Protein Engineering of Extracellular Superoxide...
Transcript of Protein Engineering of Extracellular Superoxide...
Linköping University Medical Dissertations
No. 1159
Protein Engineering of
Extracellular Superoxide Dismutase
- Characterization of Binding to Heparin
and Cellular Surfaces
Ing-Marie Ahl
Division of Cell Biology
Department of Clinical and Experimental Medicine
Faculty of Health Sciences
Linköping University
581 85 Linköping
Sweden
Linköping 2010
© Ing-Marie Ahl, 2010
ISBN: 978-91-7393-473-2
ISSN: 0345-0082
Paper I has been reprinted with permission from:
Biochemistry 2009, 48, 9932-9940. © 2009 American Chemical Society
Paper III has been reprinted with permission from:
Protein Expression and Purification 2004, 37, 311-319. © 2004 Elsevier Inc.
Cover: ECSOD binding to HepG2 cells
Printed by LiU-Tryck, Linköping, Sweden, 2010
Gör det du kan, med det du har, där du är.
Theodore Roosevelt
MAIN SUPERVISOR
Lena Tibell, Associate Professor
Department of Science and Technology
Linköping University
Linköping, Sweden
COSUPERVISOR
Bengt-Harald “Nalle” Jonsson, Professor
Department of Physics, Chemistry and Biology
Linköping University
Linköping, Sweden
FACULTY OPPONENT
Steen Vang Petersen, Associate Professor
Department of Medical Biochemistry
Aarhus University
Aarhus, Denmark
MEMBERS OF THE EXAMINATION BOARD
Ingemar Rundquist, Professor
Department of Clinical and Experimental Medicine
Linköping University
Linköping, Sweden
Sophia Hober, Professor
AlbaNova University Center
Royal Institute of Technology
Stockholm, Sweden
Arne Lundblad, Professor Emeritus
Uppsala, Sweden
HOST AT THE PUBLIC DISSERTATION
Peter Påhlsson, Professor
Department of Clinical and Experimental Medicine
Linköping University
Linköping, Sweden
TABLE OF CONTENTS
TABLE OF CONTENTS
LIST OF PAPERS 1
ABSTRACT 2
POPULÄRVETENSKAPLIG SAMMANFATTNING 4
ABBREVIATIONS 5
BACKGROUND 7
The essentiality and toxicity of oxygen 7
Various kinds of radicals 7
Sources of free radicals 8
Reactive oxygen species 9
Free radical reactions 11
Oxidative damage 11
Diseases and pathological conditions 12
Myocardial ischemia-reperfusion injury 12
Defense systems 13
Antioxidative molecules 14
Antioxidative enzymes 15
Superoxide dismutase 16
Four types of SOD 16
The substrate 17
ECSOD 18
The biological role of ECSOD 21
The potential therapeutic use of ECSOD 21
PseudoECSOD 22
Glycosaminoglycans 22
Heparin and heparan sulfate 22
Heparin-protein interactions 23
AIMS 25
TABLE OF CONTENTS
MAIN METHODS AND PROTEIN CONSTRUCTS 26
Isothermal titration calorimetry 26
Phage display 27
Ischemia-reperfusion model 28
Protein constructs 29
SUMMARIZING DISCUSSION 31
CONCLUSIONS 37
ACKNOWLEDGMENT 38
REFERENCES 41
LIST OF PAPERS
LIST OF PAPERS
This thesis is based on the following papers, which will be referred to by their Roman
numerals as follows:
Paper I Thermodynamic Characterization of the Interaction between the
C-Terminal Domain of Extracellular Superoxide Dismutase and Heparin
by Isothermal Titration Calorimetry
Ing-Marie Ahl, Bengt-Harald Jonsson and Lena A. E. Tibell
Biochemistry, 2009, 48:9932-9940
Paper II Analysis of Effects of Mutations in the C-Terminal Domain of
Extracellular Superoxide Dismutase by Isothermal Titration Calorimetry
and Phage Display
Ing-Marie Ahl, Bengt-Harald Jonsson and Lena A. E. Tibell
In manuscript, 2010
Paper III Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide
dismutases in Escherichia coli yields protein with high copper contents
Ing-Marie Ahl, Mikael J. Lindberg and Lena A. E. Tibell
Protein Expression and Purification, 2004, 37:311-319
Paper IV Cell Association and Protective Effects of PseudoECSOD
– a progress report
Ing-Marie Ahl, Sally K. Nelson, Camilla Enström, Ann-Charlotte Ericson and
Lena A. E. Tibell
In manuscript, 2010
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ABSTRACT
ABSTRACT
Accumulating evidence indicates that oxygen free radicals are involved in many diseases
and pathological conditions, such as aging, inflammation, reperfusion damage of ischemic
tissue and various cardiovascular diseases. Extracellular superoxide dismutase (ECSOD) thus
plays a major role in the maintenance of cells by providing protection against these toxic
substances in the extracellular space. Various animal studies have shown that ECSOD has the
ability to protect against many of these disorders, and interest has therefore evolved in the
potential therapeutic use of the enzyme.
However, despite strenuous efforts, large-scale production of the enzyme has not been
achieved. To overcome this problem, a mimic of the enzyme, PseudoECSOD, has previously
been constructed. This chimera is easy to produce in large amounts and has all the structural,
enzymatic and heparin-binding characteristics of ECSOD, making it a potential substitute
for ECSOD in therapeutic situations. However, the copper content of PseudoECSOD has
been shown to be rather low, and since the copper ion is very important for the catalytic
function of the enzyme, a production system that utilizes a copper chaperone for proper
insertion of copper into the active site of the enzyme was constructed. The results show that
the copper content of PseudoECSOD produced by this system is close to 100 %.
In order to use PseudoECSOD therapeutically, further investigations of its binding
capability and protective properties are needed. Therefore, the binding of ECSOD and
PseudoECSOD to heparin was investigated using isothermal titration calorimetry. The
results show that although some purely ionic interactions are important for the binding
between ECSOD and heparin, there is also a substantial contribution from non-ionic
interactions. The investigation also showed that the C-terminal domain is the only part of
ECSOD that contributes to productive binding, and that the binding of PseudoECSOD and
ECSOD to heparin is similar.
In addition, analysis of mutant proteins strongly indicated that the amino acids R210,
K211 and R214 are important for optimal binding of ECSOD to heparin, accounting for
about 30 % of the total binding energy. The structural placement of these amino acids in an
α-helix also confirms the hypothesis postulated by Margalit et al., that a common structural
motif for heparin-binding proteins may be two positively charged amino acids at a distance of
approximately 20 Å in the 3D-structure, facing opposite directions of a α-helix. The
importance of these residues was also confirmed by analysis of a phage display library of the
C-terminal domain of ECSOD.
The binding of PseudoECSOD to heparan sulfate on cell surfaces of two different cell
types, HepG2 and endothelial cells, was also investigated. The results clearly show that
PseudoECSOD binds to these cells in a very similar manner to ECSOD. To investigate the
protective properties of PseudoECSOD against ischemia-reperfusion injuries, an isolated
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ABSTRACT
rabbit heart model was used. The results indicate that the enzyme has a protective effect.
However, more experiments using the rabbit heart and other animal models are needed to
identify the optimal dose for protective purposes. The protective properties of
PseudoECSOD in human tissue should also be thoroughly investigated.
In summary, the findings in these studies, together with earlier results showing the close
resemblance of PseudoECSOD to ECSOD in structural, enzymatic and heparin-binding
properties, further support the proposition that PseudoECSOD may be a good substitute for
ECSOD to use in therapeutic interventions.
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POPULÄRVETENSKAPLIG SAMMANFATTNING
POPULÄRVETENSKAPLIG SAMMANFATTNING
Fria syreradikaler produceras konstant i människokroppen, både som biprodukter i
cellernas dagliga processer och aktivt t.ex. som försvar mot bakterier. Låga koncentrationer av
syreradikaler fungerar som signalmolekyler i cellen, men höga nivåer kan leda till skador på
cellens beståndsdelar. För att skydda sig mot dessa skadliga radikaler har cellerna i alla
syrelevande organismer utvecklat olika försvarssystem. Ett av de mest effektiva skydden mot
fria radikaler är superoxiddismutas, SOD, som är ett enzym som bryter ned
superoxidradikaler. Enzymet finns både inuti cellen (intracellulärt) och utanför
(extracellulärt). Extracellulärt superoxiddismutas (ECSOD) binder till sockerstrukturer på
cellens yta. På så sätt optimeras skyddet av cellerna mot angrepp av syreradikaler utifrån, och
denna bindning är därför viktig att studera ur medicinsk synvinkel.
I denna avhandling beskrivs bindningen mellan ECSOD och heparin i detalj genom att
använda en mätmetod kallad isoterm kalorimetri. Vi visar bland annat att de positivt laddade
aminosyrorna R210, K211 och R214 i enzymet är viktiga för optimal inbindning till negativt
laddade strukturer i heparin, och att de intar positioner i enzymets struktur som underlättar
inbindning. Resultaten visar vidare att bindningen är beroende av fler samspel mellan
oladdade atomer än man tidigare trott samt att produktiv bindning endast sker i enzymets C-
terminal.
Eftersom fria radikaler är inblandade i en lång rad sjukdomstillstånd, t.ex.
åderförkalkning, cancer, hjärtsvikt, inflammation, lungsjukdomar och åldrande, har ECSOD
potential att verka som läkemedel mot dessa. Det har dock visat sig vara svårt att producera
ECSOD i stora mängder, och vår forskargrupp har därför tidigare konstruerat ett liknande
enzym (PseudoECSOD) som har samma funktion som ECSOD, men som relativt enkelt går
att producera i stor skala. Dock har mängden koppar i detta enzym varit låg, och eftersom
kopparinnehållet är viktigt för enzymets funktion har vi nu utvecklat ett produktionssystem
för att införa koppar i enzymet. Resultatet av detta visar att PseudoECSOD får nära 100 %
kopparinnehåll.
Vi har också analyserat PseudoECSODs förmåga att binda till olika celler och vävnader,
samt dess skyddande egenskaper mot angrepp från fria superoxidradikaler. Vi har visat att
PseudoECSOD binder till båda de undersökta celltyperna, på liknande sätt som ECSOD,
och att det har potential att skydda mot radikalangrepp i isolerade kaninhjärtan. Resultaten är
mycket lovande, men fler studier, båda i djur och människa, krävs innan enzymet kan verka
som en artificiell radikalhämmare.
