pH 7.4, was mixed with IN perchloric acid
and incubated at 70 °C in a water bath for 15 minutes. One
hundred microliter of 5.43 mM acetaldehyde was added followed
by 2.0 ml of diphenylamine reagent. The tubes were vortexed
and allowed to stand at room temperature for 16-20 hr for colour
development. Absorbance was recorded at 600 nm. The DNA
concentration in the unknown sample was determined from the
standard plot constructed by employing O-IOO ig of purified calf
thymus DNA.
Protein Estimation:
Protein was estimated by the method of Lowry et al. (1951) and
Bradford (1976).
44
A. Protein estimatian by Folin-Phenol reagent:
This method of protein estimation utilises alkali (to maintain
high pH), Cu^ ions (to chelate proteins) and tartarate (to keep
the Cu^^ions at high pH).
a) Folin-ciocalteau reagent:
The reagent was purchased from Cent re for Biochemical
Technology, New Delhi (India). It was diluted with distilled
water in the ratio of 1 : 4 before use.
b) Alkaline copper reagent:
The components pf alkaline copper reagent were:
i) Two percent sodium carbonate in 0.1 M sodium hydroxide,
ii) 0.5 percent copper sulphate in 1.0 percent sodium potassium
tartarate.
The working reagent was prepared afresh by mixing components
(i) and (ii) in the ratio of 50 : 1.
c) Procedure:
To 1.0 ml of protein sample was added 5.0 ml of alkaline copper
reagent, the contents were mixed and left at room temperature
for 10 min. Then 1.0 ml of Folin-ciocalteau reagent was added
and after 30 min. the absorbance was monitored at 660 nm. The
protein content of unknown samples were determined from a
standard plot constructed against bovine serum albupiin.
45
B. Protein estimation by Bradford method:
This colorimetric assay is performed under acidic condition. The
colour change occurs when Coomassie Brilliant Blue G-250
binds strongly to protein hydrophobical ly and at posi t ively
charged groups. In the environment of these positively charged
groups, protonation is suppressed and a blue colour is observed.
Binding of dye protein causes a shift in absorption maximum of
the dye from 465 to 595 nm and it is the increase in absorption
which is read at 595 nm which is monitored.
a) Dye preparation:
One hundred milligram of Coomassie Brilliant Blue G-250 was
dissolved thoroughly in 50 ml of 95% double distilled ethanol.
To this solution was added 100 ml of 85% (v/v) orthophosphoric
acid. The resulting solution was diluted to a final volume of
one litre. It was filtered before use.
b) Protein assay:
Solution containing 0-100 ^g protein in a volume of upto 0.1
ml was pipetted into test tubes. The volume was adjusted to 1.0
ml with appropriate buffer. Five milliliter of dye solution was
added and the contents were vortexed. The absorbance was read
at 595 nm within an hour against a reagent blank prepared from
1.0 ml of buffer and 5.0 ml of dye solution.
46
Lysine est imat ion by TNBS method:
Lysine estimation of the samples was determined by using
trinitrobenzene sulphonic acid (TNBS) as described by Habeeb (1966).
Varying concentrations of lysine (0-100 [ig) were diluted with
distilled water to a final volume of 1.0 ml. To this was added 1.0 ml
of 4% N a H C 0 3 , pH 8.5 and 1.0 ml of 0 . 1 % TNBS solution. The
reaction mixture was incubated for 2 hrs at 37 °C. Subsequently, 1.0
ml of 10% SDS was added to solubilize the protein and to prevent its
precipitation on addition of 0.5 ml of IN HCl. The colour intensity
was recorded at 335 nm against a blank treated in the same manner
except that it contained 1.0 ml of water instead of lysine solution.
The lysine content in the unknown samples was determined from the
standard plot constructed against L-lysine.
Isolation of 150 kD C-protein from human heart:
Acetone powder of human heart was prepared by the method of
Horecker et al. (1955). Seventy five grams of frozen left ventric 1
was pulverized and homogenized in 5 volumes of chilled acetone. The
material was filtered through suction on a Buchner funnel using
Whatman filter paper No. 1. The semi-dry filter cake was again
homogenized as described above and was subsequently washed several
times with chi l led acetone and dried at room temperature. The
connective tissue was screened out and the remaining acetone powder
was stored at -20 °C.
4 7
Extraction of acetone powder was carried out as described by
Kurata and Tan (1976). The acetone powder was extracted with 10
volumes of PBS, pH 7.4 for 4 hrs at 4 °C with slow continuous stirring.
The solution was centrifuged at 10,000 rpm for 15 min. and the
supernatant was stored in small aliquots at -20 °C.
Gel e lectrophores is :
Polyacrylamide slab gel electrophoresis:
Polyacrylamide slab gel electrophoresis was performed under
denaturing conditions as described by Laemmli (1970). The following
stock solutions were prepared :
i) Stacking gel buffer 0 5 M Tris-HCl, pH 6 8
ii) Resolving gel buffer 3 0 M Tris-HCl, pH 8 8
iii) Reservoir buffer 0 025 M Tris, 0 192 M glycine, pH 8 3
iv) Acrylamide-Bisacrylamide (30 0 8)
Thirty grams acrylamide and 0.8 g bisacrylamide were dissolved
in distilled water to a total volume of 100 ml. The solution was stored
at 4°C in an amber coloured bottle.
v) Sample buffer
a) Six gram Tris was dissolved in 80 ml distilled water and pH
adjusted to 6.8 with o-phosphoric acid. The final volume was
brought to 100 ml.
b) To 12.5 ml of the above solution was added 1 mg bromophenol
blue and 12.5 ml glycerol. P-mercaptoethanol was added just
before use.
48
Procedure:
The glass plates (19 cm x 16 cm) soaked in chromic acid were
thoroughly washed with tap water followed by final rinsing with
distilled water and ethanol. Finally, the plates were dried and sealed
with 1% agarose with 1.5 mm thick spacers.
Recipe for 5 - 2 0 % Gradient Gel
(Total volume: 24 ml)
Solution
Acrylamide-Bisacrylamide (30 : 0.8)
Resolving gel buffer
10% SDS
1.5% ammonium persulphate
TEMED
esolving
5%
2 ml
1.5 ml
0.12 ml
0.6 ml
0.006 ml
gel
20%
8 ml
1.5 ml
0.12 ml
0.6 ml
0.006 ml
Final volume raised to 12 ml each with distilled water.
2.5%) Stacking Gel
(Total volume: 10 ml)
Acrylamide-Bisacrylamide (30 : 0.8)
Stacking gel buffer
10% SDS
1.5% ammonium persulphate
TEMED
1.25 ml
2.5 ml
0.1 ml
0.5 ml
0.0075 ml
Final volume raised to 10 ml with distilled water.
49
Recipe for 7.5% SDS-PAGE
(Total volume: 10 ml)
Acrylamide-Bisacrylamide (30 : 0.8)
Resolving gel buffer
10% SDS
1.5% ammonium persulphate
TEMED
7.5 ml
3.75 ml
0.3 ml
1.5 ml
0.015 ml
Final volume made upto 30 ml with distilled water.
The reagents were mixed and poured between glass plates. The
resolving gel was allowed to polymerize at room temperature. Later
stacking gel was layered on top followed by comb insertion and the
gel was left to polymerize at room temperature. In case of gradient
gels, a gradient was formed with the help of a gradient former (Bio-
RAD, model 385) in case of resolving gel. After ensuring complete
polymerization, the protein samples (25-50 mg) in one fourth volume
of sample buffer were electrophoresed at 70 volts for 6-8 hrs at room
temperature. The gel was stained by 0.25% Coomassie Brilliant Blue
R-250 or with silver stain reagent.
Silver staining was done by the method of Merril et al (1983).
Briefly, the gel was incubated in 40% methanol, 12% acetic acid for
45 min. followed by incubation in 50% ethanol for 30 min. Next the
gel was treated with 0.02% Hypo (sodium thiosulfate) for one min.
After washing with distilled water, the gel was placed in 0.2% silver
50
nitrate (with 0 .05% v/v formaldehyde). After subsequent washing with
distilled water, the gel was transferred to a 6% solution of sodium
carbonate (with 0 .15% v/v formaldehyde). After colour development,
the gel was washed with distilled water and the reaction was arrested
by treating the gel with 3% v/v acetic acid and 5% v/v methanol. All
the reagents used in this procedure were freshly prepared.
Gel dens i tometry:
The electrophoresed stained gels having an array of protein
bands , r ang ing from low to high m o l e c u l a r weights were
densitometrically scanned on a DU-640 spectrophotometer equipped
with a densitometer at a fixed wavelength of 550 nm.
Electroelution:
The electrophoresed 150 kD C-protein band from human heart
was carefu l ly s l i ced off from the gel and was subjec ted to
electrophoretic transfer in an electroelution chamber equipped with
BT-1 and BT-2 membranes (BIO-RAD). The electroelution was carried
out in TBS (150 mM Tris-HCl, 175 mM NaCl), pH 7.4, containing
20% methanol) at 150 volts for 5 hours at room temperature. The
electroeluted 150 kD C-protein was dialyzed against distilled water
and stored at -20 °C. Prior to storage for further s tudies , i ts
homogeneity was reassessed by PAGE.
51
CD measurements:
Circular dichroism measurements were carried out at 25°C on a
Jasco spectropolarimeter model J - 720 using a SEKONIC X - Y plotter
(model SPL - 430 A), NESLAB water bath model RTE 110 and quartz
cell of path length 0.1 cm or 1.0 cm. Other experimental conditions
were, sensitivity 5 mdeg, response time 0.5 s e c , scan speed 20 nm /
min. and aresolution of 1.0 nm. The mean residue ellipticity [9] of a
protein was calculated using the expression.
[0] = 0 / ( 1 0 n X Cp X 1)
Where 0 is the observed ellipticity in mdeg. n the number of
aminoacid residues in the protein whose molar concentration is Cp
and 1 is the path length in om.
High performance liquid chromatography (HPLC):
The electoreluted 150 KD C protein (from human heart) was
further purified by HPLC using a sephader G - 200 column. The
running buffer was PBS, pH 7.4.
Agarose gel electrophoresis of DNA:
Agarose gel was prepared by bringing 1% agarose NA to molten
state in electrophoresis buffer (0.04 M Tris acetate, pH 8.0 containing
0.002 M EDTA). Molten agarose was poured on the gel tray and
allowed to solidify for one hour at room temperature. The DNA
samples in one tenth volume of stop-mix (30% ficoll, 0 .025% xylene
52
cyanole FF and 500 mM EDTA in 10 times concentrated TAE buffer)
were electrophoresed for 2-4 hrs at 30 mA in the same buffer. The gel
was stained with ethidium bromide (0.5 \ig/m\).
Preparation of DNA - C-protein adducts:
a) Preparation of DNA - C protein photoadducts:
Calf thymus DNA, free of histones and single stranded regions
was mixed with HPLC purified C protein in the ratio of 1:2 in
PBS, pH7.4 and incubated at 2hrs. at 37°C. The complex was
subjected to UV-B irradiat ion for 30 min. at 254 nm. The
temperature during irradiation was kept constant at 25 + 1°C by
water curculation. Native DNA, unirradiated DNA - C protein
complex, unirradiated C protein, irradiated DNA and irradiated
C protein were used as controls.
b) P repa ra t ion of DNA - C - protein adduc ts in presence of
reactive oxygen species (ROS):
ROS induced photoupling of C protein to the genetic material
(DNA) was carried out in the presence of hydroxyl radical ( OH)
generated by hydrogen peroxide (1.51 M) in presence of UV - B
irradiation (254 nm) DNA - C protein complex was also formed
only in the presence of hydrogen peroxide (1.51M). Purified calf
thymus DNA and C protein were mixed in the ratio of 1:1 in
PBS, pH 7.4. Similar amounts of native DNA, unirradiated C
protein, irradiated DNA and irradiated C protein were used as
controls.
53
All the treated samples were extensively dialyzed in PBS, pH
7.4. The modif ied and unmodified samples were subjected to
electrophoresis on 1% agarose using Tris acetate EDTA buffer, pH 8.0
for 2 hrs. at 30 mA. The gel was stained with ethidium bromide (0.5
^g / ml) and was photographed under UV illumination.
Time course kinetic stsudy of formation of nDNA - C - protein
photoadduct:
The kinetic behaviour of photoaddition was assessed in order to
determine the frequency of C protein photo cross linked to DNA. The
DNA - C protein complex was subjected to UV - B irradiation (254
nm) for various time intervals (10 - 30 min). Unirradiated DNA - C
protein complex was used as control. The DNA - C protein photo
adducts were precipi ta ted in two volumes of cold ethanol . The
precipi ta te was col lec ted by centrifugation and dissolved in I.O
ml of PBS, pH 7.4. The bound prote in was e s t ima ted in the
photoadducts (Bradford et al, 1976). The lys ine con ten t in the
s amp le s was a s s e s s e d by c o l o r i m e t r i c me thod u s ing TNBS
(Habeeb, 1966).
Kinetic analysis of the photoaddition reaction was performed
by the method of Warren et al (1974) using first order rate equation.
-In ( V a - Vt) - a + kt
Where Vaand Vt are the lysine molecules photobound at infinity
(30 min.) and at a particular time t respectively. The -In (Va - Vt)
54
values were plotted against the irradiation time t and the slope yielded
the apparent first order rate constant k for the photoaddition process.
