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Investigative Ophthalmolog y & V isual Science, Vol. 30, No . 12, Decem ber 1989
Copyright © Association for Research in Vision and Ophthalmology
Protein-Related Abnormalities in Keratoconus
Noorjahan Panjwani,*j- Jim Drysdale,t Brian Clark,* John Alberta,f and Jules Baum*}:
Two-dimensional electrophoretic maps of extracts from eleven normal and eleven keratoconus corneas
were compared. Of the eleven corneas analyzed, eight were pooled and the remaining three were
analyzed individually. Several differences were demonstrated between electrophoretic patterns of
normal and keratoconus corneas. In keratoconus corneas, 1) two abnormal compon ents M W 54 kD
and 26kD) were observed; 2) three normal corneal components M W 12kD , 14k D, and 39k D) were
present in significantly h igher amounts; and 3) three normal corneal proteins M W 6 6k D , 55k D, and
13kD) were present in reduced amounts. The molecular weight and isoelectric point of one of the
normal corneal proteins that we found to be reduced in keratoconus corneas were close to that of a
subunit of prolyl-4-hydroxylase, an enzyme required for hydroxylation of proline residues of collagen.
The possibility the abnormal proteins detected in the keratoconus corneas were derived from those
normal corneal proteins which were absent or were present in reduced amounts in the keratoconus
corneas remains to be established. This study may provide protein markers for elucidation of the
biochemical abnormality in keratoconus. Invest Ophthalmol Vis Sci 30:2481-2487,1989
Keratoc onus is characterized by thinnin g and scar-
ring of the central cornea. It is slowly progressive and
either bi- or unilateral. Although the clinical and ul-
trastructural characteristics of the disease are well es-
tablished,
1
3
the biochemical defect in keratoconus
has not been elucidated. H istochemical as well as bio-
chemical approaches have not detected significant
differences in collagen types between normal and
keratoconus corneas.
4
6
It has been shown that media
incubated with keratoconus corneas, as well as stro-
mal cells in culture derived from keratoconus cor-
neas,
contain higher levels of collagenolytic activity
compared to normal controls,
7
9
a result which sug-
gests a decrease in corneal collagen content in kera-
toconus. Attempts to demonstrate reduced collagen
levels in keratoconus corneas, however, have pro-
vided conflicting results.
5
'
7
'
9
13
Cannon and Foster
14
reported abnormally high levels of lysinorleucine
crosslinks of collagen in keratoconus; these findings,
however, could not be confirmed.
15
Biochemical ap-
proaches to detect differences related to proteogly-
cans and glycosaminoglycans have also provided
variable data. Hassell et al
16
reported that the pattern
From the Departments of Ophthalmology and fBiochemistry,
Tufts University School of Medicine, and ^New England Medical
Center, B oston, Massachusetts.
Supported by the National Institutes of Health Grants
EY-07088, EY-04034, and DDK-32455.
Submitted for publication: August 1, 1988; accepted May 12,
1989.
Reprint requests: Noorjahan Panjwani, PhD, Department of
Ophthalmology, Tufts University School of M edicine, 136 Harri-
son Avenue, Box 450, Boston, MA 02111.
of proteoglycans synthesized by keratoconus corneas
in organ culture was indistinguishable from that syn-
thesized by norm al corn eas. In an earlier study, it was
reported that the hexosamine concentration in kera-
toconus corneas was about 40% greater than nor-
mal.
10
Anseth,
17
however, found that the concentra-
tion of hexosamine was identical in keratoconus and
normal corneas. Andreassen et al
12
found no differ-
ences in uronic acid concentration in normal and
keratoconus corneas; in contrast, in a recent histo-
chemical study, an abnormally high amount of poly-
anions that include primarily glycosaminoglycans
was found in keratoconus corneas when compared to
normal controls.
18
In another study, using a mono-
clonal antibody specific for keratan sulfate, it was
shown that compared to normal controls, some kera-
toconus corneas contained less keratan sulfate.