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ABBREVIATIONS
ABBREVIATIONS
AP-1 Activator protein 1
ARE Antioxidant response element
ATP Adenosine triphosphate
BH4 5,6,7,8-tetrahydrobiopterin
Ca2+ Calcium ion
cDNA Complementary DNA
CS Chondroitin sulfate
Cu2+ Copper ion
CuZnSOD Copper- and zinc-containing superoxide dismutase
CVD Cardiovascular disease
DNA Deoxyribonucleic acid
DS Dermatan sulfate
EC Enzyme Commission
ECM Extracellular matrix
ECSOD Extracellular superoxide dismutase
Fe2+ Iron ion
FeSOD Iron-containing superoxide dismutase
FITC Fluorescein isothiocyanate
GlcA β-d-glucuronic acid
GlcN β-d-glucosamine
GlcNAc N-acetylated β-d-glucosamine
GlcNS N-sulfated β-d-glucosamine
GSH Glutathione
GSSG Glutathione disulfide
H+ Hydrogen ion
HA Hyaluronic acid
H2O Water
H2O2 Hydrogen peroxide
HOCl Hypochlorous acid
•HO2 Perhydroxyl radical
HS Heparan sulfate
IdoA α-l-iduronic acid
IFN-γ Interferon-γ IgG Immunoglobulin G
IL-4 Interleukin-4
iNOS Inducible nitric oxide synthase
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ABBREVIATIONS
IR Ischemia-reperfusion
ITC Isothermal titration calorimetry
KS Keratan sulfate
LDL Low-density lipoprotein
MAPK Mitogen-activated protein kinase
Mn2+ Manganese ion
MnSOD Manganese-containing superoxide dismutase
MPO Myeloperoxidase
NAD+ Oxidized form of nicotinamide adenine dinucleotide
NADP+ Oxidized form of nicotinamide adenine dinucleotide phosphate
NADPH Reduced form of nicotinamide adenine dinucleotide phosphate
NF-κB Nuclear factor kappa beta
Ni2+ Nickel ion
NiSOD Nickel-containing superoxide dismutase
N2O3 Dinitrogen trioxide
•NO Nitric oxide
•NO2 Nitrogen dioxide
NOS Nitric oxide synthase
NOX NADPH oxidase
•OH Hydroxyl radical
ONOO - Peroxynitrite
ONOOH Peroxynitrous acid
•O2- Superoxide anion radical
pH -log [H+]
pKa -log Ka (acid dissociation constant)
R• Unspecified radical
RNS Reactive nitrogen species
ROS Reactive oxygen species
SOD Superoxide dismutase
TRITC Tetramethyl rhodamine isothiocyanate
UV Ultraviolet
XO Xanthine oxidase
XRE Xenobiotic response element
Zn2+ Zinc ion
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BACKGROUND
BACKGROUND
The essentiality and toxicity of oxygen
On the early Earth the atmospheric oxygen concentration was very low. However, about
2.45 billion years ago atmospheric oxygen levels suddenly rose in what is now termed the
Great Oxidation Event. This caused a separation between anaerobic and aerobic organisms.
The anaerobic organisms could not adapt to the new conditions across most of the globe, and
hence were restricted to niche environments, while the aerobic organisms evolved adaptive
mechanisms that paved the way for the evolution of multicellular plants and animals (1).
Today, oxygen is an essential component for the survival of aerobic organisms, since it is
the terminal acceptor of electrons during aerobic respiration, which is the main source of
energy in these organisms. However, despite its importance it also has harmful effects. At
concentrations greater than those in air, oxygen is toxic to all living creatures, due to the
oxygen free radicals that are produced in sequential, univalent reactions (Figure 1) that occur
in the metabolism of molecular oxygen (2).
Molecular oxygen 3O2 → 1O2 Singlet oxygen
↓
Superoxide radical •O2- → •HO2 Perhydroxyl radical
↓
Peroxide ion O22- → H2O2 Hydrogen peroxide
↓
O23- → H2O Water
↓
Oxene ion O- → •OH Hydroxyl radical
↓
Oxide ion O2- → H2O Water
Figure 1 Sequential, univalent reduction of molecular oxygen.
Various kinds of radicals
A free radical is any molecule, organic or inorganic, that has an odd number of, and thus
unpaired, electrons. Free radicals are highly reactive and thus transient. Due to the ubiquity of
molecular oxygen in aerobic organisms and its capability to readily accept electrons, oxygen-
centered free radicals are frequently formed via diverse mechanisms (3). Radicals and other
reactive molecules that are oxygen-centered are called reactive oxygen species (ROS). ROS
include superoxide anion radicals (•O2-), perhydroxyl radicals (•HO2), singlet oxygen (1O2),
hydrogen peroxide (H2O2), hydroxyl radicals (•OH) and hypochlorous acid (HOCl) (4). A
further group of oxygen-containing free radicals are derived from nitric oxide (•NO), but
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BACKGROUND
since they also contain nitrogen they are known as reactive nitrogen species (RNS). Besides
nitric oxide, RNS include peroxynitrite (ONOO-), peroxynitrous acid (ONOOH), nitrogen
dioxide (•NO2) and dinitrogen trioxide (N2O3) (4).
Sources of free radicals
Oxygen free radicals in humans and other aerobic organisms can originate from many
sources, both endogenous and exogenous. Examples of exogenous sources, or more strictly,
agents that induce their formation, are UV light, ionizing radiation, pesticides, heavy metals,
air pollution (5), static magnetic fields (6), redox cycling drugs and other xenobiotics from
which free radical metabolites are formed (3), tobacco smoking (7), inorganic particles such as
asbestos (8) and silica (9), and gases such as ozone (10, 11).
There are also diverse endogenous sources of reactive oxygen species, partly because
oxygen is used in a wide range of processes in normal cellular metabolism, and ROS are by-
products of many of the oxidations involved (12). Electron transport in both mitochondria
(13-16) and chloroplasts (17, 18), photorespiration in peroxisomes and β-oxidation of fatty
acids in peroxisomes and mitochondria (17, 18) are some metabolic processes that are major
sources of ROS. Free radicals are also generated in the demethylation, hydroxylation and
desaturation reactions catalyzed by cytochromes P450 and b5 in the endoplasmic reticulum and
nuclear membrane (3).
However, ROS are also actively produced in other processes that are not involved in the
normal metabolism of healthy cells, such as the respiratory burst during phagocytosis by
neutrophils and macrophages. Upon phagocytosis pathogens such as bacteria are engulfed in
internalized vesicles, phagosomes, and exposed to a variety of toxic molecules produced via
several mechanisms. One of the most important of these mechanisms is the formation of
superoxide anions in the reaction catalyzed by phagocytic nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase (NOX) (19). Superoxide can further be converted to hydrogen
peroxide (19), which subsequently can react with other radicals and transition metals such as
Fe2+, yielding highly reactive hydroxyl radicals (20). The very powerful oxidant hypochlorous
acid can also be generated from hydrogen peroxide and chloride ions by myeloperoxidase
(MPO) (19, 21). In addition, nitric oxide is produced by the action of inducible nitric oxide
synthase (iNOS). Superoxide and nitric oxide can then form the highly reactive peroxynitrite
(19). These substances have various, highly toxic effects that kill internalized pathogens (21-
23). However, although beneficial in host cell defenses they may have damaging effects on the
surrounding cells and extracellular matrix.
There are also non-phagocytic membrane-associated NAD(P)H oxidases, expressed in
various cell types, such as endothelial cells (24, 25), that generate and release low amounts of
superoxide radicals in the extracellular space (26), through electron transfer from cytosolic
NADPH to extracellular oxygen (27, 28).
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BACKGROUND
In addition, numerous enzymes are capable of generating significant amounts of free
radicals, through one-electron transfers during their normal catalytic action. For instance,
xanthine oxidase (XO) generates superoxide by catalyzing the conversion of hypoxanthine and
xanthine to uric acid (20), a process which is further increased during ischemia-reperfusion
conditions (29). Lipoxygenase produces ROS by oxidizing polyunsaturated fatty acids to
hydroperoxy fatty acid derivatives (29). ROS can also be produced by cellular oxidative
metabolic processes involving cyclooxygenase and monoamine oxidase (30). Modulation of
enzyme activity, cofactor availability, substrate concentration and oxygen tension can all affect
the rates of intracellular radical production. Thus, certain cellular metabolic states, for
example hyperoxia, ischemia and antibiotic therapy can favor free radical production (3).
Oxygen may also be transformed to superoxide radicals non-enzymatically, by reacting
with redox-active compounds such as semiubiquinone of the mitochondrial electron transport
chain (29). ROS are also formed during the autooxidation of diverse molecules such as
catecholamines (31, 32), hemoglobin (33, 34), myoglobin (35, 36), hydroquinones (37),
flavins (38) and thiols (39). In addition, under normal conditions nitric oxide synthase (NOS)
catalyzes the production of nitric oxide from the substrate L-arginine, with help from the
cofactor 5,6,7,8-tetrahydrobiopterin (BH4). However, if the availability of the substrate or the
cofactor decreases, NOS activity may switch to an uncoupled state in which the product is
superoxide rather than nitric oxide (20).
As mentioned above, transition metals play major roles in the generation of free radicals.
Iron and copper can catalyze the Fenton reaction, which generates hydroxyl radicals from
hydrogen peroxide (Reaction 1) (40-43). If superoxide is present it may participate in the
formation of further hydroxyl radicals by reducing the transition metals to their active states
(Reaction 2) (44, 45). The net reaction (Reaction 3) is then called the Haber-Weiss reaction
(46).
Fe2+/Cu+ + H2O2 → Fe3+/Cu2+ + OH- + •OH (1)
Fe3+/Cu2+ + •O2- Fe→ 2+/Cu+ + O2 (2)
•O2- + H2O2 → O2 + OH- + •OH (3)
Reactive oxygen species
The most reactive ROS are the hydroxyl radical and singlet oxygen, but as a result of
their reactivity, these species have very short half-lives and react in the vicinity of their site of
origin. The most abundant ROS are hydrogen peroxide and the superoxide anion radical.
These are less reactive and thus have longer half-lives. Hence, there is a higher probability
that they will react elsewhere from the site of their production (47). Since superoxide anion
radicals, unlike hydrogen peroxide, are charged, they do not have the ability to diffuse
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BACKGROUND
through cell membranes. However, they can cross the outer mitochondrial membrane (48) (49, 50), and the plasma membrane of various cell types (51-53) via anion channels. Another
potential mechanism is protonation of superoxide to generate the uncharged perhydroxyl
radical, which may cross membranes and then reform superoxide on the other side (54, 55).
