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, Sweden

COSUPERVISOR

Bengt-Harald “Nalle” Jonsson, Professor

Department of Physics, Chemistry and Biology


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



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



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

1

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

2

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.

3

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.

4

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

5

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

6

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

7

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).

8

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

9

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

11

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.

12

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


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


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


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


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

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


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


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


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


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


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

REFERENCES

REFERENCES

1. Sessions, A. L., Doughty, D. M., Welander, P. V., Summons, R. E., and Newman,

D. K. (2009) The continuing puzzle of the great oxidation event, Curr Biol 19, R567-

574.

2. Martinez-Cayuela, M. (1995) Oxygen free radicals and human disease, Biochimie 77,

147-161.

3. Freeman, B. A., and Crapo, J. D. (1982) Biology of disease: free radicals and tissue

injury, Lab Invest 47, 412-426.

4. Leung, P. S., and Chan, Y. C. (2009) Role of oxidative stress in pancreatic

inflammation, Antioxid Redox Signal 11, 135-165.

5. Limon-Pacheco, J., and Gonsebatt, M. E. (2009) The role of antioxidants and

antioxidant-related enzymes in protective responses to environmentally induced

oxidative stress, Mutat Res 674, 137-147.

6. Okano, H. (2008) Effects of static magnetic fields in biology: role of free radicals,

Front Biosci 13, 6106-6125.

7. Csiszar, A., Podlutsky, A., Wolin, M. S., Losonczy, G., Pacher, P., and Ungvari, Z.

(2009) Oxidative stress and accelerated vascular aging: implications for cigarette

smoking, Front Biosci 14, 3128-3144.

8. Kehrer, J. P., Mossman, B. T., Sevanian, A., Trush, M. A., and Smith, M. T. (1988)

Free radical mechanisms in chemical pathogenesis. Summary of the symposium

presented at the 1988 annual meeting of the Society of Toxicology, Toxicol Appl

Pharmacol 95, 349-362.

9. Vallyathan, V., Shi, X. L., Dalal, N. S., Irr, W., and Castranova, V. (1988)

Generation of free radicals from freshly fractured silica dust. Potential role in acute

silica-induced lung injury, Am Rev Respir Dis 138, 1213-1219.

10. Mustafa, M. G. (1990) Biochemical basis of ozone toxicity, Free Radic Biol Med 9,

245-265.

11. Pryor, W. A. (1994) Mechanisms of radical formation from reactions of ozone with

target molecules in the lung, Free Radic Biol Med 17, 451-465.

12. Touyz, R. M., and Schiffrin, E. L. (2004) Reactive oxygen species in vascular biology:

implications in hypertension, Histochem Cell Biol 122, 339-352.

13. Liu, Y., Fiskum, G., and Schubert, D. (2002) Generation of reactive oxygen species by

the mitochondrial electron transport chain, J Neurochem 80, 780-787.

41

REFERENCES

14. Loschen, G., Flohe, L., and Chance, B. (1971) Respiratory chain linked H(2)O(2)

production in pigeon heart mitochondria, FEBS Lett 18, 261-264.

15. Boveris, A., Oshino, N., and Chance, B. (1972) The cellular production of hydrogen

peroxide, Biochem J 128, 617-630.

16. Chance, B., Sies, H., and Boveris, A. (1979) Hydroperoxide metabolism in

mammalian organs, Physiol Rev 59, 527-605.

17. Miller, G., Suzuki, N., Ciftci-Yilmaz, S., and Mittler, R. (2009) Reactive oxygen

species homeostasis and signalling during drought and salinity stresses, Plant Cell

Environ.

18. Slesak, I., Libik, M., Karpinska, B., Karpinski, S., and Miszalski, Z. (2007) The role

of hydrogen peroxide in regulation of plant metabolism and cellular signalling in

response to environmental stresses, Acta Biochim Pol 54, 39-50.

19. Fang, F. C. (2004) Antimicrobial reactive oxygen and nitrogen species: concepts and

controversies, Nat Rev Microbiol 2, 820-832.

20. Madamanchi, N. R., Vendrov, A., and Runge, M. S. (2005) Oxidative stress and

vascular disease, Arterioscler Thromb Vasc Biol 25, 29-38.

21. Hampton, M. B., Kettle, A. J., and Winterbourn, C. C. (1998) Inside the neutrophil

phagosome: oxidants, myeloperoxidase, and bacterial killing, Blood 92, 3007-3017.

22. Decoursey, T. E., and Ligeti, E. (2005) Regulation and termination of NADPH

oxidase activity, Cell Mol Life Sci 62, 2173-2193.

23. Gwinn, M. R., and Vallyathan, V. (2006) Respiratory burst: role in signal

transduction in alveolar macrophages, J Toxicol Environ Health B Crit Rev 9, 27-39.

24. Munzel, T., Hink, U., Heitzer, T., and Meinertz, T. (1999) Role for

NADPH/NADH oxidase in the modulation of vascular tone, Ann N Y Acad Sci 874,

386-400.

25. De Keulenaer, G. W., Chappell, D. C., Ishizaka, N., Nerem, R. M., Alexander, R.

W., and Griendling, K. K. (1998) Oscillatory and steady laminar shear stress

differentially affect human endothelial redox state: role of a superoxide-producing

NADH oxidase, Circ Res 82, 1094-1101.

26. Lambeth, J. D. (2004) NOX enzymes and the biology of reactive oxygen, Nat Rev

Immunol 4, 181-189.

27. Klebanoff, S. J. (1980) Oxygen metabolism and the toxic properties of phagocytes,

Ann Intern Med 93, 480-489.

42

REFERENCES

28. Chanock, S. J., el Benna, J., Smith, R. M., and Babior, B. M. (1994) The respiratory

burst oxidase, J Biol Chem 269, 24519-24522.

29. Droge, W. (2002) Free radicals in the physiological control of cell function, Physiol

Rev 82, 47-95.

30. Muralikrishna Adibhatla, R., and Hatcher, J. F. (2006) Phospholipase A2, reactive

oxygen species, and lipid peroxidation in cerebral ischemia, Free Radic Biol Med 40,

376-387.

31. Adibhatla, R. M., Hatcher, J. F., and Dempsey, R. J. (2003) Phospholipase A2,

hydroxyl radicals, and lipid peroxidation in transient cerebral ischemia, Antioxid Redox

Signal 5, 647-654.

32. Misra, H. P., and Fridovich, I. (1972) The role of superoxide anion in the

autoxidation of epinephrine and a simple assay for superoxide dismutase, J Biol Chem

247, 3170-3175.

33. Misra, H. P., and Fridovich, I. (1972) The generation of superoxide radical during the

autoxidation of hemoglobin, J Biol Chem 247, 6960-6962.

34. Winterbourn, C. C., McGrath, B. M., and Carrell, R. W. (1976) Reactions involving

superoxide and normal and unstable haemoglobins, Biochem J 155, 493-502.

35. Wallace, W. J., Houtchens, R. A., Maxwell, J. C., and Caughey, W. S. (1982)

Mechanism of autooxidation for hemoglobins and myoglobins. Promotion of

superoxide production by protons and anions, J Biol Chem 257, 4966-4977.