19
Thus,
in spite of a num ber of well-designed studies,
the exact biochemical abnormality responsible for
the development of keratoconus has yet to be eluci-
dated. A major obstacle in the study of keratoconus
has been the unavailability of specific biochemical
markers with which investigations can be designed.
In various nonocular studies, comparison of two-di-
mensional gel electrophoretic patterns of normal and
diseased tissue have led to the identification of the
disease-related proteins.
20
'
21
Two-dimensional elec-
trophoresis is a powerful research tool pe rmitting res-
olution of up to 1,000 proteins from a single human
cell type.
22
To our knowledge, two-dimensional elec-
trophoretic patterns of the complete extracts of nor-
mal or keratoconus corneas have not been compared
to date. The purpose of this study is to compare two-
2481
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INVESTIGATIVE OPHT HALM OLOG Y VISUAL SCIENCE / Dec em be r 19 89
Vol. 30
dimensional maps of extracts of normal and kerato-
conus corneas and thereby identify protein markers
that may be specific to the disease.
Materials and Methods
Tissue
Human keratoconus buttons were obtained at the
time of keratoplasty. With the exception of the kera-
toconus, the patients undergoing keratoplasty were
clinically normal; there was no evidence of general-
ized connective tissue disorders. The diseased corneas
were rinsed three times with saline and kept frozen at
-80°C or in liquid N
2
until used. Normal central
human corneas were excised with an 8-mm trephine
from frozen eyes supplied by the Lions Eye Bank
(Seattle, WA) an d were rinsed three times with saline
prior to use. For the normal eyes, the average time
period between death and enucleation was approxi-
mately 7 hr, and between enucleation and freezing,
approximately 6 hr. Eleven normal corneas and
eleven keratoconus buttons were used. Within each
group, eight corneas were pooled and the remaining
three were investigated individually. Ages of the nor-
mal corneal donors and of the patients with kerato-
conus are shown in Table 1. Information concerning
the extent of scarring in keratoconus buttons is also
shown in Table 1.
Extraction of Proteins from Normal and
Keratoconus Corneas
The epithelium was scraped off the corneas while
they were still frozen. Th e corneas were then minced
into six to eight pieces with a scalpel, on dry ice. The
minced tissue was thawed directly in ice-cold 4 M
guanidine hydrochloride (1-1.5 ml/cornea), and ex-
traction was carried out for 16-18 hr on a wrist action
shaker in the presence of a solution of protease inhibi-
tors,
composed of raM phenylmethylsulfonylfluor-
ide,
1 mM benzamidine hydrochloride, 100 mM 6-
aminohexanoic acid, and 10 mM N-ethylmaleimide.
The extract was collected by decantation, and the
residue was reextracted as described above for an ad-
ditional 6-8 hr. Both supernatants were pooled, dia-
lyzed extensively against cold water, and lyophilized.
The lyophilized material was suspended in cold dis-
tilled water and aliquots were used for protein deter-
mination and two-dimensional gel electrophoresis.
Protein Determination
The protein concentration in the corneal extracts
was determined by the bicinchoninic acid (BCA)
method
23
(Pierce Chemical, Rockford, IL) using bo-
vine serum albumin as a standard. Protein yield in
the extract ranged from 400-650 /Ltg/comea.
Two Dimensional Gel Electrophoresis
Two-dimensional electrophoresis was performed
according to the method of O'Farrell
22
by Kendrick
Laboratories (Madison, WI) as follows. Isoelectric fo-
cusing using ampholines at 1.5%, pH 5-7; 1.5%, pH
5-8; and 1.0%, pH 3.5-10 (LKB Instruments, Balti-
more, MD) was carried out in 135-mm X 2-mm
tubes at 400 V for 12 hr followed by 800 V for 30
min. Forty ng of an isoelectric focusing internal stan-
dard, vitamin D-dependent calcium-binding protein,
MW 27,000 and pi 5.2, was added to the samples.
The final tube gel pH gradient was estimated by a
surface pH electrode (Biorad, Richmond, CA) and by
colored acetylated cytochrome pi markers (Calbio-
chem-Behring, La Jolla, CA) run in an adjacent tube.