As mentioned above, superoxide may also be directly produced by NAD(P)H oxidases in the
extracellular space (26). Further, since the superoxide radical can induce cascade reactions, it
may have effects far from the site of production and action of the original radical, which is the
main reason for its high toxicity.
Under physiological conditions, in which the concentration of superoxide is low, the
favored reaction is the dismutation reaction yielding hydrogen peroxide (Reaction 4).
However, during pathophysiological states, when excess superoxide is produced, it frequently
reacts with nitric oxide, yielding the highly reactive oxidant peroxynitrite (Reaction 5) (12).
The rate of this reaction is approximately three times faster than the SOD catalysis (56).
2 •O2- + 2 H+ O→ 2 + H2O2 (4)
•O2- + •NO ONOO → - (5)
Hydrogen peroxide is produced mainly from the dismutation of superoxide anions (47).
This reaction (Reaction 4) may be either spontaneous or catalyzed by superoxide dismutase
(SOD) (57).
Since hydrogen peroxide is not a radical, it is much more stable than superoxide. Further,
it is lipid soluble and can thus cross mitochondrial membranes (58), peroxisomal membranes
(16, 59) and plasma membranes (12, 60). Both hydrogen peroxide and superoxide radicals can
act either as oxidants or reductants, thus they can affect a very wide range of molecules.
ROS were first considered to have no biological function. However, there is now
abundant evidence that these partly reduced oxygen metabolites are critical for the normal
operation of a wide spectrum of biological processes (3). They participate as second
messengers in the modulation of cell signalling cascades and regulation of important cellular
event, such as cell proliferation and differentiation (61-64). Hence, there are several pathways
that employ oxidants as effectors in diverse processes.
For example, ROS affect the redox state of cells, which in turn influences the activity of
various redox-sensitive proteins, and gene transcription can be induced by ROS (65, 66).
Thus, reactive oxygen species are implicated in many intracellular signaling pathways leading
to changes in gene transcription and protein synthesis and hence cell function (12).
10
BACKGROUND
Free radical reactions
As the name implies, ROS are very reactive and may react with virtually every cellular
component, causing serious damage, sometimes sufficiently severe to lead to cell death. Free
radicals act through various mechanisms, but the most common is abstraction of univalent
atoms, such as hydrogen atoms (Reaction 6) or halides (Reactions 7). Another important
mechanism is addition to unsaturated bonds, such as those in fatty acids (3).
R• + XH → RH + X• (6)
R• + CCl4 → RCl + Cl3C• (7)
The formation of a radical is called initiation. This may occur, for example, through
electron transfer from transition metals to oxygen species, or via single electron oxidations or
reductions, as in electron transport chains. Propagation of free radical reactions may then
proceed indefinitely, causing series of damaging events, or may be terminated by collision of
pairs of radicals or by a variety of free radical scavengers (3).
Oxidative damage
Cellular targets at risk of free radical damage depend on the nature of the radical and its
site of generation (3). Free radicals are capable of damaging many cell components such as
proteins, carbohydrates, lipids and nucleic acids (2, 20). The most severe kinds of damage
caused by ROS are lipid peroxidation, oxidation/denaturation of proteins and
modifications/mutations of DNA.
Proteins containing the amino acids tryptophan, tyrosine, phenylalanine, histidine,
methionine and cysteine can undergo free radical-mediated amino acid modifications. Highly
reactive radicals such as hydroxyl radicals may also attack peptide bonds and the amino acids
lysine and proline, which are resistant to most other oxidative attacks. Enzymes that depend
on attacked amino acids for catalytic activity will be inhibited, and if the amino acids are
important for the structure of the protein, denaturation may occur. The susceptibility of
proteins to free radical damage thus depends on their amino acid composition, the location of
susceptible amino acids and their importance for the activity and/or conformation of the
protein, and whether the damaged protein can be repaired. The cellular location of proteins
and the nature of the free radical also influence the extent of protein damage. Free radicals
can also cross-link proteins into dimers or larger aggregates, by inducing the formation of
disulfide bonds between different proteins or irreversible reactions between free radical-
damaged amino acid residues (3).
The unsaturated bonds of membrane cholesterol and fatty acids can readily react with free
radicals and undergo peroxidation. This process, which yields lipid radicals, can become
autocatalytic after initiation (3), leading to severe damage to the membranes. Lipid
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BACKGROUND
peroxidation is mainly caused by hydroxyl and perhydroxyl radicals (2). The presence of
shortened fatty acids after lipid peroxidation may seriously affect membrane permeability and
microviscosity (67). The increased membrane permeability due to lipid peroxidation and
oxidation of structurally important proteins can cause the breakdown of transmembrane ion
gradients, loss of secretory functions and inhibition of integrated cellular metabolic processes
(3).
Free radical reactions with DNA will result in damaged nucleic acids, and hence
mutations if the damage is not repaired. Radical attacks can also result in strand scission.
Most nucleic acid destruction occurs through alterations in both the bases and the
deoxyribose sugars, which are often cross-linked following radical attack. The components of
single-stranded DNA that are most susceptible to free radical action are the thymine and
cytosine bases, followed by the guanine and adenine bases and finally the deoxyribose sugar.
However, for double-stranded DNA, the deoxyribose moiety is modified more frequently
than the bases, due to its exposed location in the helix. The hydroxyl radical is the major
oxidant in these reactions (2, 3).
Carbohydrates are also targets for free radicals; monosaccharides may be oxidized and
polysaccharides may be depolymerized. Hence, glycosylated proteins are especially sensitive to
oxidative damage and various studies have shown that the glycocalyx of endothelial cells can
be damaged by free radical attacks (68, 69).
Diseases and pathological conditions
Pathological events often result in overproduction of ROS/RNS. If the antioxidative
system cannot cope with this change, an imbalance between the prooxidants and antioxidants
will occur, resulting in increased bioavailability of ROS/RNS and a state of
oxidative/nitrosative stress (12, 70).
The involvement of oxidative stress has been confirmed in a number of diseases and
pathological conditions, including cardiovascular diseases (CVD) (20) such as atherosclerosis,
heart failure, diabetes and hypertension (56), myocardial (71, 72) and cerebral (73, 74)
ischemia-reperfusion injury, cancer (75-77), various lung diseases (78-80) and neurological
disorders (81), inflammation and aging (82).
Myocardial ischemia-reperfusion injury
Ischemia-reperfusion (IR) injuries may occur in the myocardium following blood
restoration after a critical period of coronary occlusion (83), since the restoration of blood
flow is accompanied by a burst of oxygen free radicals (84). The radical burst is due to
activation of membrane oxidases, mitochondrial uncoupling and neutrophil activation (85).
Xanthine dehydrogenase, which normally utilizes NAD+ as an electron acceptor, is converted
under ischemia-reperfusion conditions into xanthine oxidase which uses oxygen as substrate.
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BACKGROUND
During the ischemic period, excessive ATP consumption leads to the accumulation of
hypoxanthine and xanthine, which upon subsequent reperfusion and influx of oxygen are
metabolized by xanthine oxidase to yield massive amounts of superoxide and hydrogen
peroxide (86). Since the production of free radicals is proportional to oxygen tension, the rise
in ROS production during recirculation is related to over-oxygenation during reperfusion
(87). Since IR can cause irreversible tissue damage, early restoration of blood flow is essential
to reduce the injury. However, injuries are known to progress even after reperfusion (88).
The pathophysiology of IR-induced injury is associated with various events, such as
reperfusion arrhythmias (71, 89), microvascular damage due to inadequate oxygen delivery to
the microcirculation during reperfusion (90, 91), myocardial stunning (92, 93), inflammatory
reactions, changes in arteriolar resistance caused by alterations in oxygen metabolism (94) and
changes in tissue homeostasis that lead to necrosis and/or programmed cell death (85, 89).
Ischemia-reperfusion can lead to tissue injuries that are serious complications in organ
transplantation, myocardial infarction, and stroke (95-97).
Two main hypotheses have been proposed to explain the pathogenesis of ischemia-
reperfusion injury (98-100), namely oxidative stress, which is a well established
etiopathogenic factor of reperfusion-induced injury and its consequences (29, 101, 102), and
overload of Ca2+. These mechanisms are probably related, and oxygen radicals have been
shown to cause myocardial calcium overload by damaging the sarcoplastic reticulum that
normally sequesters and stores intracellular calcium ions (72).
Reperfusion of the ischemic tissue leads to the rapid accumulation of neutrophils at the
site of injury (72), where they are the principal effector cells of reperfusion damage (29).
These leukocytes adhere to the vascular endothelium, transmigrate to the extravascular space,
and can adversely affect viable tissue through the release of proteolytic enzymes and the
generation of reactive oxygen species, such as superoxide anion and hypochlorous acid (72).
Thus, inhibition of neutrophil adhesion to the endothelium attenuates the process (103), and
antioxidant treatment ameliorates both leukocyte adhesion and leukocyte-mediated heart
injury in the postischemic period (104).
Ischemia and reperfusion in the rat heart has been found to be associated with activation
of redox-responsive transcription factors, such as NF-κB, AP-1 and various MAPKs (105,
106), which may account for inflammatory responses and apoptotic cell death in the affected
tissue (107, 108).
Defense systems
To protect themselves from these toxic molecules, aerobic organisms have evolved
antioxidative defense systems, which include both enzymes and chemical scavengers that trap
the oxygen derivatives and thus regulate the concentration of ROS. The defense systems can
be divided into primary defenses that decrease the free radical concentration and prevent
13
BACKGROUND
initiation of radical chain reactions, and secondary defenses that trap propagator radicals and
diminish the damage they cause by reducing oxidized proteins and repairing DNA.
The primary defenses include antioxidant enzymes, like SOD, catalase and glutathione
peroxidases and molecules such as ascorbic acid, glutathione (GSH), uric acid, taurine and
hypotaurine. The secondary defenses include enzymes such as glutathione transferases with
peroxidase activity that protect against lipid peroxidation, and thioredoxin and other
oxidoreductases that act as antioxidants by facilitating the reduction of oxidized protein
groups such as thiol. Metal sequestering proteins (109), macroxyproteinase and other
proteolytic enzymes that degrade irreparably damaged proteins and DNA repair enzymes can
also be included in the secondary defense systems, as can molecules like α-tocopherol, β-
carotene and bilirubin (2).
These molecules act in various ways to scavenge various prooxidants and keep them at
acceptable levels both inside and outside the cell. Many of them have proven to be able to
decrease the damage caused by oxidative stress in cellular and ex vivo animal models (110).