36. Brantley, R. E., Jr., Smerdon, S. J., Wilkinson, A. J., Singleton, E. W., and Olson, J.

S. (1993) The mechanism of autooxidation of myoglobin, J Biol Chem 268, 6995-

7010.

37. Watanabe, N., and Forman, H. J. (2003) Autoxidation of extracellular hydroquinones

is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells,

Arch Biochem Biophys 411, 145-157.

38. Ballou, D., Palmer, G., and Massey, V. (1969) Direct demonstration of superoxide

anion production during the oxidation of reduced flavin and of its catalytic

decomposition by erythrocuprein, Biochem Biophys Res Commun 36, 898-904.

39. Misra, H. P. (1974) Generation of superoxide free radical during the autoxidation of

thiols, J Biol Chem 249, 2151-2155.

40. Torti, F. M., and Torti, S. V. (2002) Regulation of ferritin genes and protein, Blood

99, 3505-3516.

43

REFERENCES

41. Meneghini, R. (1997) Iron homeostasis, oxidative stress, and DNA damage, Free

Radic Biol Med 23, 783-792.

42. Crichton, R. R., Wilmet, S., Legssyer, R., and Ward, R. J. (2002) Molecular and

cellular mechanisms of iron homeostasis and toxicity in mammalian cells, J Inorg

Biochem 91, 9-18.

43. Neyens, E., and Baeyens, J. (2003) A review of classic Fenton's peroxidation as an

advanced oxidation technique, J Hazard Mater 98, 33-50.

44. Harrison, P. M., and Arosio, P. (1996) The ferritins: molecular properties, iron

storage function and cellular regulation, Biochim Biophys Acta 1275, 161-203.

45. McCord, J. M. (1996) Effects of positive iron status at a cellular level, Nutr Rev 54,

85-88.

46. Kehrer, J. P. (2000) The Haber-Weiss reaction and mechanisms of toxicity, Toxicology

149, 43-50.

47. Halliwell, B., and Gutteridge, J. M. (1984) Oxygen toxicity, oxygen radicals,

transition metals and disease, Biochem J 219, 1-14.

48. Han, D., Antunes, F., Canali, R., Rettori, D., and Cadenas, E. (2003) Voltage-

dependent anion channels control the release of the superoxide anion from

mitochondria to cytosol, J Biol Chem 278, 5557-5563.

49. Vanden Hoek, T. L., Becker, L. B., Shao, Z., Li, C., and Schumacker, P. T. (1998)

Reactive oxygen species released from mitochondria during brief hypoxia induce

preconditioning in cardiomyocytes, J Biol Chem 273, 18092-18098.

50. Lemasters, J. J. (2007) Modulation of mitochondrial membrane permeability in

pathogenesis, autophagy and control of metabolism, J Gastroenterol Hepatol 22 Suppl 1,

S31-37.

51. Lynch, R. E., and Fridovich, I. (1978) Permeation of the erythrocyte stroma by

superoxide radical, J Biol Chem 253, 4697-4699.

52. Hawkins, B. J., Madesh, M., Kirkpatrick, C. J., and Fisher, A. B. (2007) Superoxide

flux in endothelial cells via the chloride channel-3 mediates intracellular signaling, Mol

Biol Cell 18, 2002-2012.

53. Ikebuchi, Y., Masumoto, N., Tasaka, K., Koike, K., Kasahara, K., Miyake, A., and

Tanizawa, O. (1991) Superoxide anion increases intracellular pH, intracellular free

calcium, and arachidonate release in human amnion cells, J Biol Chem 266, 13233-

13237.

54. De Grey, A. D. (2002) HO2*: the forgotten radical, DNA and cell biology 21, 251-257.

44

REFERENCES

55. Liu, S. S. (1999) Cooperation of a "reactive oxygen cycle" with the Q cycle and the

proton cycle in the respiratory chain--superoxide generating and cycling mechanisms

in mitochondria, Journal of bioenergetics and biomembranes 31, 367-376.

56. Fukai, T., Folz, R. J., Landmesser, U., and Harrison, D. G. (2002) Extracellular

superoxide dismutase and cardiovascular disease, Cardiovasc Res 55, 239-249.

57. McCord, J. M., and Fridovich, I. (1969) Superoxide dismutase. An enzymic function

for erythrocuprein (hemocuprein), J Biol Chem 244, 6049-6055.

58. Turrens, J. F., Freeman, B. A., and Crapo, J. D. (1982) Hyperoxia increases H2O2

release by lung mitochondria and microsomes, Arch Biochem Biophys 217, 411-421.

59. Jones, D. P., Eklow, L., Thor, H., and Orrenius, S. (1981) Metabolism of hydrogen

peroxide in isolated hepatocytes: relative contributions of catalase and glutathione

peroxidase in decomposition of endogenously generated H2O2, Arch Biochem Biophys

210, 505-516.

60. Schroy, C. B., and Biaglow, J. E. (1981) Use of an oxidase electrode to determine

factors affecting the in vitro production of hydrogen peroxide by Ehrlich cells and 1-

chloro-2, 4-dinitrobenzene, Biochem Pharmacol 30, 3201-3207.

61. Griendling, K. K., Sorescu, D., Lassegue, B., and Ushio-Fukai, M. (2000)

Modulation of protein kinase activity and gene expression by reactive oxygen species

and their role in vascular physiology and pathophysiology, Arterioscler Thromb Vasc Biol

20, 2175-2183.

62. Chiarugi, P., and Cirri, P. (2003) Redox regulation of protein tyrosine phosphatases

during receptor tyrosine kinase signal transduction, Trends Biochem Sci 28, 509-514.

63. Reth, M. (2002) Hydrogen peroxide as second messenger in lymphocyte activation,

Nat Immunol 3, 1129-1134.

64. Sauer, H., Wartenberg, M., and Hescheler, J. (2001) Reactive oxygen species as

intracellular messengers during cell growth and differentiation, Cell Physiol Biochem 11,

173-186.

65. Gillespie, M. N., Pastukh, V., and Ruchko, M. V. (2009) Oxidative DNA

modifications in hypoxic signaling, Ann N Y Acad Sci 1177, 140-150.

66. Haddad, J. J. (2002) Antioxidant and prooxidant mechanisms in the regulation of

redox(y)-sensitive transcription factors, Cell Signal 14, 879-897.

67. Hochstein, P., Jain, S. K., and Rice-Evans, C. (1981) The physiological significance

of oxidative perturbations in erythrocyte membrane lipids and proteins, Progress in

clinical and biological research 55, 449-465.

45

REFERENCES

68. Rubio-Gayosso, I., Platts, S. H., and Duling, B. R. (2006) Reactive oxygen species

mediate modification of glycocalyx during ischemia-reperfusion injury, Am J Physiol

Heart Circ Physiol 290, H2247-2256.

69. Beresewicz, A., Czarnowska, E., and Maczewski, M. (1998) Ischemic preconditioning

and superoxide dismutase protect against endothelial dysfunction and endothelium

glycocalyx disruption in the postischemic guinea-pig hearts, Mol Cell Biochem 186, 87-

97.

70. Raju, S. V., Barouch, L. A., and Hare, J. M. (2005) Nitric oxide and oxidative stress

in cardiovascular aging, Sci Aging Knowledge Environ 2005, re4.