Characterization of adduct by nuclease SI digestion:
The formation of DNA - 150 KD C - pro te in adduct was
characterized by SI nuclease digestion followed by gel electrophoresis.
a) One percent agarose:
Agarose (.3g) in 30 mix TAE buffer, pH 8.0 was brought to
molter state and after cooling to 50° C was poured onto a
horizontal gel tray of GNA - 100 electrophoresis apparahis
(Pharmacia Sweden) and was left at room temperature for
complete solidificatin.
b) Electro buffer:
40 mM Iris - acetate and 2mM EDTA, pH 8.0 (TAE buffer)
c) Treatment of adduct with SI nuclease:
One jig of DNA - 150 kD C -protein adduct and riative DNA
were treated with SI nuclease (20 units of S1 nuclease per jig of
adduct) in acetate buffer for 30 minutes at 37°C. The reaction
was stopped by the addition of one tenth volume of 0.2 M EDTA,
pH 8.0.
d) Sample p repara t ion :
The treated and untreated samples in low ionic strength and at
concentrations of less than 1 jig / ml were loaded. The sample
55
contained one tenth volume of 30% Ficoll (Pharmacia), 0.025%
bromophenol blue, 0.5 M EDTA and lOX electrophoresis buffer.
e) Running conditions:
30 mA for 2 hours.
f) Staining:
The electrophoresed gel was stained with ethidium bromide (0.5
M-g / ml).
Urea - SDS - PAGE-:
Urea - SDS - PAGE was performed according to the method of
Swank and Munkres, (1971). The following stock solutions were
prepared :
i) Acrylamide : Bisacrylamide : 12.5 g acrylamide
(10 : 1) 1.25 g bisacrylamide
dissolved and made upto 50
ml with distilled water.
ii) Gel buffer stock 1% SDS
1.0 M H^PO^ (adjusted to
pH 5.0 with Tris base)
iii) Reservoir buffer stock 0 .1% SDS
0.3M H,PO,
(adjusted to pH 6.8 with
Tris base)
56
iv) Sample buffer 1% SDS
8 M Urea
1% 2-mercaptoethanol
O.IM H,PO,
(adjusted to pH 6.8 with Tris
base) Bromophenol blue
added to 0.002% (w/v)final
concentration.
RECIPE
12.5% Resolving gel
Acrylamide : Bisacrylamide (1:10) 15.0 ml
Gel buffer stock 3.0 ml
Urea 14.4 g
6% ammonium persulfate 0.3 ml
(freshly prepared)
TEMED 0.02 ml
The volume is made upto 30 ml with distilled water.
3.75% Stacking gel
Acrylamide : Bisacrylamide (1:10) 1.5 ml
Gel buffer stock 0.3 ml
Urea 1.44 g
6% ammonium persulfate 0.03 ml
(freshly prepared)
57
TEMED 0.002 ml
The final volume is made upto 10 ml with distilled water.
Procedure:
A low concentration gel of 3.75% was layered over 12.5% gel,
with the same buffer composition. This is done to avoid breakage of
sample well divis ions which become very br i t t le due to high
crosslinking of resolving gel. The gels were run for 6-8 h at 70 volts.
Stainer:
After electrophoresis, the gels were stained for one hour in:
methanol : water : glacial acetic acid in ratio of 5 : 5 : 1
Coomassie Brilliant Blue R 250
Destainer:
Destaining is by diffusion in
12.5% isopfopanol
10% acetic acid.
Urea SDS-PAGE was also pe r fo rmed by doing va r ious
alterations.
UREA - SDS-PAGE
(with varying concentrat ions of urea)
A gradient gel of 1 M to 10 M urea was polymerized
5b
RECIPE
Resolving gel (12.5%)
Acrylamide : Bisacrylamide (1:10)
Gel buffer stock
Urea
6% ammonium persulfate
(freshly prepared)
TEMED
1 M Urea
: 2 ml
0.4 ml
0.24 g
0.04 ml
0.0027 ml
10 M Urea
2 ml
0.4 ml
2.4 g
0.04 ml
0.0027 ml
The final volume is made upto 4 ml each with distilled water.
Stacking gel (3.75%)
Acrylamide : Bisacrylamide (1:10)
Gel buffer stock
Urea
6% ammonium persulfate
(freshly prepared)
TEMED
0.3 ml
0.06 ml
0.36 g
0.02 ml
0.0016 ml
The final volume is made upto 2 ml with distilled water.
UREA - SDS-PAGE
(with varying concentrations of acrylamide : bisacrylamide)
A 5-20% gradient gel of acrylamide : b i sacry lamide was
polymerized.
Gel buffer stock
Urea (8 M)
6% ammonium persulfate
(freshly prepared)
TEMED
0.4 ml
1.92 g
0.04 ml
0.0027 ml
59
RECIPE
Resolving gel (5-20%)
5% 20%
Acrylamide : Bisacrylamide(l:10) 0.6 ml 2.4 ml
0.4 ml
1.92 g
0.04 ml
0.0027 ml
The final volume is made upto 4 ml each with distilled water.
Stacking gel (3.5%)
Acrylamide : Bisacrylamide : 0.3 ml
Gel buffer stock : 0.06 ml
Urea : 0.36 g
6% ammonium persulfate : 0.02 ml
TEMED : 0.0016 ml
The final volume is made upto 2 ml with distilled water.
Absorption - temperature scan:
Thermal native to coil transition analysis of native Cardiac C
protein and native IgG was carried out in order to assess / probe their
comparative thermal stability in view of the established fact that both
of them possess similar domains.
Native C pro te in and nat ive IgG were sub jec ted to heat
denaturation on a Shimadzu UV-240 spectrophotometer coupled with
60
a temperature programme and controller assembly (Hasan and Ali,
1990). The samples were melted from 30°C to 100°C at a rate of 1°C/
min. Prior to scanning, the samples were temperature-equilibrated at
30°C for 10 minutes. The change in absorbance at 280 nm was recorded
with increasing temperature. Percent denaturation was calculated using
the following equation :
% denaturation = AT-ASO X 100 Amax-A30
where.
AT = Absorbance at a temperature T°C
Amax = Final maximum absorbance on the completion of
denaturation
A30 = Initial absorbance at 30 °C.
Thermodynamic analysis of 150 kD cardiac protein as well as
native IgG:
The thermal transition of proteins from native to denatured state
was characterized as a single variable, fo, the fraction of protein in
the denatured state. The percent loss in absorbance was used as
experimental variable to follow the transition. At any given point along
the transition curve, the observed percent change in absorbance was
related to the fraction of protein in the denatured state, by the
following expression
fo = (A)obs - (A)N ( A ) D - (A)N
61
where, (A)obs, (A)N and (A)D represent the percent loss in
absorbance in any observed, native and fully denatured s ta tes ,
respectively. Since each experimental value of (A)obs will give a
unique value of (A)D, the later was used to construct transition curves
for the thermal denaturation of proteins.
Since the unfolding/denaturation of proteins is a more than one
step process due to various factors, the initial and final states of
thermal denaturation of proteins were related to apparent equilibrium
constant, defined as
Kapp = (A)obs - (A)N ( A ) D - (A)obs
The free energy change, AG^, for the thermal reaction (native
coil conformation) was calculated by using the relationship
AGD = - RT InKapp
where, Kapp is the equilibrium constant, R is the gas constant
(8.314 J/mol/deg) and T is the absolute temperature.
Immunization schedule:
Heal thy female rabbi t and ra t s were immunized with
electrophoretically pure 150 kD C-protein antigen emulsified in an
equal volume of Freund's complete/incomplete adjuvant as described
earlier (Hasan and Ali, 1990). Injection dose of 100 \ig and 50 ng 150
kD C-protein was given to rabbit and rats, respectively.
62
The first injection eliciting primary response was given in
Freund's complete adjuvant, followed by three successive boosters
which were given after two weeks at weekly intervals in Freund's
incomplete adjuvant. The volume of the emulsion to be injected was
kept as 1.0 ml and 0.5 ml for rabbit and rats, respectively.
Blood was drawn from experimental animals by cardiac puncture
after the third booster and the antibody activity in the immune serum
was evaluated by ELISA . Prior to immunization, pre-immune serum
was c o l l e c t e d for employ ing it as c o i r e s p o n d i n g con t ro l in
immunological studies.
Isolation of IgG by affinity chromatography:
Protein A Sepharose CL-4B (1.5 g) was employed as affinity
matrix. First ly, it was swelled in 0.01 M PBS, pH 7.4 at room
temperature for 12 hrs. The swelled matrix (about 5.0 ml) was washed
with PBS and packed in a column of 0.9 cm x 15 cm. The packed
column was washed with 0.1 M sodium citrate, pH 3.0 in order to
elute any bound material. This was followed by equilibration of the
packed matrix with 5 volumes PBS, pH 7.4. Serum diluted with equal
volume of PBS, pH 7.4 was loaded onto the column and allowed to
bind at a flow rate of 20 ml/hr. Unbound protein was eliminated with
PBS and absorbance of effluent monitored till a negative absorbance
was obtained at 280 nm The bound IgG was eluted with 0.58% acetic
acid in 0.85% sodium chloride. To prevent the effect of acidic elution
63
buffer on IgG, fractions were collected in 1 M Tris-HCl, pH 8.5. The
fractions containing IgG were monitored at 251, 260, 278 and 280
nm. The fractions with the appropriate ratio for mammalian IgG (2.5-
3.0) were dialyzed in 0.01 M PBS, pH 7.4.
To check the purity of IgG, the samples were subjected to 7.5%
SDS-PAGE.
Immunoaffinity purification of ant ibodies:
a) CNBr activation of Sepharose 4B:
15 ml of supplied Sepharose 4B was suspended in distilled water,
and then washed with 300 ml of cold distilled water on a sintered
glass funnel (porosity G-2). Moist gel (about 10 g) in 10 ml of
2.0 M sodium carbonate was kept in ice - NaCl bath placed on a
magnet ic s t i rrer . One gram cyanogen bromide in 0.8 ml
acetonitrile was slowly added to the gel with gentle stirring.
The reaction was allowed for 12 min. and after that the reaction
product was filtered on a sintered glass funnel, washed with 400
ml of cold 0.01 M sodium bicarbonate and resuspended in 10 ml
of the same buffer. The unreacted CNBr was treated with ferrous
sulphate to convert it into harmless ferrocyanide. All steps were
carried out in a fumehood chamber (Ali, 1984).
b) Poly-L-lysine coupling to activated Sepharose 4B:
The method descr ibed by Wilchek (1973) was fo l lowed.
Immediately after CNBr activation of gel, equal volume of Poly-
64
L-lysine (100 mg in 10 ml of 0.1 M sodium bicarbonate) was
added to the activated gel and kept at 4 °C for 12 hrs with slow
stirring. The buffer was drained out and the gel was washed
successively with 100 ml each of (i) cold distilled water, (ii)
0.1 N HCl, (iii) 0.1 M sodium bicarbonate and (iv) distilled water
till neutral. Finally the gel was suspended in 40 ml of 0.15 M
acetate buffer, pH 4.5. The extent of Poly-L-lysine depletion
due to its covalent coupling with activated Sepharose 4B was
calculated by TNBS estimation of lysine in the drained effluent
as described by Habeeb (1966).
c) Affinity purifiction of ant ibodies:
DNA-[polylysyl-Sepharose 4B] was prepared as described by
Nicotara et al. (1982) with a slight modification. Previously
prepared 20 ml po ly ly sy l -Sepha rose 4B was packed and
equilibrated with 25 ml acetate buffer in a mini column (1.5 cm
X 5 cm). Purified DNA (12.5 ml of 100 \ig/m\) in acetate buffer
was passed through the gel and unbound material was eliminated
with PBS, pH 7.4. Heat decomplemented SLE serum dialyzed
against and diluted (1 : 10) with PBS was passed through the
column. Unbound proteins were washed with PBS and the bound
antibodies were eluted with linear ionic strength gradient of
0.15-3.0 M sodium chloride in 0.01 M phosphate buffer. pH 7.4.
The fractions were monitored at 251 nm. 260 nm, 278 nm and
280 nm.
65
d) Regeneration of affinity column:
The column was r e g e n e r a t e d severa l t imes by wash ing
successively with 50 ml each of the following (i) distilled water,
(ii) 0.1 N HCl, (iii) 0.1 M sodium bicarbonate, and (iv) distilled
water till neutral.
Enzyme linked immunosorbent assay (ELISA):
The antibodies were detected and quantitated by ELISA using
polystyrene flat bottom microtiter plates as solid phase (Alam and Ali,
1992) . The method described by Ali et al. (1985) and Arif et al. (1994)
were followed for the assay.
Buffers:
i) Tris buffered saline (TBS), pH 7.4 :
10 mM Tris, 150 mM NaCl, pH 7.4
ii) Tris buffered saline Tween-20 (TBS-T), pH 7.4 :
20 mM Tris, 144 mM NaCl, 2.68 mM KCl and 500 ^1 Tween-20,
pH 7.4.
iii) Bicarbonate buffer, pH 9.6 :
15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6.
iv) a) Substrate buffer, pH 9.6 :
(for a n t i - human / anti rabbit IgG alkaline phosphate conjugate)
15 mM sodium carbonate, 35 mM sodium bicarbonate and 2 mM
magnesium chloride, pH 9.6.
substrate : 0.5 mg/ml of p-nitrophenyl phosphate
66
b) Substrate buffer, pH 5.0 :
(for anti rat) IgG horse radish peroxidase conjugate)
50 mM citric acid, 50 mM di-sodium hydrogen orthophosphate
dihydrate (Na2HP0^.2H20)
substrate : 0.5 mg/ml of o-phenylene diamine.