However, a molecule demonstrated to have antioxidant properties in vitro might have
different or additional properties in a more complex system (111), and no clear benefit of
antioxidant therapy has yet been demonstrated in clinical intervention studies (112). The
failure to protect cells against oxidative stress in vivo may be due to the bioavailability and
metabolism of the agent (113). Hence, the concept of an antioxidant in vitro should not be
extended to cells, organs, animals or populations until the evidence has been obtained (111).
Antioxidants can have beneficial and/or toxic actions on cells depending on the situation and
therefore their use should always be made with a full appreciation of the situation, by
clarifying the molecular details of the sources of reactive oxygen species, their nature and their
regulation. Increasing evidence indicates that the production of reactive oxygen species is
precisely regulated and their downstream targets are specific (112).
Antioxidative molecules
Ascorbic acid is another name for vitamin C. The biologically active substance is the
ascorbate ion, which is a cofactor of many proteins and also has antioxidative properties. It
acts as a reducing agent that reverses oxidation and keeps iron and copper in their reduced
states. Ascorbate is water-soluble and thus is active in the cytoplasm and other aqueous
solutions (114, 115).
Glutathione can act as a cosubstrate for glutathione peroxidase, thereby reducing
peroxides, or it can react directly with oxygen radicals. GSH also reduces dehydroascorbate,
which is formed in the reactions of ascorbic acid, back to its active form.
Uric acid is a very efficient antioxidant that traps free radicals and protects against
oxidation in plasma. Taurine and hypotaurine are present in many biological fluids where
they have a protective function against free radical damage.
14
BACKGROUND
α-tocopherol is a lipid-soluble antioxidant that belongs to the vitamin E family. This
molecule protects cell membranes from oxidation damage by reacting with lipid radicals
produced in lipid peroxidation chain reactions. Scavenging of the radicals terminates the
chain reactions and prevents further damage. The oxidized α-tocopheroxyl radical produced
in the process is recycled back to its reduced form via reduction by other antioxidants such as
ascorbate, retinol and ubiquinol. Although this is a very important activity of this molecule,
the significance of its antioxidant properties at physiological concentrations is unclear.
Instead, the main role of α-tocopherol may be in cell signaling (116). A novel, natural form of
vitamin E, α-tocopheryl phosphate, can substitute for and is more potent than a-tocopherol
in in vitro and animal models (117).
β-carotene belongs to the vitamin A family of fat-soluble vitamins that play roles in
diverse functions in the body, for example vision, gene transcription, immune responses and
hematopoiesis. It also has antioxidant properties by acting as a very efficient singlet oxygen
scavenger and inhibitor of lipid peroxidation.
Bilirubin, which is actually a waste product from the breakdown of hemoglobin, breaks
radical chain reactions by reacting with the attacking radical.
Antioxidative enzymes
Catalase (EC 1.11.1.6) scavenges hydrogen peroxide in biological systems by catalyzing
its decomposition to water and oxygen (Reaction 8) (2).
2 H2O2 O→ 2 + 2 H2O (8)
Catalase is a tetramer, each subunit of which contains one porphyrin heme group, which
is needed in the reaction catalyzed by the protein. The enzyme is located mainly in the
peroxisomes and has one of the highest turnover numbers of all known enzymes (118).
Glutathione peroxidase (EC 1.11.1.9) is the general name of an enzyme family with
peroxidase activity, the biochemical function of which is to reduce lipid hydroperoxides to
their corresponding alcohols and free hydrogen peroxide to water. There are several
isoenzymes with different cellular locations, both intracellular and extracellular, and different
substrate specificity. Glutathione peroxidase 1 is the most abundant form. It is a
homotetrameric protein that is located in the cytoplasm of nearly all mammalian tissues. Its
main substrate is hydrogen peroxide and its catalytic action is dependent on selenium (119).
The enzyme requires glutathione as a cosubstrate (Reaction 9), and the oxidized form of
glutathione (GSSG) is subsequently reduced back to GSH by glutathione reductase (Reaction
10) (2).
2 GSH + H2O2 GSSG + 2 H→ 2O (9)
GSSG + NADPH + H+ → 2 GSH + NADP+ (10)
15
BACKGROUND
Thioredoxin act as an antioxidant by facilitating the reduction of oxidized proteins by
cysteine thiol-disulfide exchange. It is ubiquitous and essential for life in mammals (120).
Superoxide dismutase
Superoxide dismutase (EC 1.15.1.1) was first isolated in 1939 by Mann and Keilin (121),
but the catalytic function of the enzyme was discovered by McCord and Fridovich in 1969
(57). The main function of superoxide dismutases is to scavenge the superoxide anion radicals
generated in various physiological processes, by catalyzing the disproportionation of the
radical to hydrogen peroxide and molecular oxygen (Reaction 4). How scavenging of
superoxide radicals by SOD to yield the more potent oxidant hydrogen peroxide can be
beneficial to biological system may be confusing. However, one accepted explanation of this
paradox is that SOD prevents the formation of peroxynitrite, which is a far more toxic
substance than hydrogen peroxide. Further, hydrogen peroxide is readily scavenged by
catalase.
These enzymes, which are members of the oxidoreductase family, are metalloenzymes
that are present in both eukaryotes and prokaryotes. They are located in a wide variety of
intracellular compartments and are also active in the extracellular space. Aerobic organisms
contain more SOD than aerotolerant organisms, while strict anaerobes have no detectable
SOD activity (122).
Four types of SOD
Depending on the prosthetic metals in the active site, SODs are divided into four types:
CuZnSOD (Cu2+ and Zn2+), MnSOD (Mn2+), FeSOD (Fe2+) and NiSOD (Ni2+). In vitro,
one can distinguish between these types of SOD by examining the inhibiting effects of
cyanide and hydrogen peroxide, since they all show different inhibition patterns (123-127).
However, these reactions do not occur under physiological conditions.
The four types of SOD fall into three different phylogenetic families: the CuZnSODs
and the Fe/MnSODs and NiSOD. However, SOD enzymes harboring Ni2+ and Fe2+ or Fe2+
and Mn2+ in their active sites has also been reported in cyanobacteria (128). There is no
homology between the three families, but FeSODs and MnSODs show a great deal of amino
acid sequence and structural homologies (129, 130). CuZnSOD, MnSOD and FeSOD are all
intracellular enzymes, but there is also an extracellular enzyme called ECSOD, which belongs
to the CuZnSOD family. The ECSOD gene is 40-60% homologous to CuZnSOD, but
shows minimal homology with MnSOD (131).
Manganese superoxide dismutase (MnSOD), which was first isolated by Keele et al in
1970 (132), is present in the cytosol of prokaryotes and the mitochondrial matrix of
eukaryotes (133-135), where it scavenges the superoxide radicals that originate from the
16
BACKGROUND
respiratory chain reactions. MnSOD is often induced by oxygen (135). In higher eukaryotes,
yeast and some bacterial species the enzyme exists as a homotetramer, while in other
organisms it is usually dimeric (136).
Iron superoxide dismutase (FeSOD), which was first isolated by Yost et al. in 1973 (137),
is found in prokaryotes (133, 137, 138), simple eukaryotes (135) and in chloroplasts of plants
(139), where it protects against the action of superoxide generated by electron leakage to
oxygen at the reducing site of Photosystem I (140). The enzyme is either homodimeric or
homotetrameric.
Nickel superoxide dismutase (NiSOD) has to date been found in various bacteria and
cyanobacteria (125, 135, 141). In most organisms, the active form is homohexameric (135,
141), but it remains monomeric in the absence of nickel (126). The enzyme does not show
any sequence homology to the other SODs and have a novel SOD fold (135).
CuZnSOD is mainly found in the cytosol of eukaryotes (142), but the protein is also
present in various organelles, such as the intermembrane space of mitochondria (134),
peroxisomes (142, 143), nucleus (142) and chloroplasts (144). The enzyme has also been
reported in the periplasm of bacteria (145, 146).
CuZnSOD is the major intracellular SOD (109). Most CuZnSODs are homodimers,
with each monomer containing an active site, but monomeric isoforms have been reported in
bacteria (147). Although the function of cytosolic CuZnSODs is known, there is still
uncertainty regarding the biochemical pathways in which they participate (140).
The substrate
The substrate for SOD is the superoxide anion radical, which is formed by a one-electron
reduction of molecular oxygen. It is very unstable and undergoes spontaneous dismutation.
However, at physiological pH, SODs catalyze the reaction 104 times faster (3). The
protonated form of the superoxide radical is the perhydroxyl radical (•HO2.), which is very
reactive and a stronger oxidant than superoxide but has low abundance at physiological pH
(2). However, the protic microenvironment near cell surfaces, which are polyanionic, may
favor formation of perhydroxyl radicals, due to the locally lower pH (3). Since perhydroxyl
radicals can cross the plasma membrane, this may open the door for superoxide radicals for
action in the cytosol.
Superoxide has been shown to affect vascular physiology and pathophysiology, mainly
through its interaction with nitric oxide (148), and to activate certain signaling pathways,
leading to altered gene expression (61, 149). The radical has also been proposed to serve as a
signal molecule in growth regulation and development, partly by altering the cellular
responses to growth factors and vasoconstrictor hormones. Superoxide radicals are also
implicated in inflammatory diseases, ischemia-reperfusion injury, cancer, and the aging
process (148).
17
BACKGROUND
ECSOD
Extracellular superoxide dismutase (ECSOD) was discovered by Marklund and
coworkers in 1982 (124). It is the predominant SOD in extracellular fluids such as lymph,
synovial fluid and plasma (150, 151) and the only known antioxidant enzyme that scavenges
superoxide anion radicals specifically in the extracellular space (152). The primary location of
ECSOD in tissues is in the extracellular matrix (ECM) and on cell surfaces, where it is found
at 20 times the concentration present in plasma (109). 90-99 % of the total amount of
ECSOD in mammals is located in the extravascular space of tissues (153, 154). The
expression of ECSOD in different tissues varies between different species, but the
concentrations are generally highest in blood vessels, heart, lung, kidney, pancreas, placenta,
while very little is expressed in brain (155, 156). In the vascular system ECSOD is mainly
expressed in the arterial walls and vascular smooth muscle cells (157-159). ECSOD is also
synthesized and secreted by a variety of fibroblast and glial cell lines (153, 160).
The enzymatic, structural and heparin-binding characteristics of ECSOD have been
thoroughly investigated and also compared to the intracellular CuZnSOD enzyme (161).