71. Dhalla, N. S., Elmoselhi, A. B., Hata, T., and Makino, N. (2000) Status of

myocardial antioxidants in ischemia-reperfusion injury, Cardiovasc Res 47, 446-456.

72. Park, J. L., and Lucchesi, B. R. (1999) Mechanisms of myocardial reperfusion injury,

Ann Thorac Surg 68, 1905-1912.

73. Chan, P. H. (2001) Reactive oxygen radicals in signaling and damage in the ischemic

brain, J Cereb Blood Flow Metab 21, 2-14.

74. Floyd, R. A. (1990) Role of oxygen free radicals in carcinogenesis and brain ischemia,

Faseb J 4, 2587-2597.

75. Klaunig, J. E., and Kamendulis, L. M. (2004) The role of oxidative stress in

carcinogenesis, Annu Rev Pharmacol Toxicol 44, 239-267.

76. Valko, M., Izakovic, M., Mazur, M., Rhodes, C. J., and Telser, J. (2004) Role of

oxygen radicals in DNA damage and cancer incidence, Mol Cell Biochem 266, 37-56.

77. Valko, M., Rhodes, C. J., Moncol, J., Izakovic, M., and Mazur, M. (2006) Free

radicals, metals and antioxidants in oxidative stress-induced cancer, Chem Biol Interact

160, 1-40.

78. Bowler, R. P., and Crapo, J. D. (2002) Oxidative stress in airways: is there a role for

extracellular superoxide dismutase?, Am J Respir Crit Care Med 166, S38-43.

79. Kinnula, V. L., and Crapo, J. D. (2003) Superoxide dismutases in the lung and human

lung diseases, Am J Respir Crit Care Med 167, 1600-1619.

80. Rahman, I., Biswas, S. K., and Kode, A. (2006) Oxidant and antioxidant balance in

the airways and airway diseases, Eur J Pharmacol 533, 222-239.

81. Delanty, N., and Dichter, M. A. (1998) Oxidative injury in the nervous system, Acta

Neurol Scand 98, 145-153.

46

REFERENCES

82. Chung, H. Y., Kim, H. J., Kim, J. W., and Yu, B. P. (2001) The inflammation

hypothesis of aging: molecular modulation by calorie restriction, Ann N Y Acad Sci

928, 327-335.

83. Goldhaber, J. I., and Weiss, J. N. (1992) Oxygen free radicals and cardiac reperfusion

abnormalities, Hypertension 20, 118-127.

84. Chen, J. K., and Chow, S. E. (2005) Antioxidants and myocardial ischemia:

reperfusion injuries, Chang Gung Med J 28, 369-377.

85. Gori, T., Lisi, M., and Forconi, S. (2006) Ischemia and reperfusion: the endothelial

perspective. A radical view, Clin Hemorheol Microcirc 35, 31-34.

86. Granger, D. N. (1988) Role of xanthine oxidase and granulocytes in ischemia-

reperfusion injury, Am J Physiol 255, H1269-1275.

87. Menger, M. D., Pelikan, S., Steiner, D., and Messmer, K. (1992) Microvascular

ischemia-reperfusion injury in striated muscle: significance of "reflow paradox", Am J

Physiol 263, H1901-1906.

88. Pulsinelli, W. A., Brierley, J. B., and Plum, F. (1982) Temporal profile of neuronal

damage in a model of transient forebrain ischemia, Ann Neurol 11, 491-498.

89. Jeroudi, M. O., Hartley, C. J., and Bolli, R. (1994) Myocardial reperfusion injury: role

of oxygen radicals and potential therapy with antioxidants, Am J Cardiol 73, 2B-7B.

90. Kurose, I., Argenbright, L. W., Wolf, R., Lianxi, L., and Granger, D. N. (1997)

Ischemia/reperfusion-induced microvascular dysfunction: role of oxidants and lipid

mediators, Am J Physiol 272, H2976-2982.

91. Tsao, P. S., and Lefer, A. M. (1990) Time course and mechanism of endothelial

dysfunction in isolated ischemic- and hypoxic-perfused rat hearts, Am J Physiol 259,

H1660-1666.

92. Bolli, R., and Marban, E. (1999) Molecular and cellular mechanisms of myocardial

stunning, Physiol Rev 79, 609-634.

93. Braunwald, E., and Kloner, R. A. (1982) The stunned myocardium: prolonged,

postischemic ventricular dysfunction, Circulation 66, 1146-1149.

94. Wolin, M. S. (2000) Interactions of oxidants with vascular signaling systems,

Arterioscler Thromb Vasc Biol 20, 1430-1442.

95. Garcia, J. H., Lassen, N. A., Weiller, C., Sperling, B., and Nakagawara, J. (1996)

Ischemic stroke and incomplete infarction, Stroke 27, 761-765.

96. Gersh, B. J. (1998) Current issues in reperfusion therapy, Am J Cardiol 82, 3P-11P.

47

REFERENCES

97. Goode, H. F., Webster, N. R., Howdle, P. D., Leek, J. P., Lodge, J. P., Sadek, S. A.,

and Walker, B. E. (1994) Reperfusion injury, antioxidants and hemodynamics during

orthotopic liver transplantation, Hepatology 19, 354-359.

98. Ferrari, R. (1996) The role of mitochondria in ischemic heart disease, J Cardiovasc

Pharmacol 28 Suppl 1, S1-10.

99. Griendling, K. K., and Alexander, R. W. (1997) Oxidative stress and cardiovascular

disease, Circulation 96, 3264-3265.

100. Kaplan, P., Lehotsky, J., and Racay, P. (1997) Role of sarcoplasmic reticulum in the

contractile dysfunction during myocardial ischaemia and reperfusion, Physiol Res 46,

333-339.

101. Jones, S. P., Hoffmeyer, M. R., Sharp, B. R., Ho, Y. S., and Lefer, D. J. (2003) Role

of intracellular antioxidant enzymes after in vivo myocardial ischemia and reperfusion,

Am J Physiol Heart Circ Physiol 284, H277-282.

102. Kadambi, A., and Skalak, T. C. (2000) Role of leukocytes and tissue-derived oxidants

in short-term skeletal muscle ischemia-reperfusion injury, Am J Physiol Heart Circ

Physiol 278, H435-443.

103. Thiagarajan, R. R., Winn, R. K., and Harlan, J. M. (1997) The role of leukocyte and

endothelial adhesion molecules in ischemia-reperfusion injury, Thromb Haemost 78,

310-314.

104. Serrano, C. V., Jr., Mikhail, E. A., Wang, P., Noble, B., Kuppusamy, P., and Zweier,

J. L. (1996) Superoxide and hydrogen peroxide induce CD18-mediated adhesion in

the postischemic heart, Biochim Biophys Acta 1316, 191-202.

105. Clerk, A., Fuller, S. J., Michael, A., and Sugden, P. H. (1998) Stimulation of "stress-

regulated" mitogen-activated protein kinases (stress-activated protein kinases/c-Jun

N-terminal kinases and p38-mitogen-activated protein kinases) in perfused rat hearts

by oxidative and other stresses, J Biol Chem 273, 7228-7234.

106. Maulik, N., Yoshida, T., Engelman, R. M., Deaton, D., Flack, J. E., 3rd, Rousou, J.

A., and Das, D. K. (1998) Ischemic preconditioning attenuates apoptotic cell death

associated with ischemia/reperfusion, Mol Cell Biochem 186, 139-145.