Procedure:
Polystyrene microtiter plates were incubated with 100 |ul of
protein antigen (50 ng/ml in carbonate/bicarbonate buffer, pH 9.6) for
two hours at room temperature followed by overnight incubation at
4°C. The plates were washed thrice with TBS-T and the unoccupied
sites were blocked by 150 pi of 1.5% BSA in TBS for four hours at
room temperature. Varying dilutions of sera diluted in TBS were added
to antigen coated as well as control wells. The ant igen-ant ibody
interaction was allowed to proceed for two hours at room temperature
followed by overnight incubation at 4°C and subsequently the plates
were washed four times with TBS-T in order to remove the uninteracted
antibodies. Bound antibodies were assayed by means of appropriate
anti-immunoglobulin alkaline phosphatase / anti-immunoglobulin horse
radish peroxidase conjugate using p-ni t rophenyl p h o s p h a t e / o-
phenylene diamine as substrate, respectively. The reaction was stopped
by 3N NaOH / 5 N H^SO_j, respectively and the absorbance of each
well was monitored at 410/490 nm on an ELISA microplate reader.
Each sample was coated in duplicate and the results were expressed
as a mean of Atest - Acontrol.
67
For nucleic acid antigen, the polystyrene microtiter plates were
preincubated with 100 |il of poly D-lysine solution (50 ^g/ml in
dist i l led water) for 30 minutes at RT so as to increase antigen
immobilization (Arif et al., 1994). The plates were washed thrice with
TBS and coa ted with 100 fil of nuc le ic acid a n t i g e n ( s ) at a
concentration of 2.5 ng/ml in TBS (pH 7.4) for two hours at room
temperature followed by overnight incubation at 4°C. After eliminating
the unbound antigen by washing thrice with TBS-T, the unoccupied
positively charged polylysine molecules were neutralized by saturating
the plates with 100 1 of negatively charged poly-L-glutamate (50 \ig/
ml in TBS, pH 7.4) for two hours at room temperature. The rest of the
steps were same as described above.
Inhibit ion ELISA:
The antigen binding specificity of antibody was determined by
inhibition experiments (Hasan et al., 1991). Varying concentration of
inh ib i tors (0-20 ^g/ml) were mixed with a constant amount of
antiserum/IgG. The mixture was incubated for two hours at 37°C
followed by overnight incubation at 4°C. The resulting immune
complex was employed in the immunoassay instead of serum. The rest
of ihe steps were as in Direct binding ELISA. The resul ts were
expressed as percent inhibition
% Inhibition - 1 - A inhibited x 100 A uninhibited
68
Band shift assay:
The antibody binding to ROS modified and unmodified DNA -
C p ro t e in a d d u c t s / p h o t o a d d u c t s were ana lyzed by a l t e red
electrophoretic mobility on agarose gel (Sanford et. al., 1988). A
constant amount of antigen (1.0 ng) was allowed to interact with
varying amounts of antibody (0-150 ng). The interaction was allowed
to proceed for 2 hrs at 37°C and overnight at 4°C. This resulted in the
formation of immune complex (IC). Two microliter dye solution was
added to each assay tube and the complex was electrophoresed on 1%
agarose gel for 2 hrs at 30 mA using Tris-acetate EDTA buffer, pH
8.0. The electrophoresed gels were stained with ethidium bromide (0.5
^g/ml) and were visualized and photographed under UV illuminator.
Appropriate controls were also run simultaneously.
Estimation of glucose in serum of DCM patients:
Autopak reagent kit was used for in vitro quantitative estimation
of glucose in serum (Trinder, 1969).
In this method, glucose is oxidised by glucose oxidase (GOD)
into gluconic acid and hydrogen peroxide.
Glucose + 0 , > Gluconic acid +UXf^.
Hydrogen peroxide in presence of peroxidase oxidizes the
chromogen
4- amino phenazone/H^O, + Phenolic compound + 4 amino
phena zone > Red compound + 2H O Phenolic compound to a red
69
coloured compound . The in tens i ty of red co lour p roduced is
proportional to the glucose concentration.
b) Assay procedure:
One ml of working solution was added to 10 //I of sample volume
and incubated for 15 minutes at 37°C. The samples were read at 505nm
against reagent blank.
a) Reagents:
i) Tablets: Buffer / enzymes (GOD, POD) / Chromogen
(4 aminophenazone, phenolic compound)
ii) Standard: Glucose lOOmg/dl
One tablet was dissolved in 50ml of distilled / deionized water
to prepare the working solution.
Estimation of Cholesterol in Serum of DCM Patients:
Autopak r eagen t kit was used for in v i t ro quan t i t a t i ve
determination of cholesterol in serum (Tartuttor and Gunter, 1974).
In this method, cholesterol esters are hydrolysed by cholesterol
ester hydrolase to free cholesterol and fatty acids. The free cholesterol
produced and pre existing one are oxidised by cholesterol oxidase to
A - cholestenone and hydrogen peroxide.
CEH Cholesterol esters > cholesterol + fatty acids .
Cholesterol + 0 , > A cholestenone + H ,0 , .
70
Hydrogen peroxide in the presence of peroxidases, oxidize the
chromogen (4 aminophenazone / phenol) to a red coloured compound.
H2O2+ 4 aminophenazone / phenol > Red couloured compound.
a) Reagents:
i) Buffer / Enzymes / Chromogen
ii) Phenol
iii) Standard : Cholesterol - 200mg / dl
Reagent (i) and (ii) were mixed in the ratio of 1:1 (V/V) to
prepare working solution which was stored in a dark bottle at 2 - 8° C
b) Assay procedure:
One ml of working solution is added to 10 //I of serum and the
reaction mixture was incubated for 5 min. at 3 7'^C. The samples were
read at 510 nm against reagent blank.
Estimation of uric acid in serum of DCM patients:
Autopak reagent kit was used for in vitro quantitative estimation
of uric acid in serum (Fossati et al., 1980).
In this method, uric acid is converted by uricase into allantoin
and hydrogen peroxide which in presence of peroxidase (POD)
oxidizes the chromogen (4 - aminophenazone / 3,5 dichloro hydroxy
benzene sulphonic acid) to a red coloured compound.
uricase Uric acid + O2+ H^O > allantoin +C0^+ H^O^
2H2O2+ 4 - aminophenazone + DHBS > Red coloured + H2O
compound
71
a) Reagents:
i) Buffer / Enzymes / 4 - aminophenazone / Potassium ferrocyanide.
ii) 3,5 - Dichloro - 2 - hydroxy benzene sulphonic acid / surfactant,
iii) Standard: Uric acid - 6 mg / dl
The working solution was prepared by mixing 10ml of reagent
(ii) to one vial of reagent (i) and this was stored at 2 - 8°C.
b) Assay procedure:
One ml of working solution was added to 30 ^1 of serum. The
reaction mixture was incubated at room temperature for 15 min and
read at 505 nm against reagent blank.
Estimation of C reactive protein (CRP) in serum of DCM patients:
Stangen C - PROTEST kit was used for in vitro quantitative
and semi - quantitative determination of C - reactive protein in serum
samples in the acute phase of infection (Shire et al., 1981).
Stangen C - PROTEST is a rapid latex agglutination procedure
for the direct detection and semiquantitation on slide of C - reactive
protein in human serum samples. The reagent, a smooth suspension of
latex par t i c les coated with speci f ic an t ihuman CRP ant ibody,
agglutinates in the presence of CRP in the patients serum, a positive
result is indicated by the presence of agglutination and vice versa.
72
Reagents:
i) CRP latex reagent
ii) Positive control
iii) Negative control
Assay procedure:
A) Qualitative Test:
One drop of each control and 50 |il of test serum was placed
onto separate circles on the slide. One drop of CRP reagent was
added to each sample and mixed well over the entire encircled
area by mixing sticks. The slide was rotated gently and slowly
and after 2 min., it was observed for agglutination.
The presence of a visible agglutination on or before 2 min.
indicate a content of CRP > 6mg / L. The sample positive for
CRP was titred by semiquantitative method.
B) Semi quantitative test:
Serial two fold dilutions of positive sample were prepared in
normal saline . The rest of the procedure was same as in
qualitative test (A).
The serum titer was defined as the highest dilution showing
visible agglutination of latex particles. The approximate CRP
level present in the sample was obtained by the formula:
CRP (mg / L) - S x D
73
S = Limit of sensitivity i.e. .6 mg / L
D = Highest dilution of serum sample showing agglutination.
Detection of rheumatoid factor (RF) in serum of DCM patients:
Stangen RHEUMA TEST kit was used for in vitro quantitative
detection of Rheumatoid factor in serum samples (Linker and Williams
Jr, 1985)
RHEUMATEST Kit c o n s i s t s of a smooth s u s p e n s i o n of
polystyrene la tex pa r t i c l e s coa ted with a pur i f ied human IgG
molecules. The pre diluted serum sample to be tested was mixed with
the latex particles and allowed to react. The human IgG forms complex
with rheumatoid factor present in serum and results in an agglutination,
an indicator for positive result and vice versa.
a) Reagents:
i) RF latex reagent
ii) Positive control (prediluted)
iii) Negative control (prediluted)
iv) Specimen diluent
Assay procedure:
Quantitative test:
To 50 \i\ of specimen diluent , 10 ^1 of test serum was added
and mixed well on the slide. One drop of latex reagent was added to
the test samples as well to drop each of positive and negative control.
The reaction mixture was mixed well on be entire circle and the slide
74
was rotated gently for observation of agglutination at the end of 2
min. Agglutination observed on or before 2 min. showed presence of
Rheumatoid factors in the serum.
JR^suIts
75
Isolation of 150 kD C- protein:
¥ rotein antigens of different molecular weights in the total human
heart extract (THHE) were separated on a 5 - 20% gradient SDS - PAGE.
The electrophoresed results depicted in Fig.l inidicated a wide spectrum of
protein antigens ranging from low to high molecular weights. In order to
localize and isolate the 150 kD C protein which is known to induce
myocarditis, the above electrophoresed SDS - polyacrylamide gel was sub
jected to densitometric scanning. The densitometric scanned gels showing
prominent bands of 150 kD C - protein were sliced off and subsequently
cut into small pieces and thereafter subjected to e lec t roelut ion. The
electroeluted pure 150 kD C - protein was further ascertained for its homo
geneity by PAGE. The single band of 150 kD C - protein in PAGE revealed
the inducing 150 kD C -protein antigen. In order to minimize or eliminate
the chances or probability of undetectable impurities in the isolated pro
tein antigen, the visibly pure electroeluted 150 kD C - protein was sub
jected to HPLC (Fig.2). The electroeluted and subsequently HPLC purified
150 kD cardiac C - protein was employed in the binding studies.
Ethidium bromide (Et - Br) intercalat ion assay:
The possibility of traces of DNA present in the total human heart ex
tract (THHE) could not be ruled out. Thus, in order to ascertain and there
after eliminate the possible presence of traces of DNA in THHE which could
interfere in the binding studies, agarose gel electrophoresis was performed.
Calf thymus dsDNA as a control along with THHE were loaded onto the
wells and the gel was electrophoresed. As evident from Fig. 3, visualized
76
1^0 kP Iret-n
Fig. I Electrophoretic pattern of total human heart extract (THHE) on 5 20% gradient SDS - PAGE
77
1-8
1.6 E «= 14 o OD «N 1.2 h-
^ 1.0 UJ o 2 0.8 < OQ CE 0-6 o in ^ . (O OA <
0.2
0.0 <
-
_
—
—
I j \ 1 \ 1 1 j I / 1
/ 1 / I
/ 1 / 1 1 1
1 \ / 1 j \ / 1
/ 1 1 I
/ I 1 I / 1
f / / f -^-^ ' T—^—-t--t--r-2 10 n 12 13 U 15
TIME (min.) 16 17 18
Fig. 2 Elution profile of purification of electroeluted 150 kD C protein by HPLC
78
^
Fig. 3 Agarose gel electrophoresis to show the absence of anv trace of DNA in THHE. Lane 1 (native DNA) Lane 2 (THHE)
79
after staining with Et - Br, only lane containing DNA as control showed
fluorescence upon Et - Br intercalation, whereas no fluorescence was ob
served in the lane containing THHE, thereby ruling out the possibility for
the presence of trace amount of DNA in human heart extract.
Ultraviolet and circular dichroic spectral analysis of 150 kD C - protein:
Prior to immunological characterization, the purified 150 kD cardiac
protein was subjected to ultraviolet and circular dichroic spectroscopy. The
spectral curves showed a X max at 280 nm along with a shoulder at around
289 nm. The Xmin was observed at 248 nm. The A26O / A295 ratio was
computed out to be 1.49. It showed the characteristics of a typical dimeric
protein (Fig. 4) .
