Mechanisms of cell death induction by L-amino acid oxidase, a main component of ophidian venom

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L-amino acid oxidase,

a main component of ophidian venom

Dissertation

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

(Dr. rer. nat.)

an der Universität Konstanz (Fachbereich Biologie)

vorgelegt von

Sudharsana Rao Ande Konstanz, Germany 2006

Tag der mündlichen prüfung: 12

^th

July 2006 1) Referent: Prof. Dr. Sandro Ghisla

2) Referentin: PD. Dr. Elisa Ferrando - May

Konstanzer Online-Publikations-System (KOPS)

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Acknowledgements

First I would like to show deep appreciation and gratitude to Prof. Sandro Ghisla, my doctoral father, for giving me the opportunity to carry out my research work in his lab and for giving me the chance of collaborations. Thanks for giving me the interesting topic to work with. It has always been a pleasure to work within this project and thanks a lot for your constructive criticism, advice and support during my thesis.

Prof. Peter Macheroux, thank you very much for giving me the fine opportunity to work in Graz. Many thanks for your able guidance, fruitful discussions and timely help throughout my stay.

My main part of the work would have not been successful without the timely help of PD. Dr. Elisa Ferrando-May. To her, thanks a lot for patiently teaching me the cell culture techniques, guidelines on apoptotic studies and the able guidance, which helped me thorough the field of apoptosis!

My heartfelt thanks are due also to Prof. Kai-Uwe Fröhlich for giving me the opportunity to work in his lab and getting me exposed to the new, emerging field of science, Yeast apoptosis.

Further more, I am grateful to Prof. Martin Scheffner for agreeing to be my oral examiner.

I would like to thank all the members of AG Ghisla group, especially Robert Gradinaru, Lakshmi Narayana Kaza, Phaneeswara Rao Kommoju and Susanne Feindler-Boeckh for their help during my PhD work.

My sincere thanks to all the members of AG May group, especially Patricia Grote, Daniela Hermann for their help during my PhD work.

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I would like to thank all the members of the groups of Prof. Peter Macheroux, Prof.

Fröhlich and Prof. Frank Madeo for providing me the nice environment in the lab and make me feel at home in Graz. I miss you all!

PhD journey some times become worst, if you do not have friends to share with, I would like to thank Dr. Chandra Madhav, Dr. Gopal Reddy and Dr. Gopinath Rangam for their support and timely fun to share with!

I would like to thank Dr. Michael D’silva, Dr. Chandra Madhav, Mythili and Tzong- Yuan for their help in correcting my thesis.

Tons of thanks are reserved for all the Indian gang in Konstanz and Graz!

I thank one and all who have helped me directly or indirectly during my stay in Konstanz and Graz.

I would like to thank all the members of my family for their continuous support, patience and encouragement through out my studies.

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Fig 1. 3D Structure of LAAO from Malayan pit viper. _________________________6 Fig 2. Structure of LAAO from Malayan pit viper.____________________________7 Fig 3. Represents the cell undergoing different types of cell death namely apoptosis and necrosis. (Adapted from the book Ciclo de apoptosis). ____________________21 Fig 4. Inhibition zone formed by LAAO.___________________________________33 Fig 5. Inhibition zone formed by LAAO.___________________________________34 Fig 6. Antibacterial activity of LAAO in liquid cultures. ______________________35 Fig 7. Antibacterial activity of hydrogen peroxide in liquid cultures. ____________36 Fig 8. Estimation of hydrogen peroxide.___________________________________37 Fig 9. Effect of LAAO on yeast cells. _____________________________________47 Fig 10. Accumulation of ROS in LAAO treated Yeast cells.____________________48 Fig 11. DNA fragmentation analyzed by TUNEL staining. ____________________49 Fig 12. Annexin V staining._____________________________________________49 Fig 13. Effect of hydrogen peroxide in the medium with and without leucine. _____50 Fig 14. Interaction of LAAO with yeast cells._______________________________52 Fig 15. Immunofluorescence. ___________________________________________53 Fig 16. Protease protection assay with yeast cells. __________________________54 Fig 17. LAAO induces apoptosis and necrosis in Jurkat cells __________________68 Fig 18. Time course of H2O2 production __________________________________70 Fig 19. DAAO induces necrosis in Jurkat cells. _____________________________72 Fig 20. LAAO mediates depletion of essential amino acids in the culture medium. _74 Fig 21. FCS but not essential amino acids protect from apoptosis triggered by Lpt- medium ____________________________________________________________77 Fig 22. Effect of desialylation on LAAO-dependent apoptosis. _________________79 Fig 23. Fragmentation of LAAO but not other related enzymes upon incubation with Jurkat cells _________________________________________________________81

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Abbreviations

ADP Adenosine diphosphate

AIP Apoptosis Inducing Protein APIT Aplysia punctata ink toxin ATP Adenosine triphosphate

BCA Bicinchoninic acid

BSA Bovine serum albumin

°C Degree Celsius

CAD Caspase activated deoxyribonuclease CASY Cell counter + Analyzing system

DAAO D-Amino-acid Oxidase

DEVD-AFC Asp-Glu-Val-Asp 7-amino-4-trifluoromethyl coumarin DMSO Dimethyl sulfoxide

DTT Dithiothreitol

DNA Deoxy ribonucleic acid

EDTA Ethylene diamine tetra-acetic acid FAD Flavin Adenin dinucleotide

FACS Fluorescence activated cell sorting

FCS Fetal Calf Serum

FIG1 Interleukin-four induced gene-1

FMN Flavin mononucleotide

H2O2 HydrogenPeroxide

HIV Human immunodeficiency virus

hMCAD Human Medium Chain Acyl-CoA Dehydrogenase HPLC High pressure liquid chromatography

IL-4 Interleukin-4

IgG Immunoglobulin G

kDa Kilo Dalton

L, ml, µl Liter, milliliter, microliter Lpt-medium LAAO pretreated-medium LAAO L-Amino acid Oxidase

M Molar

MW Molecular weight

NMR Nuclear Magnetic Resonance PBS Phosphate buffered saline

POD Peroxidase

ROS Reactive Oxygen Species

RPMI Rosewell Park Memorial Institute SDS Sodium Dodecyl Sulfate

SDS-PAGE SDS Polyacrylamide Gel Electrophoresis Sig-lecs Sialic acid binding lectins