Electrophoresis in the second dimension was per-
formed in 135-mm X 150-mm X 0.75-mm slab gels
containing 10% acrylamide. The following proteins
(Sigma Chemical, St. Louis, MO) were added as mo-
lecular weight standards to the agarose that sealed the
tube gels to the slab gels: myosin heavy chain (220
kD),
phosphorylase A (94 kD), catalase (60 kD), actin
(43 kD) and lysozyme (14 kD). Protein spots were
visualized by silver staining according to the proce-
dure of Oakley et al.
24
Table 1. Ages of normal corneal donors and of patients with keratoconus at the time of keratoplasty
Group
number
1
2
3
4
5
6
7
8
Cornea
normal
no rma l
normal
no rma l
keratoconus
keratoconus
keratoconus
keratoconus
Number of
corneas
8
ii
Age (yr)
28-57 (mean: 41)
47
47
47
26-41 (mean: 33)
47
52
40
Degree of
scarring
_
-
-
-
- t o + +
+
+ + +
+
of button
scarred
_
-
-
-
0-10
<5
15
<5
Scarring: — none, + mimimal, ++ moderate, +++ severe.
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No .
12
PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Pa nj w on i er a l
2 4 8 3
Two-dimensional electrophoretic maps of normal
corneas were compared with each other and with
those of keratoco nus corneas by overlapping the gels
on a light box. The spots which were present only in
the diseased corneas, or which were present in the
diseased corneas in higher concentrations, were desig-
Fig. 1. Two-dimensional
electrophorograms of ex-
tracts prepared from eight
pooled normal (N) and
eight pooled keratoconus
(K) corneas. Ninety micro-
grams of protein was ap-
plied on each isoelectric
focusing (IEF) gel, and
polypeptide spots were vi-
sualized by silver staining.
The basic side is on the
right, the acidic on the left;
the high molecular weight
region is at the top. The
final tube gel pH gradient
was estimated by a surface
pH electrode and by colored
acetylated cytochrome pi
markers run in an adjacent
tube. Electrophoresis (SDS)
in the second dimension
was performed in 10% ac-
rylamide slab gels. Myosin
heavy chain (220 kD),
phosphorylase A (94 kD),
catalase (60 kD), actin (43
kD) and lysozyme (14 kD)
were added as molecular
weight standards to the aga-
rose which sealed the tube
gels to the slab gels. These
standards appear as fine
horizontal lines. In addi-
tion, an IEF internal stan-
dard, vitamin D-dependent
calcium binding protein,
MW 27 kD and pi 5.2, was
added to the samples. This
standard is indicated by an
arrowhead in each gel. The
spots which were present
only in diseased corneas, or
which were present in the
diseased corneas in higher
concentrations, were desig-
nated as K spots; the spots
which were present in lower
intensity in the diseased
corneas as compared to
normal corneas were desig-
nated as N spots. Arrows
point to various N and K
spots or to their approxi-
mate positions when the
spots were absent in either
gel.
1 4 -
8 0
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INVESTIGATIVE OPHTHALM OLOG Y G VISUAL SCIENCE / De ce m be r 19 89
Vol. 30
CO
LU
O
<
GO
<
CD
2 3
NORMAL
5 6 7 8
KERATOCONUS
Fig.
2. Comparison of intensities of K spots (see Fig. 1) in all
normal and diseased corneas analyzed in this study. Electropho-
retic patterns no. 1 and no. 5 were obtained from extracts of eight
pooled normal and eight pooled diseased corneas, respectively; the
nated as K spots. The spots which were absent, or
which were present in lower intensity, in the diseased
corneas as compared to normal corneas were desig-
nated as N spots. Approximate intensities of the N
and K spots were measured using an Ultroscan XL
laser densitometer (LKB). The polypeptide densities
in the respective gels were normalized against two
reference proteins which had constant density ratios
to each other in all gels. The densities of these two
proteins in each gel were used to create a normaliza-
tion factor for each gel compared to an arbitrarily
selected standard gel.