Thermal native to coil transition or unfolding study of 150 kD C - protein:
Thermal denaturation of 150 kD C - protein and IgG as control were
investigated in between 30°C and 95°C. The process was characterized by
determining the percent fraction of protein in the denatured state at various
temperatures. Increase in UV absorption at 280 nm was taken as a measure
of thermal unfolding. As evident from Fig. 5, till 75° C thermal unfolding
was found to be of a very low magnitude. Only 4.4 percent of the total 150
kD C - protein underwent thermal unfolding till 75°C. Nearly half of the
150 kD C - protein (i.e. 50%) was observed to have undergone thermal un
folding at around 79.5°C. Further supplementation of thermal energy as
evident from Fig. 5, exhibited a sharp transition in the partially unfolded C
- protein to the fully unfolded state. Complete thermal unfolding was found
1 0 -
80
8 -
-10
I _L _L 210 220 230 2A0 250
WAVELENGTH(nm) 260
Fig. 4 Circular dichroic spectra of 150 kD C protein
81
80 70 60 50 TEMPERATURE(°C)
40 30
Fig: 5(A) Thermal unfolding profile of HPLC purified 150 kD human cardiac C-pro-tein.
82
1 0 0 -
z o 1-< (T TJ 1-< Z UJ
o z ixi
u a: UJ 0 .
80
60
40
20
30 50 60 70 TEMPERATURE(^C)
Fig. 5(B) Percent thermal native -> coil transition profile (% Denaturation) of HPLC purified 150 kD human cardiac C-protein.
83
to be achieved at 87°C. Thus, as evident from the thermal unfolding curves,
significant onset of unfolding was at around 77°C (around 9.19 percent
unfolding) which spanned till 87°C i.e. the temperature at which 100%
unfolding was obtained. Thus, the results indicated the effective thermal
unfolding transition breath to the degree of lO^C (i.e. between 77°C to 87°
C). Furthermore, the above data is indicative for the tremendous structural
stability of the 150kD cardiac C-protein. Similar observation were also made
for IgG (data not shown) which was used as control in view of the estab
lished fact that both IgG as well as 150 kD C - protein have somewhat simi
lar domains. It is to be pointed out that further supplementation of thermal
energy above 87°C exhibited a decrease in absorbance at 280 nm. As evi
dent from Fig.5, the absorbance at 87°C was 1.217 and that it was found to
decrease gradually above 87°C. As evident, the A28O at 95°C was observed
to be 0.565. Thus, at 95°C, nearly more than 50 percent of the absorbance
recorded at 280 nm for 150 kD C - protein in the fully unfolded state (i.e. at
87°C) was found to be lost. Thus a sharp hypochromic effect was observed
at temperatures above the unfolded state of 150 kD C - protein.
Thermodynamical analysis of 150kD C - protein:
The high molecular weight cardiac C -protein was also characterized
by means of thermodynamic analysis. The free energy of unfolding/dena-
turation (AG^) for 150kD C - protein was computed to be 12.363 K. cal.
deg -• at 303°K (30°C) i.e. prior to thermal unfolding. As evident from the
results, the free energy of denaturation / unfolding decreased linearly from
30°C (303OK) to 78°C (351°K). The AG^ at 78°C was computed to be 2.332
84
TABLE 1
THERMAL DENATURATION OFlSOkD CARDIAC CPROTEIN
TEMPERATURE
°C
30 35 40 42 45 48 50 52 55 63 65 70 72 75 77 78 79 80 81 82 83 84 85 86 87 89 90 91 92 94 95
OK
303 308 313 315 318 321 323 325 328 336 338 343 345 348 350 351 352 353 354 355 356 357 358 359 360 362 363 364 365 367 368
^D
0 000 0 008 0 022 0 036 0 038 0 041 0 046 0 048 0 051 0 059 0 067 0 076 0 086 0 122 0 254 0 689 1 222 1 619 2 029 2 216 2 468 2 665 2 743 2 754 2 762 2 110 1 792 1 570 1 343 1 132 1 132
K, App
0 000 0 008 0 022 0 030 0 039 0 042 0 048 0 051 0 054 0 063 0 072 0 082 0 095 0 138 0 341 2 217 -9 222 -2 616 -1 971 -1 822 -1 681 -1 601 -1 574 -1 570 -1 567 -1 900 -2 263 -2,754 -3 913 -8 551 0 370
AG„ = - RT In K, D App
(Kcal.deg')
12 363 9 932 9 184 8 576 8 460 7 984 8 041 7 960 7 724 7 393 7 132 6 752 5 731 3 131 2 322 -6 502 -2 823 -1 998 -1 767 -1 536 -1 397 -1 351 -1 346 -1 343 -1 932 -2 469 -3 074 -4 139 -6 547 3 041
85
TABLE 2
THERMAL DENATURATION OF IgG
TEMPERATURE
»C •'K
30 303 35 308 40 313 45 318 50 323 55 328 60 333 65 338 70 343 75 348 77 350 78 351 79 352 80 353 81 354 82 355 83 356 84 357 85 358 86 359 87 360 88 361 89 362 90 363 91 364 92 365 93 366 94 367 95 368 96 369 97 370 98 371 99 372 100 373
fo
0 000
-0 003 -0 010 -0 013 -0 020 -0 017 0 014 0 024 0 034 0 089 0 166 0 279 0 393 0 521 0 669 0 776 0 883 0 948 1 010 1 028 1 048 1 066 1 083 1 089 1 086 1 082 1 079 1 062 1 052 1 038 1 000
App
0 000
-0 003 -0 010 -0 014 -0 020 -0 017 0 014 0 025 0 036 0 098 0 198 0 388 0 648 1 086 2 021 3 462 7 529 18 333 -97 667 -37 250 -21 714 -16 263 -13 083 -12 154 -12 600 -13 083 -13 609 -17 111 -20 333 -27 364 0 290
AG„ = RT In K, D App
(K cal deg-1)
15 607
12 564
11 818
10 993
11 620
12 351
10 734
9 700
6 798
4 752
2 787
1 281
-0 246
-2 089
-3 696
-6 026
-8 707
-13 752
-10 887
-9 289
-8 440
-7 802
-7 600
-7 730
-7 867
-8 009
-8 73 5
-9 291
-10 234
1 449
86
k.cal. deg '. The large positive AG^ values obtained at the initial stages of
thermal unfolding which decreased linearly till 78°C is indicative for the
folding of C - protein by ionic and hydrophobic interactions with probably
intermittent presence of disulfide bonds. Furthermore, the results indicate
that C - protein having domains similar to IgG is not that so thermodynami-
cally compact molecule as is native DNA, where due to stable stacking of
the base pairs, a negative AG^is observed in the initial stages of thermal
melting. Further elevation of thermal energy showed negative AGjj values
for 150 kD C -protein . The AG^ values from 79«C to 94°C (i.e. 352°K -
367°K) were computed to be - 6.502 K.cal. deg ' and -6.547 K.cal. deg "'.
The AGp values above 79''C decreased linearly till 89°C (362°C) followed by
again an increase in the AG^ values till 94°C. The results are indicative for
the complete thermal unfolding of the 150 kD C - protein at around 78°C or
79°C. Due to the reason that 150 kD C - protein is having some what simi
lar domains to IgG, comparative thermodynamical analysis between the two
as also carried out. As evident from the results, IgG was found to be a
thermodynamically more compact molecule than 150 kD C - protein. These
thermodynamic parameters are depicted in Tables - 1 & 2.
Urea denaturation studies:
The 150 kD cardiac C protein and IgG were subjected to urea SDS-
PAGE at constant concentration of acrylamide (12.5%) and urea (8M), us
ing an acrylamide (5-20%) and urea (I-IOM) gradient. A more or less simi
lar picture emerged with both denaturing process where two prominent bands
of more of less same molecular weights (Fig. 6) were observed.
87
(A)
pi' n 1 r I
Fig. 6 Electrophoretic pattern of C protein and IgG on urea-SDS-PAGE (A) 12.5% urea SDS - PAGE with 8M urea (B) 12.5% urea SDS - PAGE using a linear gradient of 1-10 M urea. (C) 5 - 20% urea SDS - PAGE using a linear gradient gel of 8M urea.
Lane 1 (C protein) Lane 2 (IgG)
88
Antigenicity of 150 kD C -protein:
Antibodies against native 150 kD C-protein were raised in rabbit and
rats. The titre of antibodies was ascertained by direct binding ELISA wheras
inhibition ELISA was performed to probe the antigenic specificity of these
antibodies.
a) Evaluation of antibody response:
Direct binding ELISA was used to characterize the immune response
in rabbit and rats following immunization with 150kD C - protein.
The serum showed a high titre of greater than 1:12800 (Fig. 7 & 8).
Pre immune serum served as negative control as it did not show any
appreciable binding to 150 kD C - protein.
b) Competition ELISA for the antibodies raised against human heart
150 kD C - protein:
The antigenic specificity of the induced antibodies in rabbit and rats
against human heart 150 kD C - protein was probed by competition
ELISA. In rabbit, a maximum inhibition of 98.7 percent was obtained
in immune serum at an inhibition concentration of 10 ng/ml (Fig. 9).
Whereas the immune serum of rat exhibited a maximum inhibition of
88.1 percent at inhibitor concentration of 10 |ig/ml (Fig. 10). Fifty per
cent inhibition was achieved at inhibitor concentration of 0.0016 \xgl
ml with rabbit serum compared to 0.07 fig/ml with rat serum.
89
1.4
1.2
E 1-0 c o
»- 0.8
u
S 0.6 a o to ** 0.A
a2
•
- \
-
^ g 9 ^ =£;^=
•^•^^..^^
= 9 u_ 2.0 2.3 2.6 2.9 3.2 3.5
-LOGSERUMDILUTION 3.8 A.I
Fig. 7 Binding of anti - C protein antibody in rabbit with C protein. Immune serum (#—# ) Preimmune serum(C>-0)
2.9 3.2 3.5
LOG SERUM DILUTION
3.8 4.1
Fig. 8 Binding of anti - C protein antibody in rat with C protein. Immune serum ( # - ) Preimmune serum ( O O )
90
0.01 0.1 1.0 10,0
INHIBITOR CONCENTRATION [Mgln>\)
Fig. 9 Inhibition of anti - C protein antibody activity in rabbit. Antigen : C protein Competitor : C protein
0.01 0.1 1.0 INHIBITOR CONCENTRATION ( >j g / m I )
10.0
Fig. 10 Inhibition of anti - C protein antibody acti\il> in rat by C protein Antigen : C protein Competitor : C protein
91
c) Purification of characterization of immune IgG:
Immune IgG was isolated from serum by Protein - A - Sepharose 4B
affinity chromatography. The purity of IgG was ascertained by SDS -
PAGE under non - reducing condition (Fig. 11) with the IgG moving
as a single band.
Direct binding ELISA of the purified IgG showed strong reactivity
with the immunogen (Fig. 12). Pre immune IgG as negative control
showed negligible binding to 150 kD C - protein.
Antigenic specificities of antibodies induced against human heart 150
kD C - protein:
The fine antigenic specificity of antibodies induced against 150 kD C
- protein was ascertained by competition ELISA. The plates coated with 150
kD C protein exhibited varying degrees of inhibition against a variety of
inhibitors. C - protein exhibited maximum inhibition of 98.7 percent at con
centration of 10 ng/ml when used as an inhibitor. Fifty percent inhibition
was achieved at a concentration of 0.0016 i^g/ml of inhibition. On ROS
modification there was a decrease in inhibition of the antibody activity that
is, 38.7 percent at an inhibitor concentration 10 |Ag/ml. Fifty percent inhi
bition was not achieved in this case (Fig. 13). Among the three photoadducts
formed by the interaction of 150 kD C -protein and nDNA, the one formed
in the presence of OH (ROS DNA-C-protein photoadduct) gave maximum
inhibition to the tune of 99.2 percent. This was followed by the photoadduct
formed in the presence of H2O2 (ROS DNA-C-protein adduct) that is 87.8 per-
92
2 3 ^ 5 6 FRACTION NUMBER 1 2 3 A 5 6 7
FRACTION NUMBER
3 A
FRACTION NUMBER
Fig. 11 Elution profile of IgG with 0.58% acetic acid and 0.85% Nacl from Protein A sepharose CL - 4B column, a) DCM IgG b) SLE IgG c) Immune IgG Inset shows single band of IgG on 7.5% SDS PAGE
93
2 /. 6 8 10 CONCENTRATION OF I g G ( ; u g / m l )
Fig. 12 Binding of rabbit immune IgG with C protein Immune IgG ( • - • ) Preimmune IgG ( Q - O )
10 20 30 AO 50
CONCENTRATION OF I g G ( ; i j g / m l )
60 70
Fig. 12 Binding of DCM IgG with C protein DCM IgG ( - ) Normal human IgG ( C M D )
94
cent was observed when the photoadduct was formed only in the presence
of ultraviolet radiation (UV-B). In all these, the maximum inhibitor concen
tration was 10 ^g/ml. The same pattern was observed when the concentra
tion for fifty percent inhibition was taken into account. The photoadduct
formed in the presence of OH gave fifty percent inhibition at an inhibitor
concentration of 0.09 \ig/ml followed by the adduct formed in presence of
H202(3.4 |ag/ml) and photoadduct formed in presence of UV - B irradiation
(7ng/ml) (Fig. 14). To check the cross reactivity of the induced antibodies,
various other inhibitors were also used. Out of these histone showed maxi
mum inhibition of 7.3 percent at 20 [ig/ml which increased to 38.7 percent
on ROS modification (Fig 15). Poly - D - lysine gave a maximum inhibition
of 9.9 percent at 20 ixg/ml as compared to Poly L - Glutamate which showed
inhibition of 30.1 percent. The Poly (L + G) compelx formed after incuba
tion of Poly - D - lysine and Poly L Glutamate at 37°C for 2 hrs. showed
maximum inhibition of 25.2 percent. Fifty percent inhibition was not
achieved in all these cases (Fig. 16). In case of nucleic acids, on ROS
modification of DNA, extent of inhibition was 15.4 percent at 20 (ig/ml. RNA
gave an inhibition of 5.2 percent at 20 ng/ml. Fifty percent inhibition was
not achieved in these two cases (Fig. 17). Percent relative affinity was calcu
lated for inhibitiors in which fifty percent inhibition was achieved. Out of
the various inhibitors, photoadduct formed in the presence of OH showed
highest relative affinity of 1.8 percent followed by photoadduct in pres
ence of H2 02(0.04 %) and photoadduct in presence of UV - B irradiation
(0.02%). The results have been summarized in Table 3.