ECSOD from most species has a molecular weight of approximately 135 kDa (124), although
there are some variations (162, 163). In most species ECSOD occurs as a homotetramer (164), which is maintained through interactions between the N-terminal domains (165, 166).
However, octameric forms of human ECSOD have also been reported (167).
The ECSOD gene (SOD3) is localized to chromosome 4q21 in the human genome
(168). The gene, which is approximately 5900 base pairs (bp) long, consists of three exons and
two introns, of which exon 3 contains the 720 bp coding region. The promoter region of the
gene contains various regulatory elements, including antioxidant response elements (ARE),
AP-1 binding sites, NF-κB motifs and xenobiotic response elements (XRE) (109). The
human ECSOD cDNA encodes a 240 aa propeptide, containing an 18 amino acid (aa) long
signal peptide that is removed to yield a 222 aa mature protein (169).
The middle part of the enzyme contains the active site, which binds a copper ion that
plays a major role in the catalytic action by accepting an electron from one superoxide radical
and donating it to another. Along with two protons, the intermediate thus formed produces
hydrogen peroxide. A specific arginine in the vicinity of the active site attracts the superoxide
radical, facilitating the action of the copper. The zinc ion does not have a catalytic function;
instead it stabilizes the structure of the active site (170).
With a kcat/Km of 2 x 109 M-1s-1, CuZnSODs are among the most efficient enzymes
known. This efficiency is partly due to the electrostatic guidance of the substrate to the active
site of the enzyme (171) arising from the surface of the protein being negatively charged,
while the entrance to the catalytic pocket has a positive charge (172-174).
18
BACKGROUND
ECSOD is a glycoprotein (124) and the glycosylation site in the human enzyme is found
at Asn 89 (169). Structurally, the carbohydrate chain is of biantennary complex type with an
internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with
terminal sialic acid linked to N-acetyl-lactosamine (124) (Adachi, 1992). No specific
biological function of the carbohydrate moiety has been reported and it does not seem to
affect most biochemical properties of the enzyme, such as activity, structure, heparin-binding
or plasma half-life. However, the non-glycosylated enzyme showed a marked reduction in solubility in aqueous media (175).
An important characteristic of ECSOD that is not shared by other SOD enzymes is its
strong affinity for the highly negatively charged glycosaminoglycans heparin and heparan
sulfate. Since the isoelectric point of ECSOD is 4.5, the enzyme carries a net negative charge
at physiological pH (176). This implies that its binding to negatively charged
glycosaminoglycans is mediated by a cluster of positively charged amino acids in the enzyme.
Such a cluster is present in the hydrophilic C-terminal domain (amino acids 194-222), which
contains nine positively charged amino acids, and this cluster has been shown to be important
for interactions with heparin and heparan sulfate (177-182).
The protection it provides against superoxide radicals is optimized by the binding
between the enzyme and heparan sulfate proteoglycans on the surfaces of cells. This binding
also prolongs the physiological half-life of the enzyme (183-185), which is reported to be
about 20 hours in the vasculature (186) and about 85 hours in tissue (187). The C-terminal
may be proteolytically processed during biosynthesis (188, 189), resulting in both intact and
cleaved subunits of ECSOD. Three different subtypes of the enzyme, with different
combinations of subunits, therefore occur: A, with no affinity; B, with intermediate affinity;
and C, with high affinity for heparin/heparan sulfate (124). In the vasculature, ECSOD C
equilibrates between the plasma phase and heparan sulfate in the glycocalyx of the vessel
endothelium and in tissue ECSOD is probably distributed between heparan sulfate on the
surfaces of most cells types in the organs and the interstitial matrix. Since most blood cells do
not bind ECSOD and no binding to E. coli has been demonstrated, ECSOD C has the
potential to protect most normal cells without protecting microorganisms or interfering with
the production of superoxide radicals at the surface of activated neutrophils/macrophages
(176).
Various hypotheses regarding the mechanism whereby ECSOD binds to heparin have
been proposed. In 1996, Oury et al. reported that two heparin-binding domains of ECSOD
are linked by a disulfide bond formed by the Cys219 of the C-terminals, and postulated that
this may be of structural importance for the affinity of ECSOD to heparan sulfate (164).
However, another study found that when alanine was substituted for Cys219 there was no
change in the affinity of the enzyme for heparin (181).
19
BACKGROUND
ECSOD binding to glycosaminoglycans other than heparin and heparan sulfate is weak
(177), although Gao et al. (2008) showed that it protects hyaluronan from oxidative
fragmentation by direct binding (190). ECSOD has also been reported to bind to type I
collagen. The heparin-binding region was found to mediate the interaction with collagen and
bound ECSOD was shown to protect type I collagen against oxidative fragmentation (191).
A naturally occurring mutation in the ECSOD gene (R213G) results in lowered affinities
for both heparin and collagen (192). Because of the reduced amount of bound ECSOD to
heparan sulfate on cell surfaces, the protective effect is lowered, which in turn is a risk factor
associated with cardiovascular disease (193, 194). The lower affinity for collagen may also
increase the risk of oxidative damage of collagen and affect the integrity of the vascular wall (192).
The structure of the middle part of ECSOD was recently determined (171) and the
overall subunit fold was shown to be very similar to the corresponding structures in
CuZnSOD (195). The active site portions of the two enzymes are sequentially and
structurally highly homologous (171, 196), as is the mechanism by which they catalyze the
disproportionation of superoxide (171, 197, 198), which may explain their similar catalytic
properties, as measured by kinetic parameters (124). The structure of the N-and C-terminal
domains of ECSOD remains obscure, since attempts to determine the structure of these parts
of the enzyme have failed, possibly because they are highly flexible. However, earlier
biophysical investigations and secondary predictions have indicated that they both adopt an
α-helical conformation (166, 178), and suggestions have been made that state that the α-
helical conformation of the C-terminal may be induced or stabilized by heparin-binding (180).
The CuZnSOD variants that have been structurally characterized, including ECSOD,
are all β-proteins containing eight antiparallel β strands, arranged in a β-barrel (195). The
strands are mainly connected through hairpin turns in a flattened Greek key motif, but the
enzyme also contains three external loops, which constitute the active site. The construction
of the β-barrel makes the protein very stable (199), and all CuZnSOD enzymes display
marked physical resistance toward high temperature, pH extremes and high urea and
guanidinium chloride concentrations (123, 161).
Due to the high degree of sequence conservation among the CuZnSODs, the amino acid
residues that are ligands to the copper and zinc ions constitute a “fingerprint” that can be used
to identify members of the family. The residues that maintain the geometry of the active site
or are involved in dimer formation are also conserved to a high degree, as are the two
cysteines that form an intramolecular disulfide bridge in the protein. In ECSOD there is a
further disulfide bridge between C219 residues in neighboring subunits, stabilizing the C-
terminal part of the enzyme (164). Petersen and Enghild have also reported the occurrence of
two different disulfide bridge patterns in the human ECSOD subunit, creating one active
20
BACKGROUND
form of subunit and one inactive form (200, 201). The combination of both active and
inactive forms in the assembly of the protein regulates the enzymatic activity (202). The
folding of these two variants has been reported to occur inside the cell, which also may be
important from a regulatory perspective (203).
It has been shown that heparin and heparan sulfate can induce both ECSOD mRNA and
protein expression, and that the sulfation level of these molecules determine the degree of
expression. However, the mechanism behind these findings is not known, and may be on
receptor level or promoter level (204). Various cytokines, such as interferon-γ (IFN-γ) and
interleukin-4 (IL-4) can upregulate the ECSOD expression, possibly via stimulation of
transcription by NF-κB (205). ECSOD expression can also be regulated by some hormones
and a number of oxidizing agents (109).
The biological function of ECSOD
ECSOD plays an important role in regulating blood pressure and vascular contraction, at
least in part through modulating the endothelial function by controlling the levels of
extracellular superoxide anion radicals and nitric oxide bioavailability in the vasculature (206,
207). By regulating the bioavailability of nitric oxide, it also affects smooth muscle relaxation,
neurotransmission, and inflammation. ECSOD has also been proposed to play an important
role in neurologic, pulmonary, and arthritic diseases (78, 152).
ECSOD also modulates oxidant injury, inflammation and fibrosis in a number of lung
diseases. In hyperoxic lung injury ECSOD expression and activity is disrupted, potentially
contributing to oxidative damage and inflammation (208). ECSOD has also been shown to
be involved in protection against oxidation of low-density lipoprotein (LDL) (209, 210),
which is a major contributor to atherosclerosis.
The potential therapeutic use of ECSOD
Oxidative stress has been shown to be involved in many diseases and pathological
conditions, such as cardiovascular diseases (20), cancer (75-77) and aging (82), which affects
a large number of people. Because of the beneficial properties of SOD enzymes against the
toxic radicals that underlie oxidative stress, interest has evolved in the therapeutic use of
SODs. Most work has focused on CuZnSOD (211) and MnSOD (212), since they are
readily produced in large amounts. However, ECSOD may be a better agent to use in
therapeutic interventions, since it has been designed for extracellular function and has many
beneficial properties, such as affinity for heparan sulfate and a long physiological half-life (186, 187). In fact, various animal models have shown its ability to protect the tissue against
oxidative damage (184, 213-215). However, despite strenuous efforts, attempts to produce
recombinant human ECSOD in various prokaryotic or simple eukaryotic systems have failed,
hindering large-scale production of the protein (216, 217). This complicates genetic
21
BACKGROUND
engineering of the enzyme and has also hampered the evaluation of the potential therapeutic
value of ECSOD.
PseudoECSOD
To overcome the limitations of production of ECSOD, a chimera of human extracellular
and intracellular superoxide dismutase has been constructed. This fusion protein is called
PseudoECSOD and comprises the N- and C-terminal domains of human ECSOD fused to
human CuZnSOD (Figure 5). PseudoECSOD can be produced in large quantities in
Escherichia coli and purified with high yields. The characteristics of PseudoECSOD closely
resemble those of human ECSOD (196), which means that this protein may be used as an
ECSOD substitute in further investigations.
Glycosaminoglycans
Glycosaminoglycans (GAGs) are linear polysaccharides, with building blocks
(disaccharides) consisting of an amino sugar and a uronic acid or galactose. The family of
GAGs includes hyaluronan (HA), chondroitin sulfate (CS), dermatan sulfate (DS), keratan
sulfate (KS), heparan sulfate (HS) and heparin. All except heparin and HA are attached to a
core protein to form a proteoglycan in the active form (218).