Results
Comparison of two-dimensional maps of normal
and k eratoconus corneas revealed that
1)
two compo-
nents,
Kl and K5, were detected in keratoconus cor-
neas, but not in normal corneas (Figs. 1, 2); 2) three
components, K2-K4, were present in significantly
higher amounts in keratoconus corneas compared to
those in normal corneas (Figs. 1, 2); and 3) three
normal corneal proteins, N1-N3, were present in sig-
nificantly lower amounts in keratoconus corneas
(Figs. 1, 3). Molecular weights and isoelectric points
of
all
N and K proteins are shown in Table 2. Inten-
sities of various K and N spots are shown in Figures 2
and 3, respectively. As shown in Figure 1, N2 con-
sisted of several spots. For density measurement of
N2,
the area representing several spots was analyzed
as a whole, and the intensity of the various spots
within the N2 area was not measured individually.
The density of component N3 was not measured be-
cause in a number of gels this component was not
resolved from lysozyme, which was used as an inter-
nal standard
Fig. 1).
Difference in the intensity of N3
between normal and keratoconus corneas was more
prominent in the two-dimensional maps of individ-
ual corneas (not shown) than in those of the pooled
corneas (Fig. 1). The broad band migrating beneath
the K3 position in the two-dimensional patterns of
normal corneas comigrated with K3 in the two-di-
mensional patterns of pooled as well as two of the
three individual keratoconus corneas.
A mixed extract of keratoconus and normal cor-
neas was also analyzed to determ ine whether those N
and K components w hich were found to be present in
both normal and keratoconus comigrate. As shown
in Figure 4, components N1-N3 and K2-K4, which
remaining electrophoretic patterns were obtained from individual
corneas. Ninety m icrograms of protein was loaded on each gel, and
polypeptide densities in the respective gels were normalized against
two reference proteins as described in the text.
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No. 12
PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Po nj w on i er ol
2 4 8 5
CO
LU
O
z
<
m
O
O
3
2
1
NORMAL
KERATOCONUS
Fig. 3. Comparison of intensities of N spots (see Fig. 1) in all
normal and diseased corneas analyzed in this study. Electropho-
retic patterns no. and no. 5 were obtained from extracts of eight
pooled normal and eight pooled diseased corneas, respectively; the
remaining electrophoretic patterns were obtained from individual
corneas. Ninety micrograms of protein was loaded on each gel, and
polypeptide densities in the respective gels were normalized against
two reference proteins as described in the text.
were present in both keratoconus and normal cor-
neas, though in varying amounts, comigrate.
Among all the keratoconus corneas we studied, the
data derived from case no . 6 appeared to deviate sig-
nificantly from those of the rest of the diseased cor-
neas.
As shown in Figure 4, in the case-6 cornea,
components Kl, K2, and K5 were present only in
trace amounts. In contrast, these components were
present in substantially greater amounts in all other
keratoconus corneas studied. The cornea of case 6
was also dissimilar from the other keratoconus cor-
neas studied in that it contained a higher amount of
component
N1,
compared to all other diseased speci-
mens (Fig. 3).
Discussion
We have demonstrated that in keratoconus corneas
several abnormal proteins are present and that the
amounts of several normal corneal proteins are re-
duced. Specifically, our data indicate that in kerato-
conus corneas, two abnormal components (Kl and
K5) are present and three normal corneal compo-
nents (K2-K4) are present in significantly higher
amounts. In addition, three normal corneal proteins
(Nl ,
N2, and N3) are present in reduced amounts in
the diseased co rneas.
The molecular weight and isoelectric point of the
normal corneal component Nl, detected only in
trace amounts in keratoconus corneas, is close to tha t
of a subunit of prolyl-4-hydroxylase.
25
It remains to
be determined whether component N is a subunit of
prolyl hydroxylase, but one study has suggested an
elevated level of prolyl-4-hydroxylase activity in ker-
atoconus corneas.