95
0,001 0.01 0.1 1.0
INHIBITOR CONCENTRATION ( > j g / m l )
10.0
Fig. 13 Inhibition of rabbit immune IgG acitivity by native and ROS C protein. Antigen : C protein Competitors : C protein ( O O )
ROS C protein ( 0 - )
96
0
c 20
z o
5 /loj z 1 -
S 60 u CE UJ
80
S ...
-
u
•
1
^ ^ \ . *
1
N. O
0O1 0.1 1.0 10.0
INHIBITOR CONCENTRATION ( j ug /m l )
100
Fig. 14 Inhibition of rabbit immune IgG activity by nDNA - C protein ad-ducts Antigen : C protein Competitors: nDNA - C protein photoadduct
ROS-DNA - C protein adduct ROS-DNA - C protein photoadduct
(O-O)
97
z i*J 6 0 « j
tLl
a.
80
J_ COl 0.1 1.0 10.0
INHIBITOR CONCENTRATION ( > j g / f D l )
100
Fig. 15 Inhibition of rabbit immune IgG activity by native and ROS Histone Antigen : C protein Competitors: Histone ( Q - O )
: ROS Histone ( # - )
98
20 z g H^
E9 AO I z »-z uj AO u Q: UJ
a 80
k TM—' ^
^
•
1 1 1
0.01 0 1 1.0 10.0
INHIBITOR CONCENTRATION (> jg /m l )
100
Fig. 16 Inhibition of rabbit immune IgG acitivity by poly D lysine, poly L. glutamate and poly (L+G) complex. Antigen : C protein Competitors: Poly D - Lysine (O-O)
Poly - L Glutamate ( - ) Poly (L+G) complex (O-Q)
99
04
20 z g P S AO I z »-z iJ 60 CJ cr UJ CL
80
^
_
—
_ _ _ _ _ _ ( J > u
1
"U < X ^
0.01 0.1 1.0 10.0
INHIBITOR CONCENTRATION (jjglmi)
100
Fig. 17 Inhibition of rabbit immune IgG activity by ROS-DNAand RNA Antigen : C protein Competitors: ROS DNA ( # - # )
RNA ( O O )
100
TABLE 3
ANTIGENIC SPECIFICITY OF ANTI C-PROTEIN ANTIBODIES
Inhibitors 50% Inhibition Maximum Inhibition % Relative (Hg/ml) (at 20 Hg/ml) (%) Affinity
C-Protein 0.0016 98.7" 100
ROS C-protein a 38.7''
nDNA C-protein 7.0 52.2'' 0.02 photoadduct
ROS-DNA-C-protein 3.4 87.8 0.04 adduct
ROS-DNA-C-protein 0.09 99.2'' 1.8 photoadduct
Histone a 7.3
ROS Histone a 38.7
Poly-D-Lysine a 9.9
Poly-L-Glutamate a 30.1
Poly (L-G) Complex a 25.2
ROS DNA a 15.4
RNA a 5.2
a = Fifty percent inhibition not achieved,
b = Maximum inhibition at 10 jig/ml.
101
Binding of total human heart extract (THHE) by DCM autoantibodies:
Out of the 15 DCM sera tested, most of them showed appreciable bind
ing with total human heart extract (50 pig/ml). Sera from 9 DCM patients
gave a titre of more than > 1:6400. Three sera gave a titre of 1:3200 whereas
two gave a titre of 1:1600 (Fig. 18).
Recognition of human heart ISO kD C - protein by DCM autoantibodies:
The sera from DCM patients showed appreciable recognition towards
gel electroeluted, followed by HPLC purified human heart 150 kD C-pro-
tein. Of the 8 sera tested, 6 sera gave a titre of >r .6400 whereas two sera
gave a titre of 1:3200 (Fig. 19). The antigenic specificity of autoantibodies
against 150 kD C-protein was ascertained by competition/inhibition ELISA.
The 6 sera showing a titre of > 1:6400 were selected for inhibition studies
(Fig. 20). The degree of inhibition varied from 67.4 percent to 81.2 percent
at inhibitor concentration of 20 ng/ml. The order of specificity of these DCM
sera was ascertained by evaluation of concentration for fifty percent inhibi
tion. The concentration for fifty percent inhibition varied from 0.02 to 2.0
|ig/ml of inhibitor (Table 4).
Binding of native DNA with DCM autoantibodies:
Native ds DNA which is the main trigger for SLE autoantibodies giv
ing an antibody titer of >1:6400 showed poor recognition with DCM au
toantibodies. Of the 7 DCM sera tested, one gave a titer of 1:800, followed
by 1:400 by three sera andtKree sera gave a titre of 1:200 (Fig. 21).
1 0 2
DIRECT BINDING ELISA OF THHE WITH DCM AUTOANTIBODIES
6 6 7 8 9 10 11 Number of serum samples
Fig. 18
12 13 14 16
a
b
1 : 1 0 0
1 :200
c d e f
g
1:400 1:800 1: 1600
1:3200
1:6400
103
0.01 0.1 1.0 10
INHIBITOR CONCENTRATION {pglm\)
0.01 0.1 1.0 10.0
INHIBITOR CONCENTRATION ( p g / m l )
Fig. 19 Inhibition ELISA of DCM autoantibodies with C protein as inhibitor. Antigen : C protein Competitor : C protein Antibody : Serum 1 ( Q - O ) Serum 4 ( O O )
Serum 2 (O-O) Serum 5 0 - 3 ) Serums ( - ) Serum 6 ( - )
104
TABLE 4
COMPETITION ELISA OF 150 kD CARDIAC C-PROTEIN
WITH DCM AUTOANTIBODIES
DCM Sera 50% Inhibition Maximum Inhibition (Hg/ml) (at 20 Jlg/ml) (%)
1 0.5 75.8
2. 0.03 78.6
3. 0.24 81.2
4. 0.02 71.9
5. 2.0 67.4
6. 0.6 73.6
105
DIRECT BINDING ELISA OF DCM AUTOANTIBODIES WITH 150 KD C-PROTEIN
c o
•H
r-l •H
o O
to 2 3 4 6 6
Number of serum samples 8
"r <
F i g . 20 i DCM Sera NHS I
106
DIRECT BINDING ELISA OF DCM AUTO ANTIBODIES WITH NATIVE DNA
0.6
0.4
c 0-3 o
p 0.2 rH •H
O 0.1 O
4J NHS
i • ^ 1 2 3 4 5 Number of serum samples
DCM sera ^ ^ NHS
Fig. 21
107
Antigenic specificity of DCM autoantibodies:
DCM autoantibodies showed variation in percent inhibition with a
wide spectrum of inhibitors. In all the cases, the coating antigen was hu
man heart 150 kD C -protein (50 [ig /ml in COj"/ HCO 3- buffer pH 9.6). A
maximum inhibition of 83.9 percent was obtained at 20 |xg/ml when C pro
tein was used as inhibitor and fifty percent inhibition was observed at in
hibit concentration of 0.9 ng/ml. The same on ROS modification showed a
lesser extent of inhibition which was found to decrease drastically upto 25.8
percent at 20 jxg/ml. Fifty percent inhibition was not achieved in this cases
(Fig. 22). The degree of inhibition among the nDNA - C protein photoadducts
varied in accordance with the agents used in their formation. A maximum
inhibition to the extent of 90 percent was formed at 20 |xg/ml in case of
photoadduct formed in presence of OH as compared to inhibition of 88.6
percent at 20 jxg/ml in case of photoadduct formed in presence of H2 O2.
Fifty percent inhibition was achieved at 1.0 |ig/ml in latter. The lowest
amount of inhibition of 41.7 percent was seen in the photoadduct formed
only in presence of UV - B irradiation (Fig. 23). To check the cross reactivity
of these antibodies various inhibitors were used. When histone was used as
an inhibitor, a maximum of 9.2 percent inhibition was achieved at inhibi
tion concentration of 20 |ig/ml . On ROS modification of the same there
was an increase of upto 33.9 percent at 20 iig/ml (Fig. 24). Poly - D - Lysine
and Poly - L - Glutamate gave an inhibition of 6.6 and 40 percent at an
inhibitor concentration of 20 )ig/ml. The Poly (L + G) complex formed by
incubation of equimolar concentration of Poly D - Lysine and Poly L -
Glutamate (for 2hrs. at 37°C) gave an inhibition of 34.2 percent at 20 ng/ml
108
0.01 0.1 1.0 10.0
INHIBITOR CONCENTRATION {jjglm\)
Fig. 22 Inhibition of DCM IgG activity by native and ROS C protein Competitors: C protein (CKD) ROS C protein ( - )
109
0,01 0.1 1.0 10.0 INHIBITOR CONCENTRATION ( ; u g / m l )
Fig 2.3 Inhibition of DCM IgG activity by nDN.A - C protein adducts Antigen : C protein Competitors : nDNA- C protein photoadduct ( # - # )
ROS-DNA - C protein adduct ( ( ^ - 3 ) ROS-DNA - C protein photoadduct (Q-O)
110
z o 1 -
00
I z
z UJ a cr Q.
AO
60
80
JL 0.01 01 1.0
INHIBITOR CONCENTRATION ( ;ug /m l ) 10
Fig. 24 Inhibition of DCM IgG activity by native and ROS modified histone. Antigen : C protein Competitors: Histone ( # - # )
ROS Histone ( O O )
I l l
"i 20
g
2 40 X 2
z lu 60 u cr a
80
k
^ - - - - _ _ _ •
—
/> VP
U
1
/^ \M
#
1
f l <» -J^
• s r
1
0.01 0.1 1.0 10
INHIBITOR CONCENTRATION ( > i g / m l )
Fig. 25 Inhibition of DCM IgG antivity by poly -D-lysine, poly-L-glutamate and poly (L+G) complex. Antigen C protein Competitors: Poly D lysine ((f_o )
Poly L Glutamate (O-O ) Poly (L+G) complex (^-9) '
112
0.01 0-1 1.0 10
INHIBITOR CONCENTRATION ( > j g / m l )
Fig. 26 Inhibition nDNA of DCM IgG activity by ROS DNA and RNA. Antigen : C-protein Competitors: ROS-DNA(#-# )
RNA ( O O )
113
TABLE 5
ANTIGENIC SPECIFICITY OF DCM AUTOANTIBODIES
Inhibitors 50% Inhibition Maximum Inhibition % Relative (Hg/ml) (at 20 \lg/m\) (%) Affinity
C-Protein 0.9 83.9 100
ROS C-protein a 25.8
nDNA-C-protein a 41.7 photoadduct
ROS-DNA-C-protein 1.2 88.6 75 adduct
ROS-DNA-C-protein 1.0 90.0 90 photoadduct
Histone a 9.2
ROS Histone a 33.9
Poly-D-Lysine a 6.6
Poly-L-Glutamate a 40.0
Poly (L-G) Complex a 34.2
ROS DNA a 15.9
RNA J .-•>
a = Fifty percent inhibition not achieved.
114
(Fig. 25). The extent of inhibition was seen to be 15.9 percent on ROS modi
fication of nDNA. On the other hand, RNA gave an inhibition of 3.3 per
cent at 20 ^ig/ml (Fig. 26). The percent relative affinity was calculated for
inhibition in which fifty percent inhibition was achieved. Among the
photoadducts, the one formed in presence of OH showed elimination in an
antibody activity as high as to the extent of 90 percent followed by the
photoadduct formed in presence of H2O2 which showed 75 percent relative
affinity. In rest of the inhibitors fifty percent inhibition was not achieved.
Formation of cardiac 150 kD C protein native DNA photoadduct:
The photocoupling of purified calf thymus native DNA and HPLC
purified 150 kD C - protein (human heart) was carried out in the presence
of UV - B irradiation at 254 nm. The photocoupling was allowed to proceed
at different time intervals of exposure to UV - B radiation (Fig. 27). The
irradiated samples were extensively dialyzed against PBS, pH 7.4 and
subjected to ultraviolet spectroscopical analysis. Irradiated DNA showed a
substantial decrease in absorbance i.e. hypochromism was observed in the
entire spectral range. Ultraviolet irradiation of C-protein also exhibited
substantial hypochromism at maxima and minima (Fig. 28) . The native
DNA - C - protein photoadduct showed relative increase in hypochromicity
with increase in time of exposure to ultraviolet irradiation . Moreover, there
was a shift in maxima and minima of the photoadduct as compared to native
DNA. The maxima showed a shift from 260 nm to 255 nm whereas the shift
in minima from 230 nm to 235 nm relative to the ultraviolet absorption
characteristics of native DNA. Also, a sharp decrease in the ratio of
115
Ui O z <
o in CD <
075 -
200 300
WAVELENGTH ( n m )
400
Fig. 27 Time course kinetic profile of photoaddition of C protein to native DNA. Irradiation time (min) were. a) 10; b) 15 c) 20 and d) 30.