Among the interactions between proteins and GAGs that have been examined,
complexes of proteins binding to the structurally similar heparan sulfate or heparin molecules
are the most intensively studied, mainly because of the commercial availability of heparin.
However, the biological ligand in most cases is heparan sulfate.
Heparin and heparan sulfate
Heparan sulfate and heparin are both linear polysaccharides consisting of repeating
uronic acid-(1 4)-d-glucosamine disaccharide subunits. The former may be either α-l-
iduronic acid (IdoA) or β-d-glucuronic acid (GlcA), both of which may be O-sulfated. The
β-d-glucosamine (GlcN) may be either N-sulfated (GlcNS) or N-acetylated (GlcNAc) and
both forms may be O-sulfated at various positions (Figure 2) (219). The chemical
composition of heparin and heparan sulfate is very similar, the major differences being that
heparan sulfate has a lower degree of sulfation, a higher degree of GlcNAc residues and a
lower content of epimerized uronic acids (IdoA) (183, 218-220). In addition, heparan sulfate
chains are arranged in different domains based on their sulfate content. Variable patterns of
substitution of the disaccharide subunits with N-sulfate, O-sulfate and N-acetyl groups give
rise to a large number of complex sequences in both heparin and HS, which are structurally
the most complex members of the glycosaminoglycan family of polysaccharides (219). This
heterogeneity considerably complicates the structural determination of heparin-protein
complexes.
22
BACKGROUND
a b c d
Figure 2 Monosaccharide building blocks of heparin and heparan sulfate; a) α-L-Iduronic
acid, b) β-D-Glucuronic acid, c) N-sulfo-α-D-Glucosamine, d) N-acetyl-α-D-Glucosamine
(219).
Heparan sulfate is synthesized by virtually all mammalian cells, while heparin is only
produced by mast cells. During biosynthesis heparin chains are attached to a core protein
called serglycin, which is only found in mast cells and other hematopoietic cells. This
proteoglycan form of heparin, referred to as “macromolecular heparin”, is not secreted by
mast cells. Instead, the heparin chains are cleaved from the core protein at random points
along the chain and stored in non-covalent complexes with basic proteases within the
cytoplasmic secretory granules of the cells. When the contents of the granules are secreted,
free heparin is released (183, 219).
In contrast, heparan sulfate usually occurs as a part of a proteoglycan (PG), and rarely in
free form. There are several families of proteoglycans, each with a different core protein. The
two major subfamilies of cell-surface heparan sulfate proteoglycans (HSPG) are the syndecans
and glypicans. The syndecan core proteins are transmembrane proteins while the glypican
core proteins are attached to cell membranes by a glycosylphosphatidyl-inositol (GPI) anchor.
There are also some subfamilies of HSPG located in the extracellular matrix, e.g. perlecan,
agrin and collagen XVIII (219). HSPGs are continuously produced and secreted from a large
variety of cells and their composition often shows high degrees of tissue-specificity (183).
Heparin-protein interactions
Hundreds of heparin-binding proteins have been identified, many of which play central
roles in important cellular activities and events, e.g. blood coagulation, lipid metabolism and
cell growth. Since the functions of these proteins are often dependent on heparin-binding,
knowledge of the structural motifs involved in this kind of interaction is essential for a
molecular understanding of these processes (183).
23
BACKGROUND
Attempts to fully characterize the nature of protein-heparin interactions have been
hampered by the heterogeneity of heparin and heparan sulfate, and a lack of structural
information regarding the heparin-binding proteins. Many interactions have been described,
but the amino acid and oligosaccharide residues that mediate the interactions have not been
defined for all of them and X-ray structures have only been obtained for a few heparin-
protein complexes (221), e.g. basic fibroblast growth factor (222), antithrombin (223, 224),
acidic fibroblast growth factor (225), RANTES (226), eosinophil-granule major basic protein
(EMBP) (227) and annexin A2 (228).
The binding of proteins by HS and heparin is primarily electrostatic, involving positively
charged groups of the amino acids and negatively charged groups of the polysaccharide.
However, there are indications that specific sequences in the GAGs are involved in selective
interactions with certain proteins, and that these interactions modulate the protein activities
(219).
Heparin and heparan sulfate binding sites have been found within both α-helical and β-
sheet frameworks. Several attempts have been made to identify consensus sequences for
heparin binding in the primary sequences of the proteins (183), but none of the published
sequences are valid for all cases. However, there are often clusters of basic amino acids, e.g.
lysine and arginine, in heparin binding regions. In 1993, Margalit et al. proposed that the
common structural “motif” amongst heparin-binding proteins may be two basic amino acids,
separated by a distance of approximately 20 Å in the 3D structure and facing opposite
directions, regardless of the secondary or tertiary protein structure. However, this hypothesis
has not yet been verified by structural evidence (229).
24
AIMS
AIMS
The general aim of the studies underlying this doctoral thesis was to investigate the
requirements for high affinity binding between proteins and heparin/heparan sulfate. The
main specific objectives were to characterize the structural requirements and limitations for
binding and to identify the amino acids that are of critical importance for the interaction.
Since ECSOD has an important biological function that may be utilized therapeutically, I
have characterized this enzyme further by using derivatives in the form of fusion proteins and
mutant proteins to gain both basic knowledge, and understanding that is interesting from a
medical perspective. The investigations included:
• Detailed thermodynamic characterization of the interactions between the C-terminal
domain of ECSOD and heparin/defined heparin fragments, using isothermal titration
calorimetry (ITC). The measurements included recombinant human ECSOD, fusion
proteins and mutant proteins under various experimental conditions (Paper I and II).
• Development of a production system, utilizing a copper chaperone that incorporates
copper into the active sites of copper-containing SOD proteins. Since properly
incorporated copper is critical for the activity of these SOD enzymes, the production
system will be used in the future production of all CuZnSOD variants including the
potential therapeutic agent PseudoECSOD (Paper III).
• Analyses of the binding of PseudoECSOD to heparan sulfate on cell surfaces by in
vitro immunological techniques, and investigation of the protective properties of
PseudoECSOD against oxidative stress in an ex vivo heart model, with the
perspective to develop an artificial free radical scavenger that could be used for
purposes such as the preservation of transplanted organs (Paper IV).
25
MAIN METHODS AND PROTEIN CONSTRUCTS
MAIN METHODS AND PROTEIN CONSTRUCTS
The methods that are central in the studies that this doctoral thesis is based upon are
briefly described below.
Isothermal Titration Calorimetry
Isothermal titration calorimetry (ITC) is a technique that is used to measure the
thermodynamic parameters of interactions between molecules, based on the fact that every
reaction involves the generation or absorption of heat. The method does not require any
labeling or immobilization of the analytes (230) and is the only method available for direct
measurement of the heat change during complex formation at constant temperature (231).
Since a single experiment can provide estimates of all the key thermodynamic variables of
reactions, ITC has emerged as the primary tool for characterizing interactions in terms of
thermodynamic parameters, elucidating binding mechanisms and, in combination with
analyses of relationships between the structure and function of compounds, rational drug
design. ITC can be used for numerous applications, e.g. binding studies of antibody-antigen,
protein-peptide, DNA-protein, enzyme-inhibitor, enzyme-substrate, carbohydrate-protein,
protein-protein interactions as well as enzyme kinetics (231).
In an ITC experiment, a solution of a macromolecule of interest is placed in the sample
cell of the calorimeter, while the reference cell is filled with water or blank buffer. Before the
experiment starts, the temperature of the two cells is equilibrated and the system tends to
maintain this balance. A ligand of interest is then injected into the sample cell, and upon
interaction with the macromolecule heat is absorbed or released. Depending on the nature of
the association, the system will increase or decrease the power supplied to the sample cell in
order to keep it at the same temperature as the reference cell. This change in energy
consumption is monitored, yielding a binding isotherm, which provides estimates of the
binding constant, the enthalpy and entropy changes associated with the binding and the
stoichiometry of the interaction (230, 232). Moreover, ITC experiments performed at
different temperatures yield estimates of the constant pressure heat capacity change (ΔCp)
(231).
In the ITC experiments performed in the studies underlying this thesis (Paper I and II), a
MicroCal VP-ITC microcalorimeter (MicroCal, Northampton, USA) was used. Each
protein solution to be tested was placed in the sample cell, and the heparin/oligosaccharide
solution was loaded into the syringe injector. The acquired data were then fitted to a
theoretical titration curve, with ΔH (enthalpy change, kcal/mol), Kb (binding constant, M-1),
and n (number of binding sites/monomer) as adjustable parameters, by the Origin 5.0 non-
linear least squares program implemented in the software supplied by Microcal. A typical
binding isotherm is shown in Figure 3.
26
MAIN METHODS AND PROTEIN CONSTRUCTS
Figure 3 Binding isotherm for the interaction of ECSOD with heparin. In the top panel, the
major advantage of ITC is that instead of one of the analytes being immobilized, as in
oth
Phage Display
lay is a technique in which proteins or peptides of interest are expressed on the
surf
peaks indicate the heat released after each addition of heparin into the protein solution. In the
bottom panel, the integrated peak area is plotted as a function of molar ratio
(heparin/ECSOD). The line represents the best fit of the ITC data, which was used to calculate
the thermodynamic parameters.
A
er methods such as SPR, both of the molecules participating in the measured interaction
are in solution, hence more reliable data are obtained since this more closely resembles the in
vivo situation. The method also has high sensitivity, since only 10-100 nanomols of reactants
are needed (231).
Phage disp
ace of a bacteriophage by fusing them to the coat proteins of the phage. By using
recombinant DNA technology and random mutagenesis a “library” of phages can be
generated, each displaying a unique random peptide. The peptides of the library can then be
screened against a ligand of interest by affinity purification. Since phage display provides a
direct link between phenotype and genotype, DNA sequencing of the binding phages allows
the molecules of interest to be readily identified. The selected phages can then be amplified
and purified for further analyses (233).
27
MAIN METHODS AND PROTEIN CONSTRUCTS
In the phage display analysis performed in Paper II, a phage display system
(T7Select®Phage Display System, Novagen), based on the bacteriophage T7 and E. coli cells
(BLT5403), was used. A population of DNA fragments encoding the peptide sequences was
cloned into the T7Select vector, packaged into phages, and amplified. To find the most
efficient heparin-binding phages, several rounds of biopanning against Heparin Sepharose,
with increased salt concentration as the selective factor, were performed. Isolated phages were
then amplified and their DNA was analyzed by sequencing across the cloning region.