9
Repor ts in the literature regarding
hydroxyproline concentration in keratoconus con-
flict; some have reported reduced levels,
9
whereas
others have found identical concentrations of this
imino acid in normal and keratoconus corneas.
71 2
It is possible that the abnormal proteins we de-
tected in keratoconus corneas may have been derived
from those normal corneal proteins which appear to
be absent, or present in reduced amounts, in kerato-
conus corneas. These abnorm al pro teins may have an
altered or truncated amino acid sequence, or they
may be proteins with a normal amino acid sequence
that may have undergone abnormal processing, such
as insufficient or excessive glycosylation, sulphation,
or phosphorylation. For example, abnormal protein
K2 (MW 12kD, pi 5.35) could have been derived by
alteration of the amino acid sequence of the normal
corneal protein N3 (MW 12kD, pi 5.7), resulting in
the decrease of the isoelectric po int. It is unlikely that
the abnormal K proteins are degradation products of
N p roteins, because all extractions were performed at
low temperatures in the presence of various protease
inhibitors. How ever, because the average time period
between enucleation and freezing of the normal eyes
was about 7 hr, it is possible, although unlikely, that
K spots were detected in lesser amounts in normal
corneas as a result of proteolytic degradation.
It remains to be determined whether the abnorm al
corneal proteins we detected in keratoconus corneas
are involved in the development of the disease. It is
Table 2.
Molecular weights and isoelectric p oints
of various N and K components
Component
K l
K 2
K 3
K 4
K 5
N l
N 2
N 3
Molecular
w i ht
(kD)
54
12
39
15
28
62-72
52-60
13
Isoelectric
point (pi)
5.25
5.35
6.45
7.38
6.75
5.20 (4.9-5.3)
5.5-5.95
5.7
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INVESTIGATIVE OPHTHALMOLOGY 6 VI5UAL 5CIENCE D e c e m b e r 1989
Vol. 30
1 4 -
Fig.
4. A
two-dimen-
sional electrophorogram
of
a mixed extract of normal
and keratoconus corneas.
Equal amounts
of
kerato-
conus and normal corneal
extracts were mixed,
and a
total of 90 n protein was
applied on the isoelectric
focusing gel.
For
other
ex-
perimental details, see leg-
end for Figure I. Note that
all
N and K
components,
which
are
present
in
both
normal and keratoconus
corneas, com igrate
in the
mixed extract. IEF = iso-
electric focusing; SDS
= SDS polyacrylamide
gel
elect rophores is.
ao
possible that the abnormal electrophoretic pattern we
found in keratoc onus cor neas is at least in part due to
the nonspecific scarring associated with keratoconus.
This study may provide marker proteins for the elu-
cidation of the biochemical abnormality in kerato-
conus.
Key words: keratoconus two-dimensional gel electrophore-
sis, proteins cornea.
Acknowledgments
We thank Drs. Juan J. Arentsen Ann M. Bajart S.
Arthur Boruchoff Helen Boisjoly Elisabeth J. Cohen Jay
Krachmer Peter R. Laibson Roger F. Meyer H. Kaz
Soong Alan Sugar and Roger F. Steinert for providing
keratoconus specimens.
References
1. Chi HH, Katzin HM, and Teng
CC:
Histopathology of kerato-
conus. Am
J
Ophthalmol 42:847, 1981.
2. Teng CC: Electron microscopic study
of
the pathology
of
the
keratoconus:
I.
Am
J
Ophthalmol 55:18, 1963.
3. Pataa CL, Joyon L,
and
Roucher E: Ultrastructure
of
kerato-
conus. Arch Ophthalmol (Paris) 30:403,
1970.
4.
Newsome DA, Foidart JM , Hassell JR, K rachmer JH, Rodri-
gues MM,
and
Katz
SI:
Detection of specific collagen types in
normal
and
keratoconus corneas. Invest Ophthalmol Vis
Sci
20:738, 1981.
5. Radda TM, Menzel EJ Freyler H and Gnad HD : Collagen
types in keratoconus. Graefes Arch Klin Exp Ophthalmol
218:262, 1982.