116
(A) '
u u z < 01 a. o 1/1 10
<
0.75
(B) 0.5
UJ u z < " 0.25 (r o (/) <
300
WAVELENGTH (nm)
CO 05
iVOO 2 00
200 300 WAVELENGTH(nm)
Fig. 28 UV absorption characteristics of .A<j native nDNA ( )
300
WAVELENGTH (nm)
400
AOO
native C protein ( d DNA ( ) ^®)UV irradiated C protein (
) UV irradiateu ui^r-\. y j - u v mauiai^u ^ pi^n-m v, -/
CO Difference UV spectra of nDNA, C protein and nDNA - C protein photoadduct
native DNA ( ) native C protein ( ) nDNA - C protein photoadduct (-»—)
117
TABLE 6
UV ABSORPTION CHARACTERSTICS OF FORMATION
OF nDNA - C PROTEIN PHOTOADDUCTS
SAMPLES
native DNA
(control)
native DNA
(Irradiated)
C protein
(Control)
C protein
(Irradiated)
nDNA - C protein
Photoadduct
Control ( 0 min)
(10 min)
(15 min)
(20min)
(30min)
ABSORBANCE 260 nm
1.044
0.901
0.082
0.069
1.071
0.920
0.926
0.946
0 978
295nm
0.091
0.082
0.055
0.052
0.146
0.112
0.116
0.122
0.128
ABSORBANCE RATIO 260/295
11.47
10.99
1.49
1.33
7.34
8.21
7.98
7.75
7.64
118
260/295 was observed for the photoadduct. This decrease was relative to
increase in the time of exposure to ultraviolet irradiation. Similarly, native
DNA and 150 kD C protein individually showed a decrease in 260 / 295
ratio on ultraviolet irradiation (Table 6 ).
Format ion of nat ive DNA 150 kD cardiac C - prote in adduct s /
photoadducts in presence of ROS:
Electroeluted and electrophoretically pure 150 kD human cardiac C -
protein was covalently crosslinked to purified calf thymus native DNA in
presence of ROS i.e. U^O^and OH (generated by H^Ojin presence of UV -
B irradiation) (Fig. 29). The samples were extensively dialyzed in PBS, pH
7.4 and subjected to UV spectroscopical analysis (Fig. 30). A substantial de
crease in absorbance i.e. hypochromism was observed in the case of
photoadduct in presence of OH. A sharp decrease in the ratio of 260/295
was observed in this case. ROS-DNA also exhibited hypchromism but of a
lower magnitude than the former with reference to native DNA. The nDNA
- C protein adduct formed in presence of H2O2 exhibited high degree of
hypochromism with an increase in 260/295 ratio . Furthermore, as evident
from the (Fig. 30), ROS-C - p io t e in exhibi ted a cer ta in degree of
hyperchromism at 280 nm with reference to native C - protein. The ultra
violet spectroscopical characteristics of modified and unmodified samples
are depicted in table 7.
De tec t i on of DNA - C - p ro t e in adduc t format ion by a g a r o s e gel
electrophoresis :
Agarose gel electrophoresis was carried out to detect the adduction
119
200 300 WAVELENGTH ( n m )
400
300 NWAVELENOTH ( n m )
600
Fig. 29 UV absorption characteristics of: native C protein( ) rgxnDNA ROS C protein (---- ) ^ ^ROS DNA
r -v ROS-DNA - C protein adduct ( ) ROS-DNA - C protein photoadduct ( )
CA) (___
120
TABLE 7
UV ABSORPTION CHARACTERISTICS OF FORMATION OF
ADDUCT/PHOTOADDUCT IN PRESENCE OF ROS.
SAMPLE
native DNA
ROS-DNA
C protein
ROS-C-protein
ROS-DNA - C
adduct
ROS-DNA - C
photoadduct
protein
protein
ABSORBANCE 260nin
1.944
1.244
0.038
0.066
1.999
1.115
295nni
0.196
0.201
0.023
0.033
0.328
0.140
ABSORBANCE RATIO 1 260/295
9.92
6.19
1,65
2.00
6.10
7.96
121
M 3
Fig. 30 Electrophoretic mobility of samples (1.0 [xg each) after ROS modification. The samples were run on 1% agarose gel at 30 mA for 2 hrs. Lane 1 (native DNA) Lane 2 (ROSDNA) Lane 3 (DNA - C protein + H^O^) Lane 4 (DNA - C protein + H^^^ U V )
122
C^) \ ^
iu 1 7, i Z
CC)
Fig. 31 Nuclease SI sensitivitiy of DNA - C protein adducts. DNA - C protein adducts (1.0 pg) were treated with nuclease SI (20 u/mg of DNA) a) nDNA - C protein photoadduct b) ROS-DNA - C protein adduct c) ROS-DNA - C protein photoadduct
Lane 1 (control) Lane 2 (treated samples)
123
of DNA to C protein. The adducts were treated with SI nuclease (20 units/
Hg adduct) and the digested sample were subjected to electrophoresis . The
results showed enhanced mobility of SI treated adducts (Fig. 31). This was
seen in the case of adducts formed in the presence of ROS which include
HjO^and OH. On the other hand photoadduct formed in the presence of UV
- B irradiation alone did not show any change in mobility. The untreated
adducts were used as controls. The results indicated the generation of single
stranded portions in DNA - C- protein a d d u c t photoadduct formed by H2O2
and OH as recognised by single strand specific enzyme SI nuclease.
Kinetic of lysine photoaddition to native DNA:
The nature of photointeraction between lysine residues of C - protein
and DNA was also investigated by kinetic study. The irradiated samples
(10-30 min) were dialyzed against PBS, pH 7.4 and their UV spectra re
corded. The results showed a constant increase in absorbance at 260 nm
with the increase in irradiation time. There was a linear increase in percent
hyperchromicity with the increase in radiant energy (Fig. 32). The number
of lysine residues in the photoadduct C - protein was determined as a result
of varying period of UV irradiation (Fig. 33). One lysine molecule of the C-
protein was in the photobound state in between 7.1, 6.3, 5.4 and 3.6 nucle
otide base pairs of DNA on irradiation for 10, 15, 20 and 30 min. respec
tively (Fig. 34). The results obtained indicates a progressive increase in the
photoaddition of lysine molecules (present in C-protein) to DNA with the
increase in irradiation time. A linear regression plot of hi (Va - Vt) vs. the
irradiation time 't' gives a straight line which is characteristic of apparent
first order reaction mechanism (Fig. 35).
124
1 ^ 3 J^ S'
Fig. 32 Time course pattern of DNA - C protein photoadducts formed by UV - B irradiation . The samples (1,0 |ig each) were electrophore-sed on 1% agarose gel at 30mA for 2 hrs. Lane 1 ( O min.) Lane 2 (lOmin.) Lane 3 (15 min.) Lane 4 (20 min.) Lane 5 (30 min.)
125
0 10 20 30 40 50 60 70 80 90 100
CONCENTRATION OF L-LYSINE ( j j g / m l )
Fig. 33 Standard plot for estimation of lysine by TNBS method.
126
10 20 30
IRRADIATION TIME (min.)
Fig. 34(A) Concentration of photobound lysine at various irradiation times in native DNA - C protein photoadduct.
10
10 2 0 30 IRRADIATION TIME (min )
Fig. 34 (B) Kinetic profile of photobound lysine residues to native DNA at various irradiation t imes.
127
5 10 15 IRRADIATION TIME(min.)
Fig 35 Apparent first order profile of nDNA-C-protein photoadduct formation at various irradiation times. The values were least square fitted and the apparent first orderate constant K was computed from the
slope.
128
Isolation of DCM and SLE IgG with protein A - sepharose:
Protein A binds IgG from most mammalian species (Kronvall et al,
1970; Langone, 1978). Although IgG is the major reactive human
immunoglobulin, some other types have also demonstrated to bind with
Protein A. The interaction of Protein A with IgG occurs via Fc portion of
IgG molecules from many species.
Protein A sepharose purified IgG's from normal, DCM and SLE sera
were each found to elute in a single symmetrical peak. The absorbance
ratio (A^^g/ A^j,) of the peak fraction was found to be 2.5, typical of mam
malian IgG. The homogeneity of the peak fraction was assessed by its
single band movement in SDS - PAGE under non reducing conditions
(Fig. 10)
Affinity purification of SLE IgG on native DNA - polylysyl sepharose 4B
column:
Poly-L - lysine sepharose 4B is an amphoteric derivative of sepharose
4R which is a group specific absorbent for purification of dsDNA. Poly - L
- lysine sepharose 4B is prepared by coumpling poly - L - lysine to sepharose
4B by the cyanogen bromide method. The IgG fractions were collected us
ing a linear gradient of ionic strength 0.15 - 3.0 M sodium chloride in 0.01
M phosphate buffer, pH 7.4 (Fig. 36). Affinity purified SLE IgG was found
to be nearly free from contamination by other classes of immunoglobulins
as judged by their homogenous movement in SDS PAGE under non reducing
conditions.
1 2 9
E c o 00
o < GO a o en CD <
1.0 -
0.8-
0.6-
0.^-
0.2-
T r 1 1 1 1 1 r-HI—T—*-nr
2 ^ 6 8 10 12 U 16 23 25
FRACTION NUMBER
Fig. 36 Imraunoaffinity purification of SLE IgG on nDNA-[polylysyl sepharose 4B] column
130
Band - shift assay:
The binding of DCM, SLE and induced 150 kD C - protein antibodies
with adducts/photoadducts was reiterated by band, shift assay. One micro
gram each of nDNA - C - protein photoadduct, ROS - nDNA - C protein
adduct ROS DNA - C - protein photoadduct was incubated with varying
amounts of DCM, SLE and immune IgG for 2 hrs at 37°C. The immune com
plex thus formed was kept overnight at 4°C and later electrophoresed on
1% agarose at 30mA for 2 hrs. in tris acetate EDTA (pH 8 0) . The wells in
the gel showed increase in the amount of immune complex on increase in
IgG concentration. A retardation in mobility was also observed indicating
the strong interaction between the DNA - C - protein adducts and DCM,
SLE and immune IgG. (Figs. 37, 38, 39).