TUNEL Terminal deoxynucleotidyl transferase mediated d-UTP nick end labeling

zVAD-fmk Benzyloxycarbonyl-Val-Ala-Asp-CH2OC(O)-2,dichloro benzene, fluoro methyl ketone

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SHORT HAND SYMBOLS FOR AMINO ACIDS One letter Three letter Amino Acid

A Ala Alanine

R Arg Arginine

N Asn Asparagine

D Asp Aspartic acid

B Asx Asn or Asp

C Cys Cysteine

Q Gln Glutamine

E Glu Glutamic acid

Z Glx Gln or Glu

G Gly Glycine

H His Histidine

I Ile Isoleucine

L Leu Leucine

K Lys Lysine

M Met Methionine

F Phe Phenylalanine

P Pro Proline

S Ser Serine

T Thr Threonine

W Trp Tryptophan

Y Tyr Tyrosine

V Val Valine

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1. Literature review

1.1. Snake venom, an overview

Snake venom is a most amazing and unique adaptation of animal evolution. It is one of the most effective and efficient weapon systems of the animal kingdom. Venoms are also produced by animal species of every phylum; examples include the poison in the rounded warts of the skin of toads, the venoms of spiders, scorpions, bees, and other arthropods, and the poison of jellyfish and other coelenterates.

What is snake venom and how does it work? Venom is a prey-immobilizing substance that is used secondarily as a defense system. Venom is not composed of a single substance, but it is a cocktail of hundreds, sometimes thousands of different proteins, enzymes, peptides and chemicals. Proteins constitute the major portion of venom's dry weight – 90% or more. The makeup of these toxins varies widely from species to species. Two general types of toxins are known, neurotoxins and hemotoxins.

Neurotoxic venom targets the victim's central nervous system and usually results in heart failure and/or breathing difficulties. Cobras, mambas, sea snakes, kraits and coral snakes are examples of snakes that produce mainly neurotoxic venom.

Hemotoxic venom targets the circulatory system and muscle tissue causing excessive scarring, gangrene, permanent disuse of motor skills, and sometimes requires amputation of the affected area. The viperidae family such as rattlesnakes, copperheads, and cottonmouths are good examples of snakes that employ mostly hemotoxic venom. The venom of some snakes contains combination of both neurotoxins and hemotoxins.

There are approximately 20 types of toxic enzymes found in snake poisons known to man. That is why it is rightly called as treasure box of enzymes. Although no venomous snake has all of these toxins, most snakes employ between six to twelve of these enzymes in their venom. Each of these enzymes has its own special function.

Some aid in the digestive process, while others specialize in paralyzing the prey.

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Scientists have identified the following enzymes from snake venom and the specific purpose of each as follows:

Cholinesterase; attacks the nervous system, relaxing muscles to the point where the victim has very little control.

Amino acid oxidase; produces hydrogen peroxide, which is an active inducer of cell death. Plays a part in digestion and the triggering of other enzymes (is responsible for venom's characteristic light yellowish coloring).

Hyaluronidase; causes other enzymes to be absorbed more rapidly by the victim.

Proteinase; plays a large part in the digestive process, breaking down tissues at an accelerated rate (causes extensive tissue damage in human victims).

Adenosine triphosphatase; believed to be one of the central agents resulting in the shock of the victim and immobilizing smaller prey (Probably present in most snakes).

Phosphodiesterase; accounts for the negative cardiac reactions in victims, most notably a rapid drop in blood pressure.

The effect of any snakebite necessarily depends on the quantity and kind of toxin it contains, as well as on the resistance of the victim. Immune serum against snake venom, or antivenom, can be prepared by repeatedly injecting sub lethal doses of venom into an animal such as the horse. The immune serum thereby produced in the animal can be extracted and used to treat snakebite victims. Unfortunately about one- third of all recipients have allergic reactions to horse serum. Standard procedure calls for a test for serum sensitivity before giving antivenom to patients. Although certain

"polyvalent" antivenoms can be utilized for certain "groups" of snakes, usually each type of snake has its own specific antivenom.

Besides the obvious benefits of snake venom to produce antivenom, there have been other breakthroughs in medical research. There are many early results from research

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that gives promise on many medical fronts. Researchers in France found out that an enzyme derived from copperhead venom may hold an answer to treatment for breast cancer (Ferrer, 2001). Ingredients from the venom of a Malayan pit viper (Ancrod) has shown promise in breaking blood clots that would be very beneficial in treating stroke victims. Enzymes from cobra venom may hold the keys to finding cures for Parkinson's disease and Alzheimer's disease (Ferrer, 2001). Some viper venom seems to hold the secrets to curing osteoporosis and reducing tumors. Several venom extracts have shown properties that could be utilized in the production of anticoagulants that would be helpful in treating heart disease. Proteins from certain rattlesnakes have been utilized in producing medicine for blood pressure. Ingredients from the red- necked spitting cobra have provided clues to breaking down cell membranes that would provide treatment for leukemia and cancer. Some of the venoms of various snakes have been used medicinally, according to their specific properties, as painkillers (in arthritis, cancer, and leprosy), antispasmodics (in epilepsy and asthma).

The venom of the Russell viper has been used as a coagulant in tonsillectomies and for bleeding gums (Ferrer, 2001). It is obvious that these very complex enzymes derived from snake venom could produce potentially huge medical benefits for mankind. Out of all the enzymes in the snake venom, L-amino acid oxidases are widely studied.

1.2. L-amino acid oxidases

L-Amino acid oxidases (L-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.2.

LAAO) catalyze the oxidative deamination of L-amino acids and produce corresponding keto acids, ammonia and hydrogen peroxide. LAAO’s are not only found in snake and insect venoms, but also found in several fungal, bacterial and algal sources (Niedermann and Lerch, 1991; Nishizawa et al., 2005; Vallon et al., 1993).

LAAO’s can be distinguished as enzymes with quite strict substrate specificity to such which accept a broad substrate range. The substrate specificity of these enzymes has been mainly studied for natural L-amino acids. They generally have a dimeric structure. Most of the L-amino acid oxidases have FAD as the cofactor and hence they exhibit absorption spectra with a maximum absorption at 360 and 460 nm, as well as a

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peak at 280 nm, which is the characteristic of all the proteins (Du and Clemetson, 2002). The physiological role of LAAOs in animals points to a protecting function against natural enemies and bacterial and parasitic infections.