107. Karin, M., Liu, Z., and Zandi, E. (1997) AP-1 function and regulation, Curr Opin

Cell Biol 9, 240-246.

108. Schreck, R., Rieber, P., and Baeuerle, P. A. (1991) Reactive oxygen intermediates as

apparently widely used messengers in the activation of the NF-kappa B transcription

factor and HIV-1, Embo J 10, 2247-2258.

48

REFERENCES

109. Fattman, C. L., Schaefer, L. M., and Oury, T. D. (2003) Extracellular superoxide

dismutase in biology and medicine, Free Radic Biol Med 35, 236-256.

110. Cemeli, E., Baumgartner, A., and Anderson, D. (2009) Antioxidants and the Comet

assay, Mutat Res 681, 51-67.

111. Azzi, A., Davies, K. J., and Kelly, F. (2004) Free radical biology - terminology and

critical thinking, FEBS Lett 558, 3-6.

112. Azzi, A. (2007) Oxidative stress: A dead end or a laboratory hypothesis?, Biochem

Biophys Res Commun 362, 230-232.

113. Bagchi, D., Bagchi, M., Stohs, S. J., Das, D. K., Ray, S. D., Kuszynski, C. A., Joshi,

S. S., and Pruess, H. G. (2000) Free radicals and grape seed proanthocyanidin extract:

importance in human health and disease prevention, Toxicology 148, 187-197.

114. Niki, E. (1991) Action of ascorbic acid as a scavenger of active and stable oxygen

radicals, Am J Clin Nutr 54, 1119S-1124S.

115. Niki, E. (1991) Vitamin C as an antioxidant, World Rev Nutr Diet 64, 1-30.

116. Azzi, A. (2007) Molecular mechanism of alpha-tocopherol action, Free Radic Biol Med

43, 16-21.

117. Gianello, R., Libinaki, R., Azzi, A., Gavin, P. D., Negis, Y., Zingg, J. M., Holt, P.,

Keah, H. H., Griffey, A., Smallridge, A., West, S. M., and Ogru, E. (2005) Alpha-

tocopheryl phosphate: a novel, natural form of vitamin E, Free Radic Biol Med 39,

970-976.

118. Chelikani, P., Fita, I., and Loewen, P. C. (2004) Diversity of structures and properties

among catalases, Cell Mol Life Sci 61, 192-208.

119. Battin, E. E., and Brumaghim, J. L. (2009) Antioxidant activity of sulfur and

selenium: a review of reactive oxygen species scavenging, glutathione peroxidase, and

metal-binding antioxidant mechanisms, Cell Biochem Biophys 55, 1-23.

120. Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata,

M., Miyazono, K., and Ichijo, H. (1998) Mammalian thioredoxin is a direct inhibitor

of apoptosis signal-regulating kinase (ASK) 1, Embo J 17, 2596-2606.

121. Mann, T. a. K., D. (1939) Proc. Roy. Soc. Ser. B Biol. Sci. 126, 303.

122. McCord, J. M., Keele, B. B., Jr., and Fridovich, I. (1971) An enzyme-based theory of

obligate anaerobiosis: the physiological function of superoxide dismutase, Proc Natl

Acad Sci U S A 68, 1024-1027.

123. Marklund, S. L. (1984) Properties of extracellular superoxide dismutase from human

lung, Biochem J 220, 269-272.

49

REFERENCES

124. Marklund, S. L. (1982) Human copper-containing superoxide dismutase of high

molecular weight, Proc Natl Acad Sci U S A 79, 7634-7638.

125. Youn, H. D., Kim, E. J., Roe, J. H., Hah, Y. C., and Kang, S. O. (1996) A novel

nickel-containing superoxide dismutase from Streptomyces spp, Biochem J 318, 889-

896.

126. Choudhury, S. B., Lee, J. W., Davidson, G., Yim, Y. I., Bose, K., Sharma, M. L.,

Kang, S. O., Cabelli, D. E., and Maroney, M. J. (1999) Examination of the nickel site

structure and reaction mechanism in Streptomyces seoulensis superoxide dismutase,

Biochemistry 38, 3744-3752.

127. Parker, M. W., and Blake, C. C. (1988) Iron- and manganese-containing superoxide

dismutases can be distinguished by analysis of their primary structures, FEBS Lett 229,

377-382.

128. Priya, B., Premanandh, J., Dhanalakshmi, R. T., Seethalakshmi, T., Uma, L.,

Prabaharan, D., and Subramanian, G. (2007) Comparative analysis of cyanobacterial

superoxide dismutases to discriminate canonical forms, BMC Genomics 8, 435-444.

129. Harris, J. I., Auffret, A. D., Northrop, F. D., and Walker, J. E. (1980) Structural

comparisons of superoxide dismutases, Eur J Biochem 106, 297-303.

130. Steinman, H. M., and Hill, R. L. (1973) Sequence homologies among bacterial and

mitochondrial superoxide dismutases, Proc Natl Acad Sci U S A 70, 3725-3729.

131. Zelko, I. N., Mariani, T. J., and Folz, R. J. (2002) Superoxide dismutase multigene

family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD

(SOD3) gene structures, evolution, and expression, Free Radic Biol Med 33, 337-349.

132. Keele, B. B. J., McCord, J. M., and Fridovich, I. (1970) Superoxide dismutase from

escherichia coli B. A new manganese-containing enzyme, J Biol Chem 245, 6176-

6181.

133. Hassan, H. M. (1988) Biosynthesis and regulation of superoxide dismutases, Free


134. Weisiger, R. A., and Fridovich, I. (1973) Mitochondrial superoxide simutase. Site of

synthesis and intramitochondrial localization, J Biol Chem 248, 4793-4796.

135. Barondeau, D. P., Kassmann, C. J., Bruns, C. K., Tainer, J. A., and Getzoff, E. D.

(2004) Nickel superoxide dismutase structure and mechanism, Biochemistry 43, 8038-

8047.

136. Wagner, U. G., Pattridge, K. A., Ludwig, M. L., Stallings, W. C., Werber, M. M.,

Oefner, C., Frolow, F., and Sussman, J. L. (1993) Comparison of the crystal

50

REFERENCES

structures of genetically engineered human manganese superoxide dismutase and

manganese superoxide dismutase from Thermus thermophilus: differences in dimer-

dimer interaction, Protein Sci 2, 814-825.

137. Yost, F. J., Jr., and Fridovich, I. (1973) An iron-containing superoxide dismutase from

Escherichia coli, J Biol Chem 248, 4905-4908.

138. Gregroy, E. M., Yost, F. J., Jr., and Fridovich, I. (1973) Superoxide dismutases of

Escherichia coli: intracellular localization and functions, J Bacteriol 115, 987-991.

139. Abdel-Ghany, S. E., Burkhead, J. L., Gogolin, K. A., Andres-Colas, N., Bodecker, J.

R., Puig, S., Penarrubia, L., and Pilon, M. (2005) AtCCS is a functional homolog of

the yeast copper chaperone Ccs1/Lys7, FEBS Lett 579, 2307-2312.

140. Van Camp, W., Inzé, D., and Van Montagu, M. (1997) The regulation and function

of tobacco superoxide dismutases, Free Radic Biol Med 23, 515-520.