Recognition of human cardiac antigens by SLE autoant ibodies:
The recognitiion of antigens ranging from nucleic acids to myocarditis
autoantigen (s) by naturally occuring SLE autoantibodies by direct binding
and competition ELISA irect binding ELISA on plates coated with nucleic
acid antigens i.e. ds DNA, SS DNA and RNA as well as THHE exhibited
appreciable reactivity with SLE autoantibodies (Fig. 40). Competit ion
inhibition ELISA exhibited inhibition in antibody activity with ds DNA, ss
DNA, RNA (Fig. 41) and THHE (Fig. 42) to a tune of 75.2%, 75%, 10.5%
and 75% respectively. Fifty percent inhibition varied except in case of RNA
where it could not be achieved. Protein A-sephorose purified SLE IgG with
C-protein exhibited inhibition of 79.3% with fifty percent inhibition at 1.8
Mg/ml (Fig. 43) was employed to further ascertain the specificity. This
131
i 9. 3 "^
Fig 37(A) Binding of DCM IgG by DNA - C protein photoadduct as analyzed by gel retardation assay nDNA - C protein photoadduct (1 0 ng) was incubated with buffer ( l ane l ) and with various concentrat ions of DCM IgG (25 (ig, lane 2, 50 (ig. lane 3 and 75 Mg. lane 4 The complex was electro-phoresed for 2 hrs on 1% agarose at 30 mA
Av '-) ^
Fig. 37(B) Binding of SLE IgG by DNA - C protein photoadduct as analyzed by ge! retardation assa\ nDNA - C protein photoadduct (1 0 fjg) was incubated with buffer ( l ane l ) and with various concent ra t ions of SLE IgG (25 |ig. lane 2, 50 (jg. lane 3, 75 (ig, lane 4
The complex was electrophoresed for 2 hrs on 1% agarose at 30 iiiA
1 ^ 3 ^ 5~
fig 3 7 ( 0 Binding of anti C" proicin Immune IgG b\ nDNA - C protein photoadduct as analyzed hy gel retardation assav nDNA - C protein photoadduct (1 0 Mg) was inci'bated with buffer (lane 1 ) and with \c loiis concentrations of anti - (" protein immune IgG (25^ig. Lane 2. 50 pg, 1 anc 3, ""5 g Lane 4 and 150 g. Lane 5) The complex was electrophoresed for 2 hrs on l°o agarose at 30 m \
1 7. W^ f
Fig 38(B) Binding of SLE IgG by ROS - D \ A, -C protein adduct and analyzed b\ gel retardation assay ROS-DN A - C protein adduct (1 0 ng) was incubated with buffei (lane 1) and with \ a r ious concent ra t ions of SLE IgG (25 pg Iane2 50 ng lanel 75 pg lane and 150 (.ig ) The complex was electro-phoresed for 2 hrs on l%agaros t at iO mA
' 3 ;i 1 A ^ 0
Fig 38(A) Binding of DCM IgG by ROS - 1 ; \ A -C protein adduct and inalyzed by gel retardation assay ROS-DNA - C protein adduct ( I 0 ng) was incubated with buffer ( lane 1) and with \a r ious concentrat ions of DCM IgG (25 ng lane2, 50 pg lane"? and 1'^ \x% lane 4) The complex was electrophoresed for 2 hrs on 1% agarose at 30 mA
Fig i8(C) Binding of anti - C protein / Immune IgG b \ ROS-DN^ - C protein adduct as anaKzed bj gel retardation assay ROS-DN^ - C p r o t e i n adduct (1 0 lag) was incubated with buffer (lane 1) and with \ar ious concentrations of anti - C protein / immune IgG (25 iig lane2' '50Lig lane3''75Mg lane 4 150 Hg lane 5) The complex was electrophoresed for 2 hr on 1% agarose at 30 mA
133
1 2. ;3 ^ 5-
Fig 39(A)Binding of DCM IgG by ROS DNA -C protein phoioadduct as analyzed by gel retardation assay ROS DNA - C protein photoadduct (1 Ong) was incubated with buffer (lane 1) and with various concentrations of DCM IgG (25 Jig, lane2, 50 ^^g lane 3, 75 ng, lane 4 and 150 |ig, l aneS) The complexed was electrophoresed for 2 hrs on 1% agarose at 30 mA
i ^ 3 ^ 5'
"w' Wp, jfKt^.m^-^^
Fig 39(B) Binding of SLE IgG by ROS DNA - C protein photoadduct as analyzed by gel retardation assay ROS DNA - C prote in photoadduct (1 O^g) was incubated with buffer (lane 1) and with various concentrations of SLE IgG (25 Hg lane2 50 )ig, lane 3, 75 ng, lane 4 and 150 fig lane'>) The complexed was elec'.rophorestd for 2 hrs on 1% agarose at ' O mA
1 ^ 5 ^ r Fig 39(C) Binding of anti - C protein / immune
IgG by ROS - DNA - C p r o t e i n photoadduct as analyzed b> gel retardation assa\ ROS DNA - C protein photoadduct (1 0 \ig) was incubated with buffer (lane 1) and with \ a r ious concentration^, of anti - ( protein / immune IgG (25 g lane2 50g lane3 75 g lane 4 and 150 g lane 5) The complex was electrophoresed for 2 hr on P/o agarose at 30 mA
134
e o
< UJ o z < CD o: o (/) CO
<
1:100 1:200 1:400 1:800 1:1600
-LOG SERUM DILUTION
1:3200 1:6400
Fig. 40 Binding of SLE auto antibodies with ds DNA, ssDNA, RNA and THHE dsDNA iA-k) ss DNA ( S - ^ ) RNA ( A ^ ) THHE ( © ^ ) NHS {O-O}
135
5 lO 15
NHIBITOR CONCENTRATION,jug/ml
Fig. 41 Inhibition ELISA of SLE autoantibodies on plates coated with native DNA Competi tors: native DNA(O-O) ss DNA (A- t^)
RNA ( § . # )
136
100 5 10 15 C INHIBITORS,;jg/ml
20
Fig. 42 Inhibition ELISA of SLE autoantibodies on plates coated with ds DNA Competitor : THHE ( • - • )
137
100 5 10 15 CiNHlBlTORa,Aig/ml
20
Fig. 43 Inhibition ELISA of protein A sepharose isolated SLE IgG on plates coated with ds DNA Competitor : THHE ( • - • )
138
5 10 15
CINHIBIT0R3,>ig/ml
Fig. 44 Inhibition ELISA of i Competitor : C protein
immunoaffinity purified SLE IgG
139
revealed inhibition of 76% and fifty percent inhibition was achieved at 1.5
^g/ml (Fig. 44).
Probing mitochondrial DNA damage in DCM patients:
The sera from DCM patients showed preferentially higher binding with
native mitochondrial DNA (mt DNA) as compared to its ROS modified
conformer (Fig. 45). The specifity was ascertained by inhibition ELISA. The
six DCM sera tested showed inhibition in the antibody activity, ranging from
64.5 percent to 73.1 percent in case of native mt DNA (Fig. 46) as compared
to inhibition varying from 54.6 percent to 63.8 percent in case of ROS mt
DNA (Fig. 47) at maximum inhibitor concentration of 20 pig/ml. The range of
inhibitor concentration for fifty percent inhibito varied from 0.15 to 4.0
jxg/ml in native mt DNA. In ROS mt DNA, fifty percent inhibition was
achieved in the range of 2.0 jig/ml to 10.0 [ig/ml in the six tested DCM
sera. The results have been summarized in Table 9.
Glucose, cholesterol, uric acid estimation and evaluation of CRP and RF
content in patients with dilated cardiomyopathy (DCM):
Elevated levels of glucose that is 146.1, 115.3 and 115 mg/dl were
found in 3 of 15 DCM patients under taken in this study (Fig. 48). Amongst
them only one showed higher level of serum cholesterol that is 248 mg/dl
(Fig. 49). An increase in uric acid content in serum was found in 7 of the 9
female patients, in the range of 4.5 to 24.9 mg/dl. The males showed a varia
tion from 4.3 to 11.2 mg/dl (Fig. 50). The C reactive protein (CRP) latex
agglutination test revealed positive agglutination (>6mg/dl) in 9 out of 15
140
DIRECT BINDING ELISA OF DCM AUTO ANTIBODIES WITH NATIVE AND ROS mt DNA
3 4 6 6 Number of serum samples
[HID Native mt DNA ROS mt DNA
Fig. 46
141
0.01 0.1 1.0 10.0
INHIBITOR CONCENTRATION ( / j g / m l )
0.01 0.1 1.0 1 0 0
INHIBITOR CONCENTRATION {^glmi)
Fig. 46 Inhibition ELISA of DCM auto antibodies with native mt DNA as inhibitor
Competitor: native mt DNA (O-O ) Serum 4 ( o - o ) ( o - O ) Serum 5 ( O-O) ( ©-€)) Serum 6 ( • - • )
Antigen : native mt DNA Antibody : Serum 1
Serum 2 Serum 3
142
0.01 0.1 1,0 10.0 INHIBITOR CONCENTRATION ( ; j g / m l )
O.Ol 0.1 1.0 10.0
INHIBITOR CONCENTRATION C<ug/ml)
Fig. 47 Inhibition ELISA of DCM auto antibodies with ROS mt DNA as inhibitor. Antigen : ROS mt DNA Antibody : Serum 1
Serum 2 Serum 3
Competitor : (a—O) Serum 4 {9—0) Serum 5 (•—•) Serum 6
ROS mt DNA
{o-o) (•-») ( — )
143
TABLE 8
COMPETITION ELISA OF MITOCHONDRIAL DNA (mtDNA) WITH
DCM AUTOANTIBODIES
DCM Sera
50% In {\lg/m\)
1 1 6
2 1 1
3 1 0
4 0 15
5 4 0
6 2 4
Antigen-Inhibitoi
hibition
mt DNA r-mt DNA
Maximum Inhibition (at 20 ng/ml)%
67 6
64 5
73 1
64 9
65 6
65 2
50%
Antigen -In
In (^ig/ml)
3 0
2 4
2 2
2 0
100
5 0
ihibitor
hibition
ROS mt DNA - ROS mt DNA
Maximum Inhibition (at 20 p,g/ml)%
63 8
61 0
63 2
58 2
54 6
56 8
144
GLUCOSE LEVELS IN SERA OF PATIENTS WITH DILATED CARDIOMYOPATHY
160 Y[
E
tn O O :s 1—1
F i g . 48
4 6 6 7 8 9 10 11 12 13 14 16 Number of serum samples
Normal range-. 70-100 mg/dl
145
TOTAL SERUM CHOLESTEROL LEVELS IN PATIENTS WITH DILATED CARDIOMYOPATHY
S
c <u •p c o o
o
« r-l o x: u
1 2 3 4 6 6 7 8 9 10 11 12 13 14 16 Number of serum samples
F i g . 49 Normal range: 130.0-220.0 mg/dl
146
LEVELS OF URIC ACID IN SERA OF PATIENTS WITH DILATED CARDIOMYOPATHY
s
T3 •H
u
u
e D
0)
Fig . 50
5 6 7 8 9 10 11 12 13 14 Number of serum samples
Normal range :-Males: 3.4 - 7.0mg/dl
Females: 2.4 - 6.7 mg / dl
147
C - REACTIVE PROTEIN (CRP) LEVELS IN SERA OF PATIENTS WITH DILATED CARDIO
MYOPATHY
•p c Q) ^J C O
o a,
200
160
100
60
T 1 r
6 6 7 8 9 10 11 12 13 14 16 Number of serum samples
F i g . 51 Normal CRP content < 6mg/L
148
patients in qualitative estimation. Semiquantitative analysis of the positive
samples showed variations in CRP contents from 12 mg/dl in 4 sera, 24 mg/
1 in 2 sera to 48 , 96 and 192 mg/1 in one serum each of DCM patients
(Fig. 51). Qualitative analysis of Rheumatoid factor (RF) by RF latex agglu
tination test did not show any agglutination in any of the tested DCM sera.
B xscuBsion
149
A utoimmune diseases are characterized by the presence of circu
lating autoantibodies which are not necessarily pathogenic but represent
markers of immune mediated injury (Rosa, R and Bona, C, 1991). Autoim
mune diseases result due to humoral and / or cellular immunological reac
tions against the self components of an individual. The triggering mecha
nisms of autoimmune reaction have not yet been completely understood.
Shoenfeld and Isenberg (1989) described the Avide spectrum of autoimmune
diseases as mosaic of autoimmunity with many factors leading to various
diseases.
Dilated cardiomyopathy (DCM) is a chronic heart muscle disease of
unknown etiology characterized by dilatation and contractile dysfunction
of left and / or the right ventricle. In man, myocarditis is known to be the
precursor of DCM in many cases, although clinical and histological diag
nosis remain problematic. Since in DCM, the only affected organ is heart, it
fits into the criteria of organ specific autoimmune disease. Autoimmune
features in patients with DCM include familial aggregation (Keeling et al. ,
1995), a weak association with HLA DR4 (Carlq uist et al., 1991) and im
munoglobulin genes ( Martinetti et al., 1992), abnormal expression of HLA
class II on cardiac endothelium (Caforio et al., 1990), and increased levels
of circulating cytokines (Limas et al., 1995).
Several groups have found cardiac antibodies to various antigens in
DCM (Caforio et al . , 1992; Schultheiss et al., 1990; Limas et al., 1995).
Multiplicity of parallel autoimmune reactions directed towards a single or
gan may be due to spread sensitization (Bach, 1995). Among the various
150
cardiac antigens, 150 kD C protein has been found to be a potent antigen
causing autoimmune myocarditis in experimental animals. C protein plays
an important role in contractile activity. It is one of the major constituent
proteins which are essential to compete with natural antigens at MHC - an
tigen bindings site (Demotz et al., 1990; Harding and Unanue, 1990). More
over C protein is an intracellular member of immunoglubin superfamily
(Einheber and Fischman, 1990). Due to identical molecular weights IgG,
and C protein gave a similar pattern on 7.5% SDS - PAGE. Urea denatur-
ation studies also showed more or less similar patterns in Urea - SDS -
PAGE. When SDS -PAGE was performed with a urea gradient (1-10 M urea)
as well as acrylamide gradient (5-20%), both IgG and C protein were dena
tured to give two prominent bands each. This further gives an insight into
this theory. The HLA binding motifs are located in the variable domain and
not the constant IgG like domain (Rudensky et al., 1992; Baum et al., 1993).
The thermal behaviour of 150 kD C - protein and human IgG as con
trol displaying Tm values of unfolding at around 79.5°C is typical of a highly
stable dimeric protein molecule. The highly stable conformational state of
150 kD C - protein was evident from the observation that only 4.4% had
undergone thermal unfolding till 75°C. Thus our observation reveals for
the tremendous structural stability of the 150 kD C - protein auto antigen.
Additional evidence for the structural changes in native 150 kD C-
protein as a result of thermal energy supplementation was revealed by ana
lyzing the data and computation of thermodynamic parameters (Table 1 &
2). The thermodynamical data speculate the tremendous structural stability
151
exhibited by 150 kD C - protein. Disruption of the hydrophobic and ionic
interaction as well as disulfide bonds in the 150 kD C - protein which
ultimately resulted in unfolding was inferred from the shift in the scales of
the AGJJ values. The negative AG^ values above 79°C suggests the transi
tions of completely folded 150kD C - protein to the unfolded state. Further
more, in comparison to the already established thermodynamic behaviour of
native DNA, our results are indicative for the 150 kD C - protein to be topo-
logically less constrained than nucleic acids. This comparative analysis has
been pointed out due to the important factor of DNA acting as auto antigen
in SLE whereas 150 kD - C-protein acting as autoantigen in DCM /
myocarditis and that as per our observations, the 150 kD C - protein which
acts as autoantigen in myocarditis was found to be equally reactive with
naturally occuring SLE autoantibodies.
Photochemical reaction have been found to produce DNA - protein
crosslinks in biological systems. Proteins and nucleic acids are colourless
macromolecules which absorb in the UV range of spectrum. These two
biomolecules coexist in vivo and their interaction results in biological dam
age in organisms. The crosslinking between DNA and protein has been found
to be covalent in nature. (Oleinick et al., 1986). In this study , we have
formed photoadducts between native DNA and C - protein in UV light (254
nm). Time course kinetics revealed that the formation of photoadduct in
creased with increase in duration of exposure to UV irradiation. This was
further reiterated by the amount of total protein bound to DNA. The protein
content increased remarkably with the increase in period of exposure. The
amount of lysine residues present in C-protein were estimated to assess their
152
binding to native DNA. Time course kinetic study indicates that the forma
tion of photoadduct obeys apparent first order reaction.