6. Nakayasu
K
Tanaka
M
Konomi
H and
Hayashi
T:
Distri-
bution
of
types
I II III IV and V
collagen
in
normal
and
keratoconus corneas. Ophthalmic Res 18:1,
1986.
7. Kao WW, Vergens J, Ebert J, Sundar Raj CV, and Brown ST:
Increased collagenase and gelatinase activities
in
keratoconus.
Biochem Biophys Res Commun 107:929, 1983.
8. Rehany U, Lahav M, and Shoshan S: Collagenolytic activity in
keratoconus. Ann Ophthalmol 14:751, 1982.
9. Ihatainen A Salo T Forsius H and Peltonen L: Increase in
type I and type IV collagenolytic activity in primary cultures of
keratoconus cornea. Eur J Clin Invest 16:78, 1986.
10.
Buddecke E and W otlensak J: Saure mucopolysaccharide und
glykoproteine
der
menschlichen cornea
in
abhangigkeit
vom
lebensalter und bei keratoconus. Graefes Arch Klin Exp
Ophthalmol 171:105, 1966.
11.
Robert L, Schillinger G, Moczar M, Junq ua S, and Moczar E:
Biochemical studies of keratoconus. Arch Ophthalmol (Paris)
30-590, 1970.
12. Andreassen TT, Simonsen AH, and Oxlund H: Biochemical
properties of keratoconus and normal corneas. Exp Eye Res
31:435. 1980.
13.
Yue BYJT, Sugar J, and Benveniste K: Heterogeneity in kera-
toconus: Possible biochemical basis. Proc Soc Exp Biol Med
175:336, 1984.
14.
Cannon
DJ and
Foster CS: Collagen crosslinking
in
kerato-
conus. Invest Ophthalmol Vis Sci 17:63, 1978.
15. Oxlund H and Simonsen AH: Biochemical studies of normal
ownloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/Journals/IOVS/933145/ on 05/04/2016
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No . 12 PROTEIN-RELATED ABNORMALITIES IN KERATOCONUS / Pa nj w an i er al 2 4 8 7
and keratoconus corneas. Acta Ophthalmol (Copenh) 63:666,
1985.
16. Hassell JR, Newsome DA, Krachmer JH , and Rodrigues MM:
Macular corneal dystrophy: Failure to synthesize a mature
keratan sulfate proteoglycan. Proc Natl Acad Sci USA
77:3705, 1980.
17.
Anseth A: Studies on corneal polysaccharides. Exp Eye Res
8:438, 1969.
18. Yue BY JT, Sugar J, and Schrode K: Histochemical studies of
keratoconus. Curr Eye Res 7:81, 1988.
19.
Conrad GW, Funderburgh ML, Funderburgh JL, and Rodri-
gues MM: Keratan sulfate in keratoconus, pellucid degenera-
tion, and macular corneal dystrophy. ARVO Abstracts. Invest
Ophthalmo l Vis Sci 29(Suppl):215, 1988.
20. Harrington M G, Merril CR, Goldman D, Xu X, and McFarlin
DE:
Two-dimensional electrophoresis of cerebrospinal fluid
proteins in multiple sclerosis and various neurological diseases.
Electrophoresis 5:236, 1984.
21. Jellum E and Tho rsrud A K: Detection of cancer-related pro-
teins by two-dimensional electrophoresis. Ann NY Acad Sci
428:173,
1984.
22.
O'Farrell PH: High resolution two-dimensional electrophore-
sis of proteins. J Biol Chem 250:4007, 1975.
23. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner
FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ,
and Klenk DC: Measurement of protein using bicinchoninic
acid. Anal Biochem 150:76, 1985.
24.
Oakley BR, Kirsch DR, and Morris NR: A simplified ultra-
sensitive silver stain for detecting proteins in polyacrylamide
gels. Anal Biochem 105:361, 1980.
25. Berg RA, Kedersha NL, and Guzman NA: Purification and
partial characterization of the two nonidentical subunits of
prolyl hydroxylase. J Biol Chem 254:3111, 1979.
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