1.2.1. Substrate specificities of L-amino acid oxidases

All the LAAO’s exhibit high stereospecificity that is they are inert towards D-isomers of amino acids. AIP (apoptosis-inducing protein) a homologue of LAAO from Chub mackerel infected with the larval nematode, Anisakis simplex, has special preference to basic L-amino acids such as lysine and arginine (Murakawa et al., 2001). Achacin, which is isolated from African giant snail has shown to have special preference to basic amino acids (Ehara et al., 2002). LAAO from Malayan pit viper utilizes mainly aromatic and hydrophobic amino acids. Out of which phenylalanine, tyrosine and leucine are the best preferred amino acids (Ande et al., 2006). It is also amenable to substrate inhibition, that is high amounts of tyrosine or phenylalanine inhibits the activity of the enzyme (Ponnudurai et al., 1994). LAAO from the venom of cobra is very active against L-Lysine, L-Phenylalanine, L-Leucine, L-Tyrosine, L- Tryptophane, L-Arginine, L-Methionine, L-ornithine, L-norleucine and L-norvaline and moderately active against L-Histidine, L-cystine and L-Isoleucine. Other L-amino acids were oxidized slowly or not oxidized (Li et al., 1994). L-amino acid oxidase from the venom of Trimeresurus mucrosquamatus (Taiwan habu snake); is effective against hydrophobic amino acids such as Leu, Met, Phe, and Tyr. While basic amino acids except for Lys, were oxidized at slower rate (Wei et al., 2003). Recently a 60 kDa monomeric protein called escapin was isolated from the defensive purple ink secretion of the sea hare Aplysia californica (Yang et al., 2005). Sequence analysis suggested that this protein is a flavin-containing L-amino acid oxidase (LAAO), escapinhad high substrate specificity towards either L-arginine or L-lysine and little to nooxidase activity with other L-amino acids (Yang et al., 2005).

Other important properties of LAAO includes, freeze inactivation and inactivation due to pH change. Massey and Curti first reported these properties in 1968 for LAAO from C. adamanteus (Curti et al., 1968). Later on Macheroux et al, indicating that

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LAAO from C. rhodostoma exhibit similar properties supported these findings (Macheroux et al., 2001). As reported by Macheroux et al, there is a slight change in the absorption spectra of active and inactive protein (Macheroux et al., 2001). None of these peculiar properties were reported from other homologues of LAAOs from other sources. It will be interesting to find out whether these properties are highly specific to snake venom LAAOs and if the above mentioned properties are required for the snakes to store these enzymes in an inactive form and gets activated once introduced into the prey, when it bites.

1.2.2. Structural properties

Preliminary crystallization data were first available for the LAAO from the venom of Agkistrodon contortrix laticinctus. It was crystallized up to a resolution of 3 Å (Souza et al., 1999). NMR analysis of this protein showed that it has FMN as a cofactor (Souza et al., 1999). Among LAAOs, L-amino acid oxidase from C. rhodostoma was well studied in response to the structural properties of the enzyme (Geyer et al., 2001;

Pawelek et al., 2000). It has been crystallized up to the final resolution of 2 Å. The comparison of structure of LAAO with the related oxidase called D-amino acid oxidase was well studied (Pawelek et al., 2000). According to this paper, LAAO is a homodimeric and glycosylated flavoprotein containing FAD as the cofactor. Asn 172 and Asn 361 are the glycosylation sites (Pawelek et al., 2000). Each subunit of LAAO contains three domains: Substrate binding, helical domain and FAD binding domain (Fig 2). There is a channel present at the active site, which allows the substrate to have access to the active site. The same channel is probably used for the release of the products. Interestingly one of the glycan moieties is located in the vicinity of the channel (Pawelek et al., 2000).

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Fig 1. 3D Structure of LAAO from Malayan pit viper.

Active center of LAAO contains a long channel, which allows the substrate to enter and allow the release of products. Near the vicinity of the channel there is a glycan moiety at Asp 172 (Adapted from Pawelek et al, 2000).

Asp 172

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Fig 2. Structure of LAAO from Malayan pit viper.

LAAO from Malayan pit viper is a homodimeric and glycosylated protein. It has FAD binding domain (Red), helical domain (Blue) and substrate binding domain (Green). At the end of each glycan moiety there are sialic acids attached (Adapted from Geyer et al, 2001).

Later on, the same group has reported the complete characterisation of the glycan moiety (Geyer et al., 2001). The glycan moiety is confirmed to be a bis-sialylated, biantennary and core fucosylated dodecasaccharide (Geyer et al., 2001). The same report suggested that sialic acids attached to the enzyme might have a role in its biological properties such as induction of apoptosis and antibacterial activity.

Recently L-amino acid oxidase from Agkisrodon halyspellas venom has been crystallized up to a resolution of 2.5 Å and partially characterized (Zhang et al., 2004a). Its molecular weight was confirmed to be 60.7 kDa by MALDI-TOF mass spectroscopy. They correlated with the crystal structure of LAAO from C.

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Rhodostoma and shown that both the LAAOs have similar structure (Zhang et al., 2004a). Presently these are the two LAAOs whose crystal structures have been solved.

1.2.3. Expression of L-amino acid oxidases

Different research groups have attempted the overexpression of L-amino acid oxidases (Ogawa et al., 1999; Torii et al., 2000). But, it has always been difficult to over express proteins of this sort, as it produces hydrogen peroxide upon oxidative deamination of L-amino acids. This results in a high toxicity to cells. Even though they overexpressed, the amount of functional protein they got was very less. Over expression studies of achacin, a protein from the body surface mucus of giant snail Achacina fulica Ferussac in methylotropic yeast Pichia pastoris, yielded about 0.2 mg / liter (Ogawa et al., 1999). Due to the low amount of protein yields, characterization has been difficult. Torii et al have claimed overexpression of apoxin, a LAAO from western diamondback rattlesnake in mammalian cells in 2000 (Torii et al., 2000).

Transfection of full-length apoxin into human 293T cells generated a significant amount of apoxin that was secreted into the medium. This secreted protein was apparently active and possessed apoptotic inducing activity. However, some amount of protein was expressed inside the cells and this protein did not show any activity.

Deletion mutants affecting the FAD binding domain, the carboxy terminal domain and regions flanking the signal sequence lead to a system that did not secrete active protein into the medium. Furthermore, the protein that was formed inside the cell was found to be inactive. Based on their results they suggested that N-glycosylation is required to produce functionally active protein that can be secreted into the medium (Torii et al., 2000).