Since the C-terminal of ECSOD has an α-helical structure, the design of the peptides
was based on an optimized helical background, dominated by alanine residues to maintain
helical structure (Figure 4). A limited set of mutations at randomized positions, approximately
20 Å away from a fixed arginine residue, was introduced to test a hypothesis regarding
structural requirements for heparin-binding proposed by Margalit et al. 1993 (229).
N-AARAAAAQAAAQAXXXXXXAAAQ-C
Figure 4 Design of the peptides of the phage display library. The varied amino acids are
indicated by X and the fixed arginine residue in marked.
Ischemia-reperfusion model
To determine the protective properties of PseudoECSOD against ischemia-reperfusion
damage, an isolated rabbit heart model was used, which previously has been used to study the
effects of various potentially protective agents against ischemia-reperfusion injury (234). The
Animal Care and Use Committee in USA gave ethical permission for the animal studies
(Animal Protocol # 33901903( )1A, title: “Optimizing the Pharmacological Properties of
Superoxide Dismutase.”), and hearts from 14 rabbits were used.
The ischemia-reperfusion experiments were performed essentially as described by Nelson
et al. (234). Hearts were mounted through the ascending aorta in a non-recirculating
perfusion apparatus and perfused under 80 mmHg with oxygenated, glucose-containing
Krebs-Henseleit buffer. After 15 minutes of equilibration the hearts were subjected to 60
minutes of global ischemia followed by 45 minutes of reperfusion. The reperfusion solution
contained PseudoECSOD at three different concentrations (200, 300 and 450 U/L), and
control experiments were performed in which no exogenous SOD was added to the
reperfusion solution. Results were expressed as percent recovery of the developed tension. At
the end of the experiments grafts of the hearts were stored at -70°C for further analysis.
28
MAIN METHODS AND PROTEIN CONSTRUCTS
Protein Constructs
In the studies this thesis is based upon, various fusion proteins are used. The wild type
ECSOD protein has been included in the investigations as well as PseudoECSOD, which is a
fusion protein of the N-and C-terminal domains of human ECSOD fused to the N- and C-
terminal of human CuZnSOD, respectively. The binding properties of the fusion protein
FusCC, which comprises the protein human carbonic anhydrase II (HCA II) fused to the C-
terminal domain of human ECSOD, has also been determined. Figure 5 describes the
different protein constructs.
A
B
C
D
E
Figure 5 Schematic view of the protein constructs used in the different studies.
A) Human CuZnSOD, B) Human ECSOD, C) PseudoECSOD, D) HCA II, E) FusCC
29
30
SUMMARIZING DISCUSSION
SUMMARIZING DISCUSSION
Accumulating evidence indicates that oxygen free radicals are involved in many
pathological conditions and diseases such as aging (235, 236) reperfusion damage of ischemic
tissue (237), atherosclerosis (238) and various inflammatory conditions (239). Extracellular
superoxide dismutase thus plays a very important role in the maintenance of cells by
scavenging superoxide radicals in the extracellular matrix. Various animal studies have shown
that ECSOD protects against various pathological cardiovascular conditions such as
ischemia-reperfusion injuries (184, 213-215, 240, 241), myocardial infarction (242),
myocardial stunning (243) and oxidation of low-density lipoprotein (LDL) (209, 210), which
is a major contributor to the development of atherosclerosis.
In recent decades there has been increasing interest in the use of SOD enzymes as
therapeutic agents, and this has been explored in several investigations, largely focusing on
native CuZnSOD (211) and MnSOD (212) or chimeras of these enzymes (234, 244) since
they are easy to produce. However, ECSOD has other attractive and beneficial features for
therapeutic applications, for example its strong affinity for heparan sulfate proteoglycans on
cell surfaces and the related glycosaminoglycan heparin, which is mediated through
interactions with the C-terminal part of the enzyme. This property is an therapeutically
advantage of ECSOD, compared to other SOD enzymes, since the association to heparan
sulfate on cell surfaces increases the physiological half-life of the enzyme and facilitates
optimal protection of the cell surfaces (183-185).
ECSOD is heterogeneous in the vasculature with regard to heparin-binding and can be
divided into three subtypes: A, B and C, which have no affinity, intermediate affinity and
strong affinity, respectively. Most tissue ECSOD occurs as type C, and exists in equilibrium
between the plasma phase and endothelial cell surfaces. Since the activity of the enzyme is not
related to its affinity, type A and B have a protective function in plasma, while type C protects
the cellular surfaces.
The differences between the subtypes are due to limited proteolysis of the heparin-
binding domain, and are proposed to be of regulatory function (188, 189). The endoprotease
furin has been suggested to be responsible for the first part of this processing, by cleaving the
C-terminal domain at position R213, but an additional, as yet unidentified, carboxypeptidase
is required to remove the remaining basic amino acids (R210-K212) (245).
Previous studies of the heparin-binding properties of ECSOD have shown that the
positively charged amino acids in the C-terminal domain of the enzyme contribute to the
binding (178, 180), and that the arginine residues may be more important than the lysine
residues (179). A naturally occurring mutation in the C-terminal domain (R213G) reduces
the affinity of the protein for both heparin and collagen (192), and thus increases the plasma
concentration 10- to 30-fold (193, 194, 246). However, in this case the effect may rather be a
31
SUMMARIZING DISCUSSION
consequence of disruption of the α-helical structure than to importance of the positive charge.
ECSOD harboring this R213G mutation has also been shown to be less susceptible than the
wild type protein to proteolysis.
In order to use derivatives of ECSOD as therapeutic agents, more information regarding
its association with heparan sulfate on cell surfaces must be obtained. Therefore, in the study
reported in Paper I, ITC was used to perform a detailed characterization of the interactions
between the C-terminal domain of human ECSOD, which is rich in positively charged
amino acids, and heparin, which is structurally very similar to the highly negatively charged
heparan sulfate. The binding involves ionic interactions, but the acquired results show that
non-ionic interactions make substantial contributions to the binding affinity (Paper I).
Similar findings regarding the importance of non-ionic interactions have been reported for
other heparin-binding proteins (247-250). In the study described in Paper II corresponding
analyses were performed with the fusion protein FusCC, which consists of the well-
characterized protein human carbonic anhydrase II (HCA II) fused to the C-terminal domain
of human ECSOD (178) (Figure 5). This protein was used to elucidate the specific roles of
the amino acids in the C-terminal domain in the interaction with heparin, with no
interference from potential contributions from other parts of ECSOD to the interaction. The
interaction between heparin and the fusion protein PseudoECSOD, which comprises the N-
and C-terminal of human ECSOD fused to human CuZnSOD was also analyzed (196)
(Figure 5).
Isothermal titration calorimetry is a very convenient method since it provides information
about the thermodynamic parameters of investigated interactions. As mentioned above, in
addition to the binding constant, it also provides estimates of the enthalpy change, the
entropy change and the stoichiometry of the interaction. Further, by conducting the analyses
under various experimental conditions, such as buffer system, salt concentration and
temperature, other valuable information regarding the interaction, such as proton transfer
parameters, ionic and non-ionic contributions and the constant pressure heat capacity change
(ΔCp) were obtained.
The analyses performed at different salt concentrations indicated that 2.8 purely ionic
interactions are formed between ECSOD and heparin. A similar value (2.6) was obtained for
FusCC, indicating that the ionic components of the binding of these two proteins to heparin
are virtually identical. In both cases, the non-ionic component of the binding is substantial,
and very similar, and 2.3 and 2.1 sodium ions were released from ECSOD and FusCC,
respectively, upon binding. The similarities of these results strongly indicate that the attractive
interactions between heparin and ECSOD are confined solely to the C-terminal domain.
By performing the analyses in different buffer systems, information regarding protonation
events upon binding was obtained. For the ECSOD-heparin interaction, a value of 0.4
transferred protons was obtained, similar to the value (0.2) calculated for the binding between
32
SUMMARIZING DISCUSSION
FusCC and heparin. The positive sign of the values indicate that protons are transferred from
the buffer to the binding complex upon binding. These findings also indicate that the binding
modes of the two interactions are similar.
The only amino acid in the C-terminal domain of ECSOD capable of accepting a proton
at the experimental pH (7.4) is histidine H207, which may have a pKa near this value and
thus should be a good candidate for this assignment. The values obtained indicate that the
pKa of histidine is increased upon binding. Hence, in order to accept a proton, the resulting
positive charge of the imidazole group of histidine has to be stabilized. Since the proton
transfer occurs upon binding, the negative charges in heparin are probably responsible for this
stabilization.
A positive entropy change (ΔS) of an interaction, which could result from release of ions
or hydrated water molecules upon binding, contributes to the overall free energy change (ΔG)
in a favorable way. Positive ΔS values were obtained when ECSOD interacted with small
oligosaccharides derived from heparin, but not in the binding to long-chain heparin. This
could be due to a large negative entropy contribution associated with more degrees of freedom
being lost in heparin upon binding than for the smaller and relatively rigid oligosaccharides.
For heparin, this effect apparently exceeds the effect of the sodium ion release.
Since the short oligosaccharides bind more strongly (Kd = 0.21-0.24 μM) than heparin
(Kd =1.42 μM), it is tempting to speculate that a more ordered structure in heparin is induced
by unspecific and non-productive interactions with the protein, outside the primary binding
site, upon complex formation. This would also be consistent with the observation of a smaller
ΔCp value for the octasaccharide, which may indicate that a larger surface area is hidden in
the interaction between ECSOD and heparin than in its binding to the octasaccharide. The
observation that the enthalpy change (ΔH) of the binding is more negative for the interaction
of ECSOD with heparin than with the octasaccharide, indicative of more contacts being
made, together with the fact that the binding affinity is not increased, also supports the
suggestion that non-productive interactions occur between heparin and the central part of
ECSOD. These unproductive interactions might occur within a groove of the central part of
ECSOD, which according to molecular docking studies has been shown to be able to harbour
heparin (171). The fact that the obtained stoichiometry of the binding is smaller than
expected may be due to some binding sites in the heparin molecule being shielded from
productive binding due to the large size of the ECSOD molecule.
The interactions between FusCC and the oligosaccharide or heparin both showed
positive entropy changes upon binding and similar binding constants. Again this supports the
conclusion that the interactions are exclusively made between the C-terminal domain of
ECSOD and the saccharides.