Oxygen free radicals (OFR's) occupy a prime position in the world of
free radical biochemistry . The prima donna in this category are oxygen
itself, superoxide, hydrogen peroxide, transition metal ions and hydroxyl
radicals, where the first four interact in many ways to produce the last
(Halliwell and Gutteridge, 1990). Exogenous sources like toxic foreign com
pounds and ionizing radiations are known to increase the production of free
radicals. There are several sites for generation of reactive oxygen species
(ROS) in cells, but the main source is the autooxidation of reduced compo
nents of mitochondrial transport chain (Haiku et al., 1993).
Reactive oxygen species produce DNA - protein crosslinks in biologi
cal systems. Here we have taken two entities, hydrogen peroxide (H^
O^) and hydroxyl radical (OH) to produce crosslinks and photoadduct for
mation between native DNA and C - protein. Among the two, hydroxyl radi
cal (OH) appeared to be a better agent to induce DNA - C- protein crosslinks.
The formation of photoadduct in the above cases causes single strand breaks
in DNA as revealed by nuclease SI sensitivity assay. On the contrary, the
control and treated samples of nDNA C-protein photoadduct showed the
same pattern. It signifies that in the photoadduct, interaction may have taken
place via lysine residues. The other aminoacids in the protein can also un
dergo photointeraction to form protein-protein photoadduct. Thus, crosslinks
are again formed between nDNA C-protein and C-protein C-prote in
photoadduct which is responsible for such a pattern.
153
The recognition between nucleic acid and proteins is of great signifi
cance as it triggers most important steps of nucleic acid metabolism. This
recognition involves electrostatic interactions between phosphate groups and
positively charged groups in protein. Later more specific interactions be
tween nucleic acid bases (or other structural elements) and amino acid side
chains in proteins take place. A closer look reveals the possible recognition
of secondary structure of nucleic acids by proteins, either way recognition
is dependant of nucleotide sequence (Duguet, 1981).
A different chemistry arises when protein and DNA are irradiated to
gether or separately. Since DNA and protein are in intimate contact with
each other in vivo, it is suggested that photochemical interaction of DNA
and protein would play a significant role in the inactivation of UV irradi
ated cells under certain conditions.
The photoadducts formed showed appreciable reco. gnition with DCM,
SLE and immune sera. The reactivity of DCM and immune sera was maxi
mum with photoadduct formed by hydroxyl radical (OH) followed by the
one formed only in the presence of UVB irradiation showed least reactivity
as shown by band shift assay and completion ELISA studies. This indicates
the effectiveness of hydroxyl radical(-OH) to generate DNA protein crosslinks
which produce pathological changes.
The sera of patients with dilated cardiomyopathy (DCM) showed a
typical picture in the biochemical investigations . Quantitative determina
tion of serum cholesterol gave normal values except for one patient. The
determination of serum cholesterol is considered to be significant mostly
154
in coronary artery diseases, hyperlipoproteinemas, hypothyroidism,
nephrosis, diabetes mellitus, and various liver diseases, and has very little
role to play in dilated cardiomyopathy (DCM). Similarly patients with DCM
rarely show an increase of serum glucose. Only two of the total fifteen cases
showed a moderate increase in glucose levels. The moderate rise is associ
ated with infectious diseases, intracranial diseases and haemorrhage. There
was a significant increase in uric acid levels in serum. Serum or plasma
uric acid determination has an important diagnostic values in gout and kid
ney failure. It is also increased in acute stages of infectious diseases, se
vere uremia, toxemia of pregnancy and leukemia. DCM is a end stage car
diac disorder in which there is active degeneration of cardiac tissue leading
to heart failure. This chronic inflammatory process may be responsible for
increase in uric acid levels in DCM patients.
A significant rise was observed in the C - reactive protein (CRP) lev
els in sera of DCM patients. A majority of patients gave a high titre on semi
- quantitative determination of CRP in serum. CRP an acute phase reactant
is considered to be the most sensitive marker in the acute phase and there
fore, a universal early indicator for inflammatory, necrotic and neoplastic
diseases. CRP is a non - glycosylated cyclic pentamer which increases in
concentration in a fixed and phased sequence during inflammation and / or
active tissue infections. It is important in protection against development
of autoimmunity. It acts by clearing cell debris autoantigens recognised by
auto antibodies. The rise in CRP levels in DCM patients can be attributed
to this factor. Rheumatoid factor (RF) was absent in all the sera of DCM
155
patients. RF are credible markers in inflammatory diseases of the joints .
Hence their absence rules out the possibility of any such factor in DCM.
Autoantibodies found in patients with dilated cardiomyopathy (DCM)
exhibited remarkable binding with the HPLC purified 150kD human C-pro-
tein as is evident from competition inhibition ELISA results. Nearly all the
sera of patients with DCM showed inhibition in the antibody activity by
150 kD C-protein in the range of 64 .7% to 82.1%. Furthermore, an appre
ciable number of DCM sera exhibited inhibition in their antibody activity
by a very low magnitude of inhibitor concentration, which in turn, is fur
ther substantiative/or indicative for the remarkably high specificity of the
DCM autoantibodies towards 150kD C-protein. However, the binding to
wards 150kD C-protein was of higher magnitude with the induced anti 150
kD C-protein antibodies in comparison to the binding with DCM autoanti
bodies. The binding results are suggestive for the major involvement of
150kD C-protein as autoantigen in autoimmune DCM/myocarditis.
It is to be pointed out that native DNA was found to be of low reactiv
ity with all the sera of patients with DCM. But interestingly, mitochondrial
DNA (mt DNA) on the other hand, exhibited appreciable binding with DCM
autoantibodies, although the magnitude of binding was lower than with
150kD C-protein. Moreover, surprisingly, modification of mt DNA with ROS
showed decrease in binding with DCM autoantibodies, whereas conversely,
nDNA which was unreactive towards DCM autoantibodies, showed a bind
ing of low magnitude when modified with ROS. The results are suggestive
for the probable presence of conformational epitopes on mtDNA and to a
156
lower extent on ROS DNA which are somewhat similar to the ones present
on 150 kD C-protein.
The highly immunogenic nature of native 150kD C-protein was evi
dent from the competition results, where around 98 percent of the antibody
activity was inhibited by the immunogen. However, when native 150kD C-
protein was ROS modified, the antibody binding was lost to the extent of
more than fifty percent. The results suggest for the damage or loss of sig
nificant number of immunodominant epitopes on 150kD C-protein upon ROS
modification.
Although the induced anti-150kD C-protein antibodies were unreactive
with native DNA, a low magnitude of binding was observed when nDNA
was photoadducted with C-protein, whereas, surprisingly, ROS-DNA-C-Pro-
tein photoadduct exhibited a high degree of binding which was even higher
than the binding observed with the immunogen i.e. 150kD C-protein. This
could be attributed to the cumulative binding effect of immunogenic epitopes
on C-protein as well as somewhat similar epitopes to the immunogen on the
ROS modified DNA and hence when both ROS-DNA and C-protein were
photoadducted, a higher binding was observed. The results suggest that prob
ably apart from 150kD C-protein, the photoadducted ROS-DNA C-protein
complex could also act as an alternate autoantigen for myocarditis/DCM.
The above possibility could not be ruled out because of the fact that reac
tive oxygen species (ROS) are formed in vivo which modifies the nucleic
acid macromolecule and that the possibililty of photoconjugation of ROS-
DNA with C-protein exists. Thus, our findings could possibly throw some
light on the pathogenesis and etiology of autoimmune myocarditis/DCM.
157
The binding of induced anti-C-protein antibodies with histones, ROS-
histone, poly-D-lysine, poly-L-glutamate and poly (lysine-glutamate) com
plex was of low magnitudes thereby suggesting for the presence of a minor
sub-population of epitopes responding to the anti-C-protein antibodies. Simi
lar binding results were also observed with DCM autoantibodies against C-
protein, ROS C-protein ROS-DNA, ROS-DNA photoadducted to C-protein,
ROS-histone, histones, poly-D-lysine, poly-D-glutamate and poly (L+G)
complex respectively. The similarity in binding results further substanti
ates the above argument.
Apart from the above the systematic probe to see the correlation be
tween myocarditis autoantigen (C-protein) and autoimmune SLE autoanti
bodies gave encouraging results.
Systemic lupus erythematosus (SLE) is a multisystem prototype au
toimmune disease (StoUar, 1981) characterized by circulating autoantibod
ies that bind to cellular antigens of self and exogenous origin. Although the
correlation of SLE with immune system is well documented, neither the
origin of these autoantibodies nor the etiology of the disease is known. Re
cent studies point out that native DNA might actually be a cross reactive
antigen whereas some other structures like modified nucleic acids (Alan et
al., 1993; Ara and AH; 1993; Ara and Ali, 1992), polypeptides (Arif et al.,
1994) and phospholipids (Smeenk et al., 1987), etc. stimulate the antibody
response. Moreover, the polyspecificity of autoantibodies with a variety of
heteroantigens might be the result of immune response to substance (s) other
than native DNA and interplay of some environmental factors.
158
In the present study attempts were also made -to probe the possible
involvement of 150 kD C - protein isolated from human heart which is known
as potential autoantigen in myocarditis and DCM in humans , in autoimmune
SLE. The protein is known to have similar domain as to those of IgG and
has been implicated in the induction of autoantibodies in patients with SLE.
The 150 kD C- protein, which is a component of thick filament of skeletal
and cardiac muscles, plays an important role in the contractile activity.
Assays of anti - DNA and anti - human heart antigens in the sera of patients
with SLE and DCM respectively were carried out by employing direct
binding ELISA whereas specificity determination was accomplished by
competition ELISA. The antigen binding characteris t ics of anti - DNA
autoantibodies towards native and modified nucleic acids were in accordance
with earlier findings (Losman et al., 1993).
The most striking finding of the present study is the appreciable
recognition of THHE as well as 150 kD C - protein (human heart) by SLE
autoantibodies. As revealed by competition inhibit ion studies, the SLE
autoantibodies expressing specificity towards dsDNA, ssDNA, DNA-lysine
photoadduct and Br - DNA also showed almost similar recognition of human
heart proteinic antigen (s) . Interestingly, as evident from the binding results
the human heart extract antigens reactive with the DCM autoantibodies
showed strong recognition of SLE anti-DNA autoantibodies whereas on the
contrary DNA was found to be less reactive with DCM autoantibodies. This
is suggestive for the possible involvement of a common or somewhat similar
trigger/epitope (s) for immune response in autoimmune myocarditis/DCM
and autoimmune SLE, although such inferences could not lead to confirmity
at such a preliminary stage of investigation. Moreover, the high binding
159
Specificity of immunoaffinity purified anti - DNA antibodies to 150 kD C -
protein from human heart is strongly suggestive for the presence of
immunodominant epitopes on 150 kD C- protein for SLE anti - DNA anti
body binding. The binding results are indicative for the presence of unique
conformational epitopes on the 150 kD C - protein whose domains are some
what similar to IgG. It is to be pointed out that recently nucleosome spe
cific antibodies have been indicated in lupus prone mice. The monoclonal
antibody was found to react with nucleosome core particle but not histone-
histone and histone-DNA complexes. The core particles having ordered as
sociation of histones and DNA were found to express a unique conforma-
tional autotpitope (s). Only the pathogenic T - helper cells of lupus prone
mice responded to nucelosomal antigens and their stimulation was specific
(Chandra et al., 1993). Therefore, it appears that pathogenic anti - DNA
autoantibodies are generated through some conformational epitope (s) of
nucleic acids or proteins / polypeptides.
Thus, in view of the above argument, it appears from our data that
150 kD C-protein having domains similar to those of IgG and being involved
in the induction of autoimmune myocarditis may provide some unique con
formational epitopes which could possibly act as an alternate autoantigen
or trigger in the production of anti-DNA autoantibodies in SLE. Our pre
liminary investigation is supportive for the above argument. The mecha
nism or the role of myocarditis inducing 150 kD human heart C-protein in
the stimulation of immune system for the production of antibodies in SLE
remains to be ascertained.
160
In conclusion, it would be inferred from this study.that:
(1) 150kD C-protein is having somewhat similar domains to human IgG
as indicated by urea SDS-PAGE.
(2) 150kD C-protein was thermodynamically more stable than human IgG
although both of them had somewhat similar domains.
(3) The C-protein is a potent immunogen inducing hightiter polyclonal
antibodies with major reactivity towards HPLC purified immunogen.
(4) Nearly all the DCM sera tested exhibited elevated C-reactive protein
(CRP) and uric acid levels and that all of them showed remarkable
recognition towards electroeluted.followed by HPLC purified 150kD
C-protein.
(5) The kinetics of formation of DNA-C-protein photoadduct at various
irradiation time obeyed the apparent first order reaction.
(6) Out of the various adducts/photoadducts formed by crosslinking na
tive DNA with C-protein, the one formed in the presence of'OH (ROS
-DNA-C-protein photoadduct) showed highest reactivity with DCM,
SLE and immune IgG.
(7) Nearly all the DCM sera tested, showed preferentially higher binding
with native mitochondrial DNA than its ROS modified conformer.
(8) The most striking finding is the appreciable recognition of THHE as
well as 150 kD cardiac C-protein by immunoaffinity purified anti-DNA
SLE autoantibodies.
'^titxtntts
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