A recent report deals with the expression of an L-amino acid oxidase from Rhodococcus opacus in E. coli and Streptomyces lividans (Geueke and Hummel, 2003). This LAAO, when over expressed in E. coli went into the insoluble fractions and was found to be inactive. But when over expressed in Streptomyces lividans, they could get significant amount of protein in soluble fractions. These results indicate that

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Streptomyces lividans is an important hosts for the expression of toxic proteins that require glycosylation such as LAAO (Geueke and Hummel, 2003).

Recently, IL-4 Induced gene-1 or FIG 1 found in B-lymphocytes grabbed the attention of the researchers. The sequence of this gene is highly similar to that of apoxin (Chavan et al., 2002). Furthermore its expression is limited to the pre immune tissues and genetically maps to a region of susceptibility to autoimmune disease. Its expression is found in antigen presenting cells such as dendritic cells and macrophages (Chavan et al., 2002). Fusion of full-length gene of IL-4 induced gene to EGFP and ds-red and subsequent transfection into NIH3T3 cells resulted in significant protein amounts in the insoluble fractions. Interestingly, no significant protein amounts were found in the supernatant or in the soluble fractions of the cells.

These findings were proved to be coherent as they found the significant activity of this protein in insoluble fractions but not in supernatant and soluble fractions (Mason et al., 2004). By the immunofluorescence studies it was shown that IL-4 induced protein preferentially localizes to lysosomes.

A recent study was reported on the expression of a homologue of LAAO called rebeccamycin L-amino acid oxidase (Nishizawa et al., 2005). It is from the strain Lechevalieria aerocolonigenes ATCC 39243. This LAAO has 27% identity to an LAAO from Scomber japonicus and is a member of the family of FAD-dependent oxidase enzymes. This enzyme was stably expressed in E. coli. Its expression was possible in E. coli, because it was co- expressed with the aid of a plasmid called pG- JKE8. This plasmid is responsible for the prevention of aggregation and degradation of the enzyme. Overexpression of just the full length of this protein without the plasmid yielded poor amount of rebeccamycin oxidase (Nishizawa et al., 2005). It is also important to note that this protein is unglycosylated and hence it could have been feasible for overexpression.

The experiments described above dealing with the expression of LAAO indicate that this endeavor might be difficult due to the intrinsic properties of the enzyme, i.e. its production of hydrogen peroxide and its probable degradation/depletion of essential

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amino acids within the host cell. These last factors might turn out to be lethal for the cell.

1.2.4. Antimicrobial activity of LAAO

One of the important features of LAAO is its antimicrobial activity. Skarnes first reported the antibacterial nature of LAAO in 1970 (Skarnes, 1970). Therein it was stated that LAAO from C. adamanteus is active against both gram positive and gram- negative bacteria. One of the interesting points in this article concerned D-amino acid oxidase (DAAO). DAAO is an enzyme similar to LAAO and produces the same products hydrogen peroxide and ammonia upon oxidative deamination of D-amino acids. However, it was shown to have no antibacterial activity. They used mainly D- serine as the substrate for DAAO, at all the used concentrations of D-serine, it was proved not to be antibacterial (Skarnes, 1970). These findings showed that there could be different mechanisms for antimicrobial activity by LAAO.

Later on Stiles et al in 1991 made quite extensive studies on the antibacterial effects of snake venoms (Stiles et al., 1991). They tried venoms from different snakes such as C. scutulatus, C. adamanteus, Psuedechis australis and Echis carinatus. All these venoms proved to be antibacterial. More specifically they could isolate the antibacterial components from the snake venom of Psuedechis australis (Australian king brown or mulga snake) and these antimicrobial components were found to be LAAO 1 and LAAO 2 (Stiles et al., 1991). They also concluded that the majority of antibacterial effects seen in the elapid venoms were due to the L-amino acid oxidase.

They used different aeromonas strains of bacteria, which are infectious agents in humans, reptiles and amphibians. Compared to a tetracycline drug, which is generally used for the infections caused by these aeromonas strains, the antibacterial effects of LAAO 1 and LAAO 2 are 70 and 17.5 times more effective respectively than tetracycline (Stiles et al., 1991). In the above reports, it has been shown that hydrogen peroxide generated by the enzyme played a key role in the antibacterial activities.

Addition of catalase, a prominent scavenger of hydrogen peroxide prevented the antibacterial activity (Skarnes, 1970; Stiles et al., 1991).

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Further studies were made on the antibacterial activity of achacin; a glycoprotein isolated from the African giant snail and is a close homologue of LAAO (Ogawa et al., 1999). It was shown to be active against gram positive and gram-negative bacteria.

It appears to attack the plasma membrane of the bacteria and was shown to induce extensive filamentation in E. coli (Otsuka-Fuchino et al., 1993). The report by Ehara et al, showed that achacin inhibits the growth of S. aureus and E. coli. At minimum inhibitory concentrations, achacin could produce about 0.2 to 0.4 mM of hydrogen peroxide. But the minimum inhibitory concentrations of hydrogen peroxide required to kill the bacteria was 0.7 to 1 mM. These results indicate that hydrogen peroxide produced by achacin was not sufficient to kill the bacteria. However, they showed that achacin specifically binds to the growth phase specific bacteria and this binding is responsible for exerting maximum antibacterial activity even though it produces low amounts of hydrogen peroxide which is not sufficient to kill the bacteria (Ehara et al., 2002). So, hydrogen peroxide produced in the medium and the local concentrations of hydrogen peroxide generated by binding may have a cumulative effect and that helped achacin to have antimicrobial activity. These findings are similar to those of Suhr et al (1999), wherein it is shown that LAAO binds to the cells and generates a local concentration of hydrogen peroxide.

Another factor they looked at was, whether sialic acid (Neu5Ac) could inhibit the interaction of achacin to the bacteria. According to this study, concentrations up to 2 mg/ml could not block the interaction of this enzyme to the bacterial cells. At higher concentrations of Neu5Ac, it inhibited the enzyme activity of achacin (Ehara et al., 2002).

Other possible reason for the antibacterial activity of LAAO has been put forward by Mitsuru et al in 2003 (Mitsuru et al., 2003). According to them, aplysianin A, a homologue of LAAO induced antibacterial effect on Bacillus subtilis. This effect was mainly contributed to the hydrogen peroxide generated by this enzyme. Interestingly, aplysianin A in the presence of catalase could induce antibacterial activity. The reason for the latter effect was shown to be rapid depletion of L-arginine from the medium

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(Mitsuru et al., 2003). This shows that also depletion of amino acids might play a role in the antibacterial activity of the LAAO.