141. Wuerges, J., Lee, J. W., Yim, Y. I., Yim, H. S., Kang, S. O., and Djinovic-Carugo, K.

(2004) Crystal structure of nickel-containing superoxide dismutase reveals another

type of active site, Proc Natl Acad Sci U S A 101, 8569-8574.

142. Crapo, J. D., Oury, T., Rabouille, C., Slot, J. W., and Chang, L. Y. (1992)

Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells, Proc

Natl Acad Sci U S A 89, 10405-10409.

143. Keller, G. A., Warner, T. G., Steimer, K. S., and Hallewell, R. A. (1991) Cu,Zn

superoxide dismutase is a peroxisomal enzyme in human fibroblasts and hepatoma

cells, Proc Natl Acad Sci U S A 88, 7381-7385.

144. Asada, K., Urano, M., and Takahashi, M. (1973) Subcellular location of superoxide

dismutase in spinach leaves and preparation and properties of crystalline spinach

superoxide dismutase, Eur J Biochem 36, 257-266.

145. Kroll, J. S., Langford, P. R., Wilks, K. E., and Keil, A. D. (1995) Bacterial [Cu,Zn]-

superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not

so rare after all!, Microbiology 141 ( Pt 9), 2271-2279.

146. Puget, K., and Michelson, A. M. (1974) Isolation of a new copper-containing

superoxide dismutase bacteriocuprein, Biochem Biophys Res Commun 58, 830-838.

147. Battistoni, A., and Rotilio, G. (1995) Isolation of an active and heat-stable

monomeric form of Cu,Zn superoxide dismutase from the periplasmic space of

Escherichia coli, FEBS Lett 374, 199-202.

148. Buetler, T. M., Krauskopf, A., and Ruegg, U. T. (2004) Role of superoxide as a

signaling molecule, News Physiol Sci 19, 120-123.

51

REFERENCES

149. Brandes, R. P. (2003) Role of NADPH oxidases in the control of vascular gene

expression, Antioxid Redox Signal 5, 803-811.

150. Marklund, S. L., Holme, E., and Hellner, L. (1982) Superoxide dismutase in

extracellular fluids, Clin Chim Acta 126, 41-51.

151. Marklund, S. L., Bjelle, A., and Elmqvist, L. G. (1986) Superoxide dismutase

isoenzymes of the synovial fluid in rheumatoid arthritis and in reactive arthritides, Ann

Rheum Dis 45, 847-851.

152. Nozik-Grayck, E., Suliman, H. B., and Piantadosi, C. A. (2005) Extracellular

superoxide dismutase, Int J Biochem Cell Biol 37, 2466-2471.

153. Marklund, S. L. (1984) Extracellular superoxide dismutase in human tissues and

human cell lines, J Clin Invest 74, 1398-1403.

154. Marklund, S. L. (1984) Extracellular superoxide dismutase and other superoxide

dismutase isoenzymes in tissues from nine mammalian species, Biochem J 222, 649-

655.

155. Folz, R. J., and Crapo, J. D. (1994) Extracellular superoxide dismutase (SOD3):

tissue-specific expression, genomic characterization, and computer-assisted sequence

analysis of the human EC SOD gene, Genomics 22, 162-171.

156. Qin, Z., Reszka, K. J., Fukai, T., and Weintraub, N. L. (2008) Extracellular

superoxide dismutase (ecSOD) in vascular biology: an update on exogenous gene

transfer and endogenous regulators of ecSOD, Transl Res 151, 68-78.

157. Oury, T. D., Chang, L. Y., Marklund, S. L., Day, B. J., and Crapo, J. D. (1994)

Immunocytochemical localization of extracellular superoxide dismutase in human

lung, Lab Invest 70, 889-898.

158. Oury, T. D., Day, B. J., and Crapo, J. D. (1996) Extracellular superoxide dismutase in

vessels and airways of humans and baboons, Free Radic Biol Med 20, 957-965.

159. Stralin, P., Karlsson, K., Johansson, B. O., and Marklund, S. L. (1995) The

interstitium of the human arterial wall contains very large amounts of extracellular

superoxide dismutase, Arterioscler Thromb Vasc Biol 15, 2032-2036.

160. Marklund, S. L. (1990) Expression of extracellular superoxide dismutase by human

cell lines, Biochem J 266, 213-219.

161. Tibell, L., Aasa, R., and Marklund, S. L. (1993) Spectral and physical properties of

human extracellular superoxide dismutase: a comparison with CuZn superoxide

dismutase, Arch Biochem Biophys 304, 429-433.

52

REFERENCES

162. Ookawara, T., Kizaki, T., Ohishi, S., Yamamoto, M., Matsubara, O., and Ohno, H.

(1997) Purification and subunit structure of extracellular superoxide dismutase from

mouse lung tissue, Arch Biochem Biophys 340, 299-304.

163. DiSilvestro, R. A., Yang, F. L., and David, E. A. (1992) Species-specific

heterogeneity for molecular weight estimates of serum extracellular superoxide

dismutase activities, Comp Biochem Physiol B 101, 531-534.

164. Oury, T. D., Crapo, J. D., Valnickova, Z., and Enghild, J. J. (1996) Human

extracellular superoxide dismutase is a tetramer composed of two disulphide-linked

dimers: a simplified, high-yield purification of extracellular superoxide dismutase,

Biochem J 317 ( Pt 1), 51-57.

165. Tibell, L. A., Skarfstad, E., and Jonsson, B. H. (1996) Determination of the structural

role of the N-terminal domain of human extracellular superoxide dismutase by use of

protein fusions, Biochim Biophys Acta 1292, 47-52.

166. Stenlund, P., Andersson, D., and Tibell, L. A. (1997) Subunit interaction in

extracellular superoxide dismutase: effects of mutations in the N-terminal domain,

Protein Sci 6, 2350-2358.

167. Due, A. V., Petersen, S. V., Valnickova, Z., Ostergaard, L., Oury, T. D., Crapo, J.

D., and Enghild, J. J. (2006) Extracellular superoxide dismutase exists as an octamer,

FEBS Lett 580, 1485-1489.

168. Hendrickson, D. J., Fisher, J. H., Jones, C., and Ho, Y. S. (1990) Regional

localization of human extracellular superoxide dismutase gene to 4pter-q21, Genomics

8, 736-738.

169. Hjalmarsson, K., Marklund, S. L., Engstrom, A., and Edlund, T. (1987) Isolation

and sequence of complementary DNA encoding human extracellular superoxide

dismutase, Proc Natl Acad Sci U S A 84, 6340-6344.

170. Fridovich, I. (1986) Superoxide dismutases, Adv Enzymol Relat Areas Mol Biol 58, 61-

97.

171. Antonyuk, S. V., Strange, R. W., Marklund, S. L., and Hasnain, S. S. (2009) The

structure of human extracellular copper-zinc superoxide dismutase at 1.7 A resolution:

insights into heparin and collagen binding, J Mol Biol 388, 310-326.

172. Getzoff, E. D., Tainer, J. A., Weiner, P. K., Kollman, P. A., Richardson, J. S., and

Richardson, D. C. (1983) Electrostatic recognition between superoxide and copper,

zinc superoxide dismutase, Nature 306, 287-290.