The analysis of the binding between the octasaccharide and ECSOD at different salt
concentrations showed that 3.6 sodium ions were released upon binding and that 4.5 ionic
33
SUMMARIZING DISCUSSION
interactions were formed between the two molecules. The free energy of interaction at 1 M
NaCl differs only by 1.9 kJ/mol between heparin and the octasaccharide, indicating that the
contributions from non-ionic interactions to their binding are very similar. The observation
that the binding of the octasaccharide involves additional ionic interactions compared to the
binding to heparin can be explained by differences between them in charge density and the
distribution of negative charges, which allow formation of additional ion pairs, or by the small
size of the octasaccharide, which may allow conformational rearrangements that favor the
formation of more ionic bonds. The affinity of ECSOD for the octasaccharide is stronger
than for heparin, which may be due to the additional ionic interactions or, alternatively, it
may be coupled to an entropic contribution in contrast to the entropic cost observed for
heparin. It is possible that the purification procedure for the heparin fragments has led to an
enrichment of fragments that have a higher charge density than the average heparin molecule.
Since heparin was the source of the oligosaccharide fragments, long-chain heparin must
contain regions with corresponding binding affinity. However, attempts to resolve distinct
binding sites with different affinities in heparin failed, probably because the differences are
too small to be resolved by ITC.
The analyses at different temperatures provided information about the ΔCp associated
with the binding, which may also be correlated to the change in solvent accessible surface
area. In the case of ECSOD binding to heparin a ΔCp of -644 J K-1 mol-1 was calculated,
while the value calculated for the interaction between ECSOD and a heparin octasaccharide
was -306 J K-1 mol-1. Both of these values are negative, which is common for interactions
between proteins and ligands involving specific hydrophobic binding (251-253), exposure of
polar groups (254), dehydration, cavity formation and/or a change in water ordering (255,
256). These results are consistent with the results obtained from the analyses at different salt
concentrations, which showed that there is a substantial contribution to binding from non-
ionic interactions. These interactions might increase the specificity of the binding, following
initial electrostatic attraction between the positive amino acids in the C-terminal and the
negative groups of the glycosaminoglycan.
The smaller ΔCp value obtained for the saccharide supports the hypothesis that ECSOD
forms interactions with heparin beyond these involved in the binding, thus burying more
surface area. The ΔCp value obtained for the ECSOD-heparin interaction should also be
compared to the value calculated for the binding between FusCC and heparin (+611 J K-1
mol-1). In this case the ΔCp was positive, indicative of electrostatic interaction (257), charge
neutralization (258) and exposure of non-polar groups upon binding (254). Since the ionic
component of the interaction and the protonation event in ECSOD and FusCC binding are
similar, the difference in ΔCp is probably due to differences in the non-ionic component.
These results supports the suggestion that long chain heparin inevitably forms non-productive
interactions with the central domain of ECSOD.
34
SUMMARIZING DISCUSSION
Investigations of a set of mutant proteins, based on the structure of FusCC, showed that
residues R210, K211 and R214 in the human ECSOD sequence are important for heparin
binding since the binding affinities for heparin were 2-4 fold lower when these amino acids
were replaced with alanine. These three amino acids may be the ones involved in the ionic
interactions detected in the ITC measurements at different salt concentrations. Since
structural data of the C-terminal domain is incomplete, definitive evidence regarding the
specific amino acids that interact with heparin is not available. However, earlier studies also
indicated that the positive amino acids in the RK-cluster (amino acids 210-215) are important
for binding to heparin (178-180).
Interestingly, when the human ECSOD C-terminal was visualized in an α-helical wheel,
the R210, K211 and R214 residues were found at one side of the helix, and R202 faced the
opposite direction. This supports the hypothesis made by Margalit et al. in 1993, which
postulates that a common structural motif for heparin-binding proteins may be two positive
amino acids at a distance of approximately 20 Å in the 3D-structure, facing opposite sides of
the α-helix. This putative binding area also contains the residue H207, the binding properties
of which have been shown to change upon heparin binding.
In order to further investigate the binding properties of ECSOD, a phage display library
based on an α-helical structure was constructed. To find the most efficient heparin binders,
the library was screened against Heparin Sepharose with increasing salt concentration as the
selective factor. Results from these studies indicate that position R214 in the human ECSOD
sequence may play an important role in heparin binding, since 57 % of the selected phages
that were sequenced had an arginine in a position that corresponds to R214 in human
ECSOD. These results support the findings from the ITC measurements, indicating that this
amino acid is of importance for strong affinity for heparin.
Since ECSOD is difficult to produce in large scale, a mimic of the enzyme called
PseudoECSOD, which closely resembles ECSOD in structural, enzymatic and heparin-
binding properties, has previously been constructed. This enzyme is easily produced in large
amounts and purified with high yield in E. coli (196). In the study described in Paper IV the
binding between PseudoECSOD and heparan sulfate on cell surfaces of human umbilical
cord vessels was investigated. The binding capability of the human cell line HepG2 was also
determined by immunological techniques. The results clearly show that PseudoECSOD
binds to the cell surfaces of both the umbilical cord tissue and the HepG2 cell line, in a
similar way to recombinant human ECSOD (Paper IV).
The use of PseudoECSOD as a substitute for ECSOD in therapeutic situations also
requires detailed investigations of its actions in vivo. The copper content of proteins
belonging to the CuZnSOD family is critical for optimal enzymatic activity. However, earlier
attempts to produce the proteins, including PseudoECSOD, in various expression systems
seldom yielded fully metalized end products, despite addition of supplementary copper to the
35
SUMMARIZING DISCUSSION
growth medium. This could be due to the fact that high copper concentrations are toxic to
cells and mechanisms have evolved which reduce their copper contents. Hence, in yeast and
mammals, copper-trafficking systems and chaperones are needed for the proper incorporation
of copper into proteins (152). Thus, in order to overproduce CuZnSOD variants in E. coli a
production system was developed in which a yeast copper chaperone (yCCS) is coexpressed
with the enzymes to incorporate copper into the active sites. The resulting proteins, including
PseudoECSOD, have full copper contents (Paper III).
Finally, the protective properties of PseudoECSOD against ischemia-reperfusion injuries
in an isolated rabbit heart model were investigated. The results indicate that the enzyme has
protective abilities in vivo (Paper IV).
36
CONCLUSIONS
CONCLUSIONS
The studies this doctoral thesis is based upon have shown that ITC is an excellent
technique for thermodynamic investigations of protein-carbohydrate interactions. Future
investigations may include ITC analyses of the interactions between ECSOD, or other
proteins of interest, with heparan sulfate, which is the biological ligand for ECSOD.
However, the ITC measurements have provided important new findings about the interaction
between ECSOD and heparin, including the following:
• Although the ionic interactions are important in the interaction between ECSOD
and heparin, the contribution of non-ionic interactions to the interaction is
substantial, and may provide binding specificity.
• Residues R210, K211 and R214 seem to be very important for the binding,
accounting for about 30 % of the total binding energy. The importance of these
amino acids was confirmed by results from both the ITC measurements and the
phage display analysis. The structural placement of these residues in an α-helix,
also supports the hypothesis made by Margalit et al.
• The C-terminal domain is the only part of ECSOD that contributes to the
productive heparin-binding.
• The ITC experiments have also confirmed that FusCC is a good model protein to
use in studies of binding by ECSOD, since the results clearly show that the only
interactions made between FusCC and heparin are those with the C-terminal.
In addition, a system that can be used to produce CuZnSOD enzymes with properly
incorporated copper has been developed. This production system, which yields fully metalized
proteins, will be of great importance for studies of CuZnSOD variants, especially those with
reduced stability in their apo state such as ALS-associated CuZnSOD mutants.
In summary, the results further support the proposition that PseudoECSOD may be a
good substitute for ECSOD in therapeutic situations, since it seems to bind heparan sulfate
on surfaces of HepG2 cells and, especially, endothelial cells in a very similar manner to
ECSOD. The ITC measurements indicate that the binding affinities of PseudoECSOD and
ECSOD to heparin are similar. Together with earlier results, showing the close resemblance
of PseudoECSOD to ECSOD in structural, enzymatic and heparin-binding properties, the
findings in these studies indicate that PseudoECSOD may be a promising molecule to use in
therapeutic interventions. However, although the protective properties of PseudoECSOD
against ischemia-reperfusion damage were confirmed, more experiments using the rabbit
heart and other animal models are needed to identify the optimal dose. The protective
properties of PseudoECSOD in human tissue should also be thoroughly investigated.
37
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS
This thesis has been accomplished through hard work by me, but also through the help, big
or small, from different persons in my surroundings. In particular, I would like to thank:
Lena Tibell, my main supervisor, for giving me the opportunity to perform my doctoral
studies in her small research group. It has not always been easy, but you never gave up hope
on me.
Bengt-Harald “Nalle” Jonsson, my cosupervisor, for interesting discussions and for always
being helpful.
Louise Levander, my colleague at the office and in the laboratory, for always being friendly
and helpful, for listening to my problems and for making me realize that it is possible to fulfill
a doctoral thesis despite lack of results.
Johan Olausson, my other laboratory colleague, for making the everyday life much easier
with your sense of humor and never-ending stories.
Anna Berg, my office-mate that gave the never-ending student as face.
All the other persons at Division of Cell Biology, level 10, present now or who has left the
building for other missions, for the chats at the coffee breaks about everything in life.
Especially I would like to thank Lena Cedergren and Anna Lardfelt in the facility
compartment for always being helpful with my laboratory issues. You make a great work!
Karin Enander and Peter Stenlund for helping me with the SPR measurements, even
though the results were not as good as expected.
Laila Villebeck and Sofia Håkansson for help during my time the Department of Physics,
Chemistry and Biology and for our nice stay in Italy.
All other persons at IFM that I had contact with both during my undergraduate and doctoral
studies.
Sally Nelson, Swapan Bose and all the others in Denver, for making my visit in America so
great.
38
ACKNOWLEDGMENTS
Luminita Damian at MicroCal for your help with the ITC-measurements and answers to
my never-ending questions, both during the course and afterward.
All of the participant at the Free Radical Summer School in Spetses, Greece, especially my
room-mate Laura Vera.
Jenny Larsson, Mirjam Granat, Gabriella Klockare and Jessica Frisk, my colleagues
during different parts of my education. I hope we can stay in touch.
The people engaged in SDok, SFS-dk and Eurodoc, for interesting discussions, and for
making think of something else than research.
My family, you were always interested in my research, even if you never understood what I
was doing.
Magnus, my partner in life. Thank you for putting up with me during hard times when the
house was a mess and when I came home at 4 o’clock in the morning after a long working
day. I love you!
Jesper and Fanny, my lovely kids, you show me the meaning of life.
39
40
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