A new LAAO from Crotalus durissus cascavella venom has been isolated recently (Toyama et al., 2006). It presented high sequence similarities with other snake venom LAAO’s such as Calloselasma rhodostoma and Crotalus adamanteus. This showed antibacterial properties against gram positive and gram-negative bacteria (Toyama et al., 2006). Antibacterial activity exhibited by the enzyme was mainly contributed for the hydrogen peroxide produced by this enzyme. In the presence of catalase, antibacterial activity was completely suppressed. Hydrogen peroxide produced by this enzyme induced bacterial membrane rupture and consequently promoted extravasation of plasmatic content to the out side of the cells. This was the first report that indicated that LAAO can cause the bacterial membrane rupture (Toyama et al., 2006).

From all the above literature reports it appears that LAAO’s exhibit antibacterial properties. The primary attribute to this effect was, for sure, production of hydrogen peroxide. In this context its worth mentioning the recent work by Zhang et al, where it is shown that LAAO causes antibacterial activity through binding to the cell surface as previously reported by Ehara et al, in 2002. One more interesting fact they put forward was the antibacterial action of DAAO. They could find out that DAAO could elicit antibacterial effect and at the same time it interacts with the cell surface (Zhang et al., 2004b). This is contradictory to the work by Skarnes (1970), wherein it was reported that DAAO did not elicit any antibacterial effect.

Apart from the antibacterial effects, LAAO’s have been reported to possess antiviral activity (Zhang et al., 2003). According to them, LAAO from Trimeresurus stejnegeri has the potential to inhibit the HIV-1 virus. It inhibited the replicative action and infection of virus. But, addition of catalase did not result in the complete inhibition of the antiviral activity. At the same time, exogenous hydrogen peroxide did not show any antiviral activity (Zhang et al., 2003). These results once again pinpoint the

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specific action of LAAO that appears to be different from that of exogenous hydrogen peroxide.

Leishmania species are responsible for causing leishmaniasis or sleeping sickness in human beings. This disease affects around 12 million people annually worldwide. The snake venom of Bothrops moojeni was shown to inhibit the growth of Leishmania species. LAAO that was present in this venom was shown to be the causative agent.

Hydrogen peroxide produced by LAAO was the sole cause for this killing and can be completely rescued by the addition of catalase (Tempone et al., 2001).

In conclusion, LAAOs from different organisms exhibit different properties such as antibacterial activity, antiviral activity and they are also active against intracellular parasites such as Leishmania species. More detailed understanding on the mechanisms of these effects caused by LAAO will help to gives us more clues and if possible making it a wonderful drug in the field of therapeutic approaches.

1.2.5. Induction of cell death by L-amino acid oxidases

There are different forms of cell death that occur in higher organisms. Well-studied cell death processes include apoptosis and necrosis. L-amino acid oxidases are shown to induce cell death in a variety of mammalian cells. The first report by Araki et al, shows that LAAO induces apoptosis in vascular endothelial cell lines. Later on Suhr and Kim demonstrated that LAAO present in the snake venom as the causative agent for apoptotic cell death. Later on several publications revealed that LAAO induces apoptotic cell death. For example, LAAO has been shown to induce apoptosis in mouse lymphocytic leukemia, human T cell leukaemia cell lines (Suhr and Kim, 1996; Suhr and Kim, 1999) human promyelocytic leukaemia cell and human embryonic kidney cells (Torii et al., 1997; Torii et al., 2000). Hydrogen peroxide produced by this enzyme was the causative agent for cell death. The report by Suhr and Kim shows that apoptotic cell demise caused by LAAO is different from the apoptotic effects caused by external hydrogen peroxide (Suhr and Kim, 1999). They could also show that LAAO interacts with the cell surface and thereby generates

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hydrogen peroxide locally and this seems to be the governing factor for cytotoxic effects. They came to the conclusion based on experiments using different cell lines whereby the intensity of effects varied from cell line to cell line. This is one of the reports in early 1990’s that gave a better understanding of the mechanism of cytotoxic action of L-amino acid oxidase.

Ali et al in 2000, provided evidence that LAAO from leaf nosed viper could cause cell death in monocytic cell line derived from the blood of a patient with monoblastic leukaemia. Purified LAAO showed typical markers of apoptosis such as chromatin condensation, nuclear fragmentation (Ali et al., 2000). Later on in 2001, AIP (apoptosis-inducing protein) purified and cloned from Chub mackerel infected with the larval nematode, Anisakis simplex, has been shown to induce apoptosis in various mammalian cells including human tumor cell lines (Murakawa et al., 2001).

Murakawa et al, reported two different mechanisms of action of this homologue of LAAO: One is dependent on hydrogen peroxide produced by the enzyme and the other one, a slower cell death, which is proved to be independent of hydrogen peroxide (Murakawa et al., 2001). The first mechanism was solely due to the rapid production of hydrogen peroxide as the addition of hydrogen peroxide scavengers like catalase could completely abolish the cell death. This cell death was shown to be having typical markers of apoptosis such as, DNA fragmentation and exposure of phosphatidyl serine on the outer membrane. zVAD-fmk, a broad-spectrum caspase inhibitor could completely inhibit this apoptotic process.

The other cell death was a slower process and took place after 24 h, even in the presence of hydrogen peroxide scavengers like catalase. This cell death was shown to occur in the hydrogen peroxide resistant cell lines also. They observed that depletion of L-lysine from the medium was the actual cause for this cell death. AIP mainly uses lysine from the medium, which was its best substrate and there by lysine gets depleted slowly from the medium. This was shown to be the cause as the addition of lysine into the medium could abolish the cell death. This also showed the features of apoptosis.

Both these processes were inhibited by Bcl-2 over expressed cell lines, indicating that

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apoptosis by LAAO is caused by mitochondrial dependent pathways (Murakawa et al., 2001).