53

REFERENCES

173. Marino, M., Galvano, M., Cambria, A., Polticelli, F., and Desideri, A. (1995)

Modelling the three-dimensional structure and the electrostatic potential field of two

Cu,Zn superoxide dismutase variants from tomato leaves, Protein Eng 8, 551-556.

174. Desideri, A., Falconi, M., Polticelli, F., Bolognesi, M., Djinovic, K., and Rotilio, G.

(1992) Evolutionary conservativeness of electric field in the Cu,Zn superoxide

dismutase active site. Evidence for co-ordinated mutation of charged amino acid

residues, J Mol Biol 223, 337-342.

175. Edlund, A., Edlund, T., Hjalmarsson, K., Marklund, S. L., Sandstrom, J., Stromqvist,

M., and Tibell, L. (1992) A non-glycosylated extracellular superoxide dismutase

variant, Biochem J 288 ( Pt 2), 451-456.

176. Marklund, S. L., and Karlsson, K. (1990) Extracellular-superoxide dismutase,

distribution in the body and therapeutic applications, Adv Exp Med Biol 264, 1-4.

177. Karlsson, K., Lindahl, U., and Marklund, S. L. (1988) Binding of human extracellular

superoxide dismutase C to sulphated glycosaminoglycans, Biochem J 256, 29-33.

178. Tibell, L. A., Sethson, I., and Buevich, A. V. (1997) Characterization of the heparin-

binding domain of human extracellular superoxide dismutase, Biochim Biophys Acta

1340, 21-32.

179. Stenlund, P., Lindberg, M. J., and Tibell, L. A. (2002) Structural requirements for

high-affinity heparin binding: alanine scanning analysis of charged residues in the C-

terminal domain of human extracellular superoxide dismutase, Biochemistry 41, 3168-

3175.

180. Lookene, A., Stenlund, P., and Tibell, L. A. (2000) Characterization of heparin

binding of human extracellular superoxide dismutase, Biochemistry 39, 230-236.

181. Sandstrom, J., Carlsson, L., Marklund, S. L., and Edlund, T. (1992) The heparin-

binding domain of extracellular superoxide dismutase C and formation of variants

with reduced heparin affinity, J Biol Chem 267, 18205-18209.

182. Adachi, T., and Marklund, S. L. (1989) Interactions between human extracellular

superoxide dismutase C and sulfated polysaccharides, J Biol Chem 264, 8537-8541.

183. Conrad, H. (1998) Heparin-Binding Proteins, Academic Press.

184. Sjoquist, P. O., and Marklund, S. L. (1992) Endothelium bound extracellular

superoxide dismutase type C reduces damage in reperfused ischaemic rat hearts,

Cardiovasc Res 26, 347-350.

54

REFERENCES

185. Karlsson, K., Edlund, A., Sandstrom, J., and Marklund, S. L. (1993) Proteolytic

modification of the heparin-binding affinity of extracellular superoxide dismutase,

Biochem J 290 ( Pt 2), 623-626.

186. Karlsson, K., Sandstrom, J., Edlund, A., Edlund, T., and Marklund, S. L. (1993)

Pharmacokinetics of extracellular-superoxide dismutase in the vascular system, Free


187. Karlsson, K., Sandstrom, J., Edlund, A., and Marklund, S. L. (1994) Turnover of

extracellular-superoxide dismutase in tissues, Lab Invest 70, 705-710.

188. Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo,

J. D. (1999) The heparin-binding domain of extracellular superoxide dismutase is

proteolytically processed intracellularly during biosynthesis, J Biol Chem 274, 14818-

14822.

189. Olsen, D. A., Petersen, S. V., Oury, T. D., Valnickova, Z., Thøgersen, I. B.,

Kristensen, T., Bowler, R. P., Crapo, J. D., and Enghild, J. J. (2004) The intracellular

proteolytic processing of extracellular superoxide dismutase (EC-SOD) is a two-step

event, J Biol Chem 279, 22152-22157.

190. Gao, F., Koenitzer, J. R., Tobolewski, J. M., Jiang, D., Liang, J., Noble, P. W., and

Oury, T. D. (2008) Extracellular superoxide dismutase inhibits inflammation by

preventing oxidative fragmentation of hyaluronan, J Biol Chem 283, 6058-6066.

191. Petersen, S. V., Oury, T. D., Ostergaard, L., Valnickova, Z., Wegrzyn, J., Thøgersen,

I. B., Jacobsen, C., Bowler, R. P., Fattman, C. L., Crapo, J. D., and Enghild, J. J.

(2004) Extracellular superoxide dismutase (EC-SOD) binds to type i collagen and

protects against oxidative fragmentation, J Biol Chem 279, 13705-13710.

192. Petersen, S. V., Olsen, D. A., Kenney, J. M., Oury, T. D., Valnickova, Z., Thøgersen,

I. B., Crapo, J. D., and Enghild, J. J. (2005) The high concentration of Arg213-->Gly

extracellular superoxide dismutase (EC-SOD) in plasma is caused by a reduction of

both heparin and collagen affinities, Biochem J 15, 427-432.

193. Marklund, S. L., Nilsson, P., Israelsson, K., Schampi, I., Peltonen, M., and Asplund,

K. (1997) Two variants of extracellular-superoxide dismutase: relationship to

cardiovascular risk factors in an unselected middle-aged population, J Intern Med 242,

5-14.

194. Juul, K., Tybjaerg-Hansen, A., Marklund, S., Heegaard, N. H., Steffensen, R.,

Sillesen, H., Jensen, G., and Nordestgaard, B. G. (2004) Genetically reduced

55

REFERENCES

antioxidative protection and increased ischemic heart disease risk: The Copenhagen

City Heart Study, Circulation 109, 59-65.

195. Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C.

(1982) Determination and analysis of the 2 A-structure of copper, zinc superoxide

dismutase, J Mol Biol 160, 181-217.

196. Stenlund, P., and Tibell, L. A. (1999) Chimeras of human extracellular and

intracellular superoxide dismutases. Analysis of structure and function of the

individual domains, Protein Eng 12, 319-325.

197. Hart, P. J., Balbirnie, M. M., Ogihara, N. L., Nersissian, A. M., Weiss, M. S.,

Valentine, J. S., and Eisenberg, D. (1999) A structure-based mechanism for copper-

zinc superoxide dismutase, Biochemistry 38, 2167-2178.

198. Tainer, J. A., Getzoff, E. D., Richardson, J. S., and Richardson, D. C. (1983)

Structure and mechanism of copper, zinc superoxide dismutase, Nature 306, 284-287.

199. Bourne, Y., Redford, S. M., Steinman, H. M., Lepock, J. R., Tainer, J. A., and

Getzoff, E. D. (1996) Novel dimeric interface and electrostatic recognition in bacterial

Cu,Zn superoxide dismutase, Proc Natl Acad Sci U S A 93, 12774-12779.

200. Petersen, S. V., and Enghild, J. J. (2005) Extracellular superoxide dismutase: structural

and functional considerations of a protein shaped by two different disulfide bridge

patterns, Biomed Pharmacother 59, 175-182.

201. Petersen, S. V., Oury, T. D., Valnickova, Z., Thøgersen, I. B., Højrup, P., Crapo, J.

D., and Enghild, J. J. (2003) The dual nature of human extracellular superoxide

dismutase: one sequence and two structures, Proc Natl Acad Sci U S A 100, 13875-

13880.

202. Petersen, S. V., Valnickova, Z., Oury, T. D., Crapo, J. D., Chr Nielsen, N., and

Enghild, J. J. (2007) The subunit composition of human extracellular superoxide

dismutase (EC-SOD) regulates enzymatic activity, BMC Biochem 15, 19-25.

203. Petersen, S. V., Kristensen, T., Petersen, J. S., Ramsgaard, L., Oury, T. D., Crapo, J.

D., Nielsen, N. C., and Enghild, J. J. (2008) The folding of human active and inactive

extracellular superoxide dismutases is an intracellular event, J Biol Chem 283, 15031-

15036.

204. Adachi, T., Hara, H., Yamada, H., Yamazaki, N., Yamamoto, M., Sugiyama, T.,

Futenma, A., and Katagiri, Y. (2001) Heparin-stimulated expression of extracellular-

superoxide dismutase in human fibroblasts, Atherosclerosis 159, 307-312.

56

REFERENCES

205. Brady, T. C., Chang, L. Y., Day, B. J., and Crapo, J. D. (1997) Extracellular

superoxide dismutase is upregulated with inducible nitric oxide synthase after NF-

kappa B activation, Am J Physiol 273, L1002-1006.

206. Gongora, M. C., Qin, Z., Laude, K., Kim, H. W., McCann, L., Folz, J. R., Dikalov,

S., Fukai, T., and Harrison, D. G. (2006) Role of extracellular superoxide dismutase

in hypertension, Hypertension 48, 473-481.

207. Jung, O., Marklund, S. L., Geiger, H., Pedrazzini, T., Busse, R., and Brandes, R. P.

(2003) Extracellular superoxide dismutase is a major determinant of nitric oxide

bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice, Circ Res 93,

622-629.

208. Oury, T. D., Schaefer, L. M., Fattman, C. L., Choi, A., Weck, K. E., and Watkins,

S. C. (2002) Depletion of pulmonary EC-SOD after exposure to hyperoxia, Am J

Physiol Lung Cell Mol Physiol 283, L777-784.

209. Laukkanen, M. O., Lehtolainen, P., Turunen, P., Aittomaki, S., Oikari, P.,

Marklund, S. L., and Yla-Herttuala, S. (2000) Rabbit extracellular superoxide

dismutase: expression and effect on LDL oxidation, Gene 254, 173-179.

210. Takatsu, H., Tasaki, H., Kim, H. N., Ueda, S., Tsutsui, M., Yamashita, K.,

Toyokawa, T., Morimoto, Y., Nakashima, Y., and Adachi, T. (2001) Overexpression

of EC-SOD suppresses endothelial-cell-mediated LDL oxidation, Biochem Biophys

Res Commun 285, 84-91.

211. Omar, B. A., Gad, N. M., Jordan, M. C., Striplin, S. P., Russell, W. J., Downey, J.

M., and McCord, J. M. (1990) Cardioprotection by Cu,Zn-superoxide dismutase is

lost at high doses in the reoxygenated heart, Free Radic Biol Med 9, 465-471.

212. Omar, B. A., and McCord, J. M. (1990) The cardioprotective effect of Mn-

superoxide dismutase is lost at high doses in the postischemic isolated rabbit heart,

Free Radic Biol Med 9, 473-478.

213. Johansson, M. H., Deinum, J., Marklund, S. L., and Sjoquist, P. O. (1990)

Recombinant human extracellular superoxide dismutase reduces concentration of

oxygen free radicals in the reperfused rat heart, Cardiovasc Res 24, 500-503.

214. Wahlund, G., Marklund, S. L., and Sjoquist, P. O. (1992) Extracellular-superoxide

dismutase type C (EC-SOD C) reduces myocardial damage in rats subjected to

coronary occlusion and 24 hours of reperfusion, Free Radic Res Commun 17, 41-47.

215. Sjoquist, P. O., Carlsson, L., Jonason, G., Marklund, S. L., and Abrahamsson, T.

(1991) Cardioprotective effects of recombinant human extracellular-superoxide

57

REFERENCES

dismutase type C in rat isolated heart subjected to ischemia and reperfusion, J

Cardiovasc Pharmacol 17, 678-683.

216. Tibell, L., Hjalmarsson, K., Edlund, T., Skogman, G., Engstrom, A., and Marklund,

S. L. (1987) Expression of human extracellular superoxide dismutase in Chinese

hamster ovary cells and characterization of the product, Proc Natl Acad Sci U S A 84,

6634-6638.

217. Hansson, L., Edlund, M., Edlund, A., Johansson, T., Marklund, S. L., Fromm, S.,

Stromqvist, M., and Tornell, J. (1994) Expression and characterization of biologically

active human extracellular superoxide dismutase in milk of transgenic mice, J Biol

Chem 269, 5358-5363.

218. Esko, J. D., Kimata, K. and Lindahl, U. (2009) Proteoglycans and Sulfated

Glycosaminoglycans, in Essentials of Glycobiology (Varki, A., Cummings, R.D., Esko,

J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W. and Etzler, M.E., Ed.)

Second ed., pp 229-248, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,

New York.

219. Rabenstein, D. L. (2002) Heparin and heparan sulfate: structure and function, Nat

Prod Rep 19, 312-331.

220. Varki, A., Cummings, R., Esko, J., Freeze, H., Hart, G. and Marth, J. (1999)

Essentials of Glycobiology, Cold Spring Harbor Laboratory Press.

221. Imberty, A., Lortat-Jacob, H., and Perez, S. (2007) Structural view of

glycosaminoglycan-protein interactions, Carbohydr Res 342, 430-439.

222. Faham, S., Hileman, R. E., Fromm, J. R., Linhardt, R. J., and Rees, D. C. (1996)

Heparin structure and interactions with basic fibroblast growth factor, Science 271,

1116-1120.

223. Jin, L., Abrahams, J. P., Skinner, R., Petitou, M., Pike, R. N., and Carrell, R. W.

(1997) The anticoagulant activation of antithrombin by heparin, Proc Natl Acad Sci U

S A 94, 14683-14688.

224. Johnson, D. J., and Huntington, J. A. (2003) Crystal structure of antithrombin in a

heparin-bound intermediate state, Biochemistry 42, 8712-8719.

225. DiGabriele, A. D., Lax, I., Chen, D. I., Svahn, C. M., Jaye, M., Schlessinger, J., and

Hendrickson, W. A. (1998) Structure of a heparin-linked biologically active dimer of

fibroblast growth factor, Nature 393, 812-817.

226. Shaw, J. P., Johnson, Z., Borlat, F., Zwahlen, C., Kungl, A., Roulin, K., Harrenga,

A., Wells, T. N., and Proudfoot, A. E. (2004) The X-ray structure of RANTES:

58

REFERENCES

heparin-derived disaccharides allows the rational design of chemokine inhibitors,

Structure 12, 2081-2093.

227. Swaminathan, G. J., Myszka, D. G., Katsamba, P. S., Ohnuki, L. E., Gleich, G. J.,

and Acharya, K. R. (2005) Eosinophil-granule major basic protein, a C-type lectin,

binds heparin, Biochemistry 44, 14152-14158.