Okinawa Habu apoxin protein-1 (OHAP-1), isolated from Okinawa Habu (Trimeresurus flavoviridis) venom is a LAAO (Sun et al., 2003). It is the first report to show that a homologue of LAAO could induce apoptosis in glioma cell lines (Sun et al., 2003). They tested on various glioma cell lines such as C6, RBR17T, and U251 cells. Incubation of LAAO with these cell lines showed typical markers of apoptosis such as DNA fragmentation and chromatin condensation (Sun et al., 2003). They aimed at measuring reactive oxygen species and expression levels of p53 protein after incubation with OHAP-1. They observed rapid enhancement of reactive oxygen species inside the cell and higher levels of p53 expression (Sun et al., 2003).. These results suggested that OHAP-1 would be a promising agent in the treatment of tumor therapy (Sun et al., 2003).

Dolabellanin, an antineoplastic protein from an ocean mollusk, has been shown to have L-amino acid oxidase activity and induced cytotoxicity on EL-4 murine lymphoma cells (Iijima et al., 2003). Hydrogen peroxide produced by this enzyme has been shown to be responsible for cytotoxic activity (Iijima et al., 2003). But addition of catalase could not completely rescue the cells from cytotoxic effect. The cytotoxic effect generated by dolabellanin was apoptosis. This was shown by measuring caspase 3 activity and DNA fragmentation (Iijima et al., 2003). In 2004, achacin a homologue of LAAO was examined for its cytotoxic effects (Kanzawa et al., 2004). Achacin was able to induce cytotoxic effects on HeLa cell lines by two different mechanisms.

When incubated with different concentrations of achacin, it could induce cell death in a concentration dependent manner. This type of cell death was faster and could not show typical characteristic features of apoptosis. But, when achacin was incubated in the presence of catalase, it could induce a slower cell death and it is observed after 48 h of incubation. By examining the cell death, it showed DNA fragmentation and PARP cleavage and caspase activity, which are typical markers for apoptosis. zVAD- fmk could inhibit the process of apoptosis. Achacin used Arg, Leu, Lys, Met, Phe and Trp as the substrates from the medium. Depletion of these amino acids has been

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shown to be the cause for the apoptotic cell death by achacin. Cells cultured in the absence of above amino acids resulted in the apoptotic effect (Kanzawa et al., 2004).

This clearly indicates that depletion of amino acids resulted in apoptosis.

Recent report by Butzke et al in 2004, showed that a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata, marine snail of the genus Aplysia triggers cell death with profound tumor specificity (Butzke et al., 2004). It was shown to be a homologue of L-amino acid oxidase. Cytotoxic effect of APIT was analyzed on both tumor and non-tumor cell lines. Jurkat, which is a typical tumor cell line and PBMC (Peripheral blood mononuclear cells) and HUVEC (Human umbilical vein endothelial cells) cell lines, which are non-tumor cell lines were used for the experiments. It induced cell death only in Jurkat cell line but not in non-tumor cell line, indicating its tumor specificity. The cell demise induced by APIT showed markers independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Addition of catalase, a scavenger of hydrogen peroxide completely rescued the cells from cytotoxic effect.

Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides.

Interestingly, knockdown of peroxiredoxin I with RNA interference, hypersensitized the cells for APIT induced cell death. From these results they concluded that knock down of periredoxin systems in a hydrogen peroxide induced stimulus could be the good therapy for fighting against the tumor cell lines (Butzke et al., 2004). Another novel member of LAAO was from Agkistrodon halys pallas venom, and it has been named as AHP-LAAO. Experimental evidences shows that, it induces apoptosis in Hela cell lines (Zhang et al., 2004a).

By closely examining the above-mentioned reports, it can be concluded that hydrogen peroxide generated by the enzymatic action of LAAO is the main cause for its cytotoxic action. A couple of publications highlight the possibility that there are also hydrogen peroxide independent actions of LAAO, namely depletion of L-amino acids by LAAO, which is the major point for the independent effects. Report by Suhr and Kim states that cytotoxic effects caused by LAAO were different from the hydrogen

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peroxide induced effects. They could show that LAAO can cause local concentrations of hydrogen peroxide around the cell by binding to the cell. It could also be that local concentrations of hydrogen peroxide, accompanied by amino acid depletion act cumulatively and can lead to the cell death. Any way, deeper insights into the mechanism of cytotoxic action of LAAO would certainly enhance the opportunities for LAAO to become a good antitumor agent.

1.2.6. Platelet aggregation

There are controversial reports on effects of snake venom on platelet functions.

Venom from variety of snakes has been tested; some snakes inhibit platelet aggregation and others potentiate the process. Nathan I et al, was the first report in 1982 to demonstrate that snake venom from Echis colorata induces impairment of platelet aggregation (Nathan et al., 1982). LAAO present in the snake venom was the shown to be the causative agent. Addition of catalase, completely inhibited the impairment of platelet aggregation (Nathan et al., 1982). This is an indication that hydrogen peroxide produced by the enzyme played an important role in the impairment of platelet aggregation.

Later on Li et al, worked on LAAO from king cobra (Li et al., 1994). They purified LAAO from king cobra and addition of this protein to the platelet rich plasma system completely induced platelet aggregation. This observation was in contrast with the findings of Nathan et al in 1982 (Nathan et al., 1982). Enzyme induced aggregation was completely abolished by the addition of catalase. There are two pathways described for platelet aggregation namely ADP release and formation of thromboxane A2 and prostaglandin endoperoxides. Using of creatine phosphate, which is an ADP scavenging system, did not inhibit the platelet aggregation induced by LAAO (Li et al., 1994). This shows that LAAO induced platelet aggregation is independent of ADP scavenging system. LAAO induced platelet aggregation was completely rescued by the addition of indometacin, aspirin, arachidonic acid (Li et al., 1994). Indometacin or aspirin is a cyclooxygenase inhibitor and this clearly indicates that LAAO mediated platelet aggregation is mediated by formation of prostaglandins or thromboxane A2

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(Li et al., 1994). Mepacrine, an inhibitor of the activation of endogenous phospholipase A2, completely inhibited the LAAO induced toxic effect (Li et al., 1994). Therefore, it clearly shows that activation of endogenous phospholipases is necessary for the aggregation process. EDTA, a chelator of calcium ions and verapamil, an inhibitor of calcium influx, inhibit LAAO induced platelet aggregation (Li et al., 1994). Prostoglandin E1 which is an activator of adenylate cyclase, nitroprusside activator of soluble guanylcyclase completely, inhibit LAAO induced aggregation effects by increasing the cyclic AMP and cyclic GMP concentrations (Li et al., 1994). Because cyclic AMP and cyclic GMP inhibit calcium mobilization, and calcium is required for platelet aggregation because it is required for thromboxane A2 synthesis (Li et al., 1994). On a whole, LAAO induces platelet aggregation by formation of hydrogen peroxide, which will in turn activate thromboxane A2 followed by activation of cyclooxygenases, which are prerequisite for platelet aggregation.