228. Shao, C., Zhang, F., Kemp, M. M., Linhardt, R. J., Waisman, D. M., Head, J. F.,

and Seaton, B. A. (2006) Crystallographic analysis of calcium-dependent heparin

binding to annexin A2, J Biol Chem 281, 31689-31695.

229. Margalit, H., Fischer, N., and Ben-Sasson, S. A. (1993) Comparative analysis of

structurally defined heparin binding sequences reveals a distinct spatial distribution of

basic residues, J Biol Chem 268, 19228-19231.

230. Cooper, M. A. (2003) Label-free screening of bio-molecular interactions, Anal

Bioanal Chem 377, 834-842.

231. Perozzo, R., Folkers, G., and Scapozza, L. (2004) Thermodynamics of protein-ligand

interactions: history, presence, and future aspects, J Recept Signal Transduct Res 24, 1-

52.

232. O´Brien, R., and Haq, I. (2004) Applications of Biocalorimetry: Binding, Stability

and Enzyme Kinetics, in Biocalorimetry 2; Applications of Calorimetry in the Biological

Sciences (Ladbury, J. E. a. D., M. L., Ed.), pp 3-34, John Wiley & Sons, Ltd.

233. Hoess, R. H. (2001) Protein design and phage display, Chem Rev 101, 3205-3218.

234. Nelson, S. K., Gao, B., Bose, S., Rizeq, M., and McCord, J. M. (2002) A novel

heparin-binding, human chimeric, superoxide dismutase improves myocardial

preservation and protects from ischemia-reperfusion injury, J Heart Lung Transplant

21, 1296-1303.

235. Knight, J. A. (2000) The biochemistry of aging, Adv Clin Chem 35, 1-62.

236. Chehab, O., Ouertani, M., Souiden, Y., Chaieb, K., and Mahdouani, K. (2008)

Plasma antioxidants and human aging: a study on healthy elderly Tunisian population,

Mol Biotechnol 40, 27-37.

237. Cuzzocrea, S., Riley, D. P., Caputi, A. P., and Salvemini, D. (2001) Antioxidant

therapy: a new pharmacological approach in shock, inflammation, and

ischemia/reperfusion injury, Pharmacol Rev 53, 135-159.

238. Harrison, D., Griendling, K. K., Landmesser, U., Hornig, B., and Drexler, H. (2003)

Role of oxidative stress in atherosclerosis, Am J Cardiol 91, 7A-11A.

59

REFERENCES

239. Salvemini, D., Doyle, T. M., and Cuzzocrea, S. (2006) Superoxide, peroxynitrite and

oxidative/nitrative stress in inflammation, Biochem Soc Trans 34, 965-970.

240. Hatori, N., Sjoquist, P. O., Marklund, S. L., Pehrsson, S. K., and Ryden, L. (1992)

Effects of recombinant human extracellular-superoxide dismutase type C on

myocardial reperfusion injury in isolated cold-arrested rat hearts, Free Radic Biol Med

13, 137-142.

241. Hatori, N., Sjoquist, P. O., Marklund, S. L., and Ryden, L. (1992) Effects of

recombinant human extracellular-superoxide dismutase type C on myocardial infarct

size in pigs, Free Radic Biol Med 13, 221-230.

242. Li, Q., Bolli, R., Qiu, Y., Tang, X. L., Guo, Y., and French, B. A. (2001) Gene

therapy with extracellular superoxide dismutase protects conscious rabbits against

myocardial infarction, Circulation 103, 1893-1898.

243. Li, Q., Bolli, R., Qiu, Y., Tang, X. L., Murphree, S. S., and French, B. A. (1998)

Gene therapy with extracellular superoxide dismutase attenuates myocardial stunning

in conscious rabbits, Circulation 98, 1438-1448.

244. Inoue, M., Watanabe, N., Morino, Y., Tanaka, Y., Amachi, T., and Sasaki, J. (1990)

Inhibition of oxygen toxicity by targeting superoxide dismutase to endothelial cell

surface, FEBS Lett 269, 89-92.

245. Bowler, R. P., Nicks, M., Olsen, D. A., Thogersen, I. B., Valnickova, Z., Hojrup, P.,

Franzusoff, A., Enghild, J. J., and Crapo, J. D. (2002) Furin proteolytically processes

the heparin-binding region of extracellular superoxide dismutase, J Biol Chem 277,

16505-16511.

246. Sandstrom, J., Nilsson, P., Karlsson, K., and Marklund, S. L. (1994) 10-fold increase

in human plasma extracellular superoxide dismutase content caused by a mutation in

heparin-binding domain, J Biol Chem 269, 19163-19166.

247. Olson, S. T., Halvorson, H. R., and Bjork, I. (1991) Quantitative characterization of

the thrombin-heparin interaction. Discrimination between specific and nonspecific

binding models, J Biol Chem 266, 6342-6352.

248. Patel, H. V., Vyas, A. A., Vyas, K. A., Liu, Y. S., Chiang, C. M., Chi, L. M., and

Wu, W. (1997) Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated

oligosaccharides as a potential target for cardiotoxin action, J Biol Chem 272, 1484-

1492.

60

REFERENCES

249. Hileman, R. E., Jennings, R. N., and Linhardt, R. J. (1998) Thermodynamic analysis

of the heparin interaction with a basic cyclic peptide using isothermal titration

calorimetry, Biochemistry 37, 15231-15237.

250. Thompson, L. D., Pantoliano, M. W., and Springer, B. A. (1994) Energetic

characterization of the basic fibroblast growth factor-heparin interaction:

identification of the heparin binding domain, Biochemistry 33, 3831-3840.

251. Gomez, J., Hilser, V. J., Xie, D., and Freire, E. (1995) The heat capacity of proteins,

Proteins 22, 404-412.

252. Spolar, R. S., Livingstone, J. R., and Record, M. T., Jr. (1992) Use of liquid

hydrocarbon and amide transfer data to estimate contributions to thermodynamic

functions of protein folding from the removal of nonpolar and polar surface from

water, Biochemistry 31, 3947-3955.

253. Niedzwiecka, A., Stepinski, J., Darzynkiewicz, E., Sonenberg, N., and Stolarski, R.

(2002) Positive heat capacity change upon specific binding of translation initiation

factor eIF4E to mRNA 5' cap, Biochemistry 41, 12140-12148.

254. Murphy, K. P., and Freire, E. (1992) Thermodynamics of structural stability and

cooperative folding behavior in proteins, Adv Protein Chem 43, 313-361.

255. Chandler, D. (2002) Hydrophobicity: two faces of water, Nature 417, 491.

256. Härd, T. (2004) Dissecting the Thermodynamics of DNA-Protein Interactions, in

Biocalorimetry 2; Applications of Calorimetry in the Biological Sciences (Ladbury, J. E. a.

D., M. L., Ed.), pp 81-91, John Wiley & Sons, Ltd.

257. Klocek, G., and Seelig, J. (2008) Melittin interaction with sulfated cell surface sugars,

Biochemistry 47, 2841-2849.

258. Ziegler, A., and Seelig, J. (2004) Interaction of the protein transduction domain of

HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic

parameters, Biophys J 86, 254-263.

61

protein engineering of extracellular superoxide...

Documents