A report by Ali et al in 2000 demonstrates that LAAO from leaf nosed viper (Eristocophis macmahoni) induced platelet aggregation in platelet rich plasma in a concentration dependent fashion (Ali et al., 2000). Addition of catalase completely rescued platelets from aggregation. Interestingly, when they used whole venom from the snake, they could observe quite opposite effects, that is inhibition of platelet aggregation (Ali et al., 2000). Takastuka et al in 2001, worked on L-amino acid oxidase from Agkistrodon halys blomhoffii and looked at its effects on platelet aggregation (Takatsuka et al., 2001). They put forward the hypothesis that LAAO did not induce platelet aggregation but, instead, it inhibited the effect. LAAO could inhibit agonist and shear stress induced platelet aggregation effects in a concentration dependent manner. Addition of catalase, an oxidative scavenger, completely quenched the impairment of platelet aggregation (Takatsuka et al., 2001). Shear stress or agonist induced platelet aggregation effects are due to the continuous interaction of platelet integrin alphaIIb/beta3 and fibrinogen. Continuous generation of hydrogen peroxide produced by LAAO, inhibited the interaction between platelet integrin alphaIIb/beta3 and fibrinogen and hence leading to the impairment of platelet aggregation (Takatsuka et al., 2001).These results in turn suggest that bite from the above snake causes prolonged bleeding from the vessels, which, in turn, leads to fatal effects.

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Sakurai et al, examined the effect of LAAO from Naja naja kaouthia venom on platelet functions (Sakurai et al., 2001). They noted that LAAO did not induce platelet aggregation at various concentrations. However, it has been shown that it could inhibit the platelet aggregation induced by ADP, collagen or ristocetin in a dose dependent manner. Aggregation induced by shear stress, has been completely inhibited by LAAO. Addition of catalase, completely quenched the event of inhibition of platelet aggregation (Sakurai et al., 2001). These results indicate that hydrogen peroxide produced by the enzyme is the main governing factor. Another report was about L-amino acid oxidase from Trimeresurus jerdonii snake venom, which has been shown to induce platelet aggregation in platelet rich plasma system. Addition of catalase rescued platelets from aggregation (Lu et al., 2002).

Stabeli et al in 2004, reported that L-amino acid oxidase from Brothropus alternus snake venom known as Balt LAAO-1 could induce platelet aggregation in platelet rich plasma and also in washed platelets in a concentration dependent manner (Stabeli et al., 2004). Recently, Toyama et al, demonstrated that LAAO from Crotalus durissus cascavella venom induced platelet aggregation in platelet rich plasma in a dose dependent manner (Toyama et al., 2006). Addition of catalase rescued platelets from undergoing aggregation. Apart from that, they observed that indometacin, which is an inhibitor of activation of endogenous phospholipase A2 could block the effect of platelet aggregation induced by LAAO (Toyama et al., 2006). Aspirin, which is a general inhibitor of cyclooxygenase pathway was able to inhibit aggregation induced by LAAO (Toyama et al., 2006). These results suggest that hydrogen peroxide generated by LAAO is the main causative agent, which in turn activates inflammatory enzymes such as thromboxane that leads to platelet aggregation.

To conclude, LAAOs from the snake venom potentiates platelet aggregation and at the same time can inhibits the aggregation process. Literature reports suggest that platelet aggregation induced by LAAO is mainly due to the production of hydrogen peroxide, as the addition of catalase, completely rescues platelets from aggregation.

There are also possibilities that produced hydrogen peroxide may activate other inflammatory enzymes that are responsible for platelet aggregation. This was evident

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as the addition of inhibitors of phospholipases could inhibit the platelet aggregation induced by LAAO. Before coming to specific conclusion, it would be worthwhile to see, if these specific inhibitors could inhibit the activity of L-amino acid oxidases.

Impairment of platelet aggregation by LAAOs has been observed in some snake venoms and produced hydrogen peroxide is the main factor for the impairment.

Oxidative scavengers completely abolish the impairment. On a whole, LAAOs are unique enzymes with efficient physiological functions. A thorough understanding of the mechanisms of their action on platelet functions could lead to a better understanding of the snake venom.

1.3. Apoptosis- Introduction

Proper development of embryo to become a full-fledged organism requires commendable strategies, efficient and co-ordinated mechanisms and actions of various cellular machinery. In the process of cellular development and throughout the development of organism into a successful adult, some cells have to die and give opportunity for the fresh cells to develop. Now, the question arises, why do only some cells need to die and why not the other cells? If they die, by which process do they die? These basic questions will move us to a better understanding of various cell death processes, their importance and molecular mechanisms involved in it.

As outlined above, basically there are two types of cell death, which take different routes to the same end. Although there are few other cell deaths types such as onkosis apoptosis and necrosis are widely studied. Apoptosis, the word was introduced by Kerry in 1972, based on the Greek word which means ‘falling of or dropping of”, in analogy to leaves falling off trees or petals dropping of flowers (Kerr et al., 1972).

Apoptosis is a highly regulated form of cell death in which the cell contains necessary information to die on its own. Once the decision to die is taken, there is a proper execution of apoptotic program, which requires co-ordinated activation and execution of several other multiple sub programs. That is why; apoptotic process is rightly called as programmed cell death.

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1.3.1. Morphological features of apoptosis and necrosis

Apoptosis and necrosis were initially characterized by the distinct differences in physical attributes of the affected cells. Necrosis normally results from a severe insult.

In this process, first the integrity of the plasma membrane is lost and then the internal organelles get affected. Thus, it leads to the spilling of cytosolic contents and organelle contents to the surrounding environment. Immune cells are attracted to this area and they will release cytokines that generate inflammatory response. But during the process of apoptosis all the changes takes place in an orderly manner. First cell shrinks; chromatin gets condensed, followed by fragmentation of DNA and then cell forms into apoptotic bodies. These apoptotic bodies are eventually taken up by the phagocytes without damaging the surrounding cells (Fig 3).

.

Fig 3. Represents the cell undergoing different types of cell death namely apoptosis and necrosis.

(Adapted from the book Ciclo de apoptosis).

There are several techniques that have been used to distinguish apoptotic cells from necrotic cells. One is exposure of phosphatidyl serine to the outer surface, which is recognized by annexin V staining. In the process of apoptosis, DNA gets cleaved into small fragments, and this can be visualized by examining the DNA of the apoptotic cells. We can also examine the cells by visualization through a fluorescence microscope after staining the cells with commercially available DNA staining dyes.

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Table 1 shows the morphological, biochemical and physiological differences between apoptosis and necrosis.

Table 1. Represents the basic differences between apoptosis and necrosis (This was adapted from Apoptosis, Cell death and Cell proliferation manual from Roche-applied sciences)

1.3.2. Significance of apoptosis

Apoptosis or programmed cell death plays a crucial role in a wide variety of physiological processes. During the fetal development, many cells are produced in excess and these will eventually undergo programmed cell death to form complete organs and tissues. In the course of development of an organism into a mature adult,

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many cells sacrifice themselves by the process of apoptosis and give rise to a matured adult. As a whole, apoptosis plays a crucial role in the development and maintaining equilibrium of the body (Meier et al., 2000). It plays an important role in the proper development of the immune system. For example, in the process of T-cell proliferation, only the matured cells are positively selected, but immature cells are taken out by the process of apoptosis (Vaux, 1993). Besides these, apoptosis plays a vital role in eliminating the dangerous cells such as tumor cells, cells infected with pathogens and cells defective in their function (Vaux, 1993). Defect in apoptosis program or deregulation in apoptotic process results in cancer, autoimmune diseases and spreading of viral infections. Excessive apoptosis could lead to development or enhancement of neurodegenerative diseases, Ischemic diseases (Stroke, myocardial infraction) and AIDS (Fadeel et al., 1999).

Due to the importance of programmed cell death in various biological processes, this phenomenon has been widely studied in mammals, insects (Richardson and Kumar, 2002), cnidarians (Cikala et al., 1999) and nematodes (Liu and Hengartner, 1999). It has been shown that, even plants can undergo programmed cell death (Solomon et al., 1999). In the recent years, it has been shown that monocellular organism like yeast can also undergo apoptosis (Madeo et al., 1999).

1.3.3. Caspases

Apoptosis is an evolutionary conserved process and requires specialized form of cellular machinery. Such machinery is a proteolytic system involving a family of proteases called caspases. About thirteen caspases have been identified in humans and most of them have been shown to have a functional role in the process of apoptosis (Earnshaw et al., 1999; Thornberry and Lazebnik, 1998). Caspases are cysteine proteases and are highly conserved through evolution. They usually possess cysteine at the active site and cleave the substrate after aspartic acid residues. Recognition of at least four amino acids NH2-terminal to the cleavage site is a necessary requirement for the efficient catalysis. Caspases have been divided into subfamilies based on their substrate specificity, extent of sequence identity and structural similarities. Because

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these bring out most of the changes that characterize the apoptotic cell death, they are often recognized as central executioners of the apoptotic pathway (Thornberry and Lazebnik, 1998).

All the caspases are expressed as proenzymes called zymogens. They contain three domains, namely an NH2-terminal domain, a large subunit and a smaller subunit. The larger subunit is usually of around 20 kDa and the smaller one around 10 kDa.

Activation of these enzymes involves cleavage between these domains that leads to formation of heterodimer by the association of large and small subunits. Two heterodimers associate to form a functional tetramer (Thornberry and Lazebnik, 1998). Till now, crystal structures of two active caspases, caspase-1 and caspase-3 have been determined (Thornberry and Lazebnik, 1998). How can caspases contribute to the complex process of apoptosis? The overall picture is not clearly understood till now. One role of caspases is the inactivation of cellular proteins that protect the living cell from undergoing apoptosis. A good example is the cleavage of I^CAD / DFF45, an inhibitor of a nuclease responsible for DNA degradation, CAD (caspase activated deoxyribonuclease) (Thornberry and Lazebnik, 1998). In the normal cells CAD is present in an inactive complex with I^CAD. During the process of apoptosis I^CAD is inactivated by caspases leaving CAD free to function as a nuclease. CAD synthesized in the absence of I^CAD in not active, indicating that the CAD- I^CAD complex is formed co-translationally and that ICAD is required for both activation and inhibition of this nuclease.

Another important role of caspases is their contribution to the direct disassembly of cell structures such as nuclear lamina (Thornberry and Lazebnik, 1998). This is a rigid structure underlying the nuclear membrane and is involved in chromatin organization.

This lamina is made up of intermediate filamentous proteins called lamins. During the process of apoptosis, the caspases and leads to the dismantling of lamina cleave these lamins. This in turn contributes to condensation of chromatin. Its also important to note that caspases inactivate or deregulate the proteins involved in various cellular mechanisms such as DNA repair and replication (Thornberry and Lazebnik, 1998).

Caspases play a vital role in regulation of the Bcl-2 family of proteins.

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Most of the biochemical and genetic evidences suggests that caspases are activated in a cascade. First of all when the cell gets the apoptotic stimulus, initiator caspases gets activated. These in turn activate the cascade of effector caspases. In the case of apoptosis due to the activation of death receptors, caspase-8 gets activated initially, which in turn activates other caspases (Susin et al., 1996). In the case of apoptosis due to cytotoxic agents, caspase-9 gets activated and in turn activates other effector caspases. So, it’s like a cascade mechanism, but depending on the initial insult, the initiator caspase will differ. Because of the crucial role of caspases in the apoptotic process, it is worthwhile to investigate the mechanisms by which these caspases gets activated in cancer cells. One disadvantage is that these mechanisms should be specific to cancer cells otherwise it will affect normal cells and leads to extensive apoptosis and hence to death also of a normal cell.

1.3.4. Bcl-2 Family proteins

Bcl-2 was first discovered in 1988 in B-cell lymphomas (Vaux et al., 1988). B-cells transfected with Bcl-2 were shown to be resistant towards apoptosis, normally induced in B-cells by IL-3 withdrawal (Vaux et al., 1988). Bcl-2 family proteins are conserved throughout evolution with homologues found in mammals, avian, fish, and amphibian species as well as in invertebrates such as C. elegans, Drosophila and marine sponges (Reed, 2000). Most of the Bcl-2 family proteins are constitutively localized to the membranes of mitochondria and some of these proteins are also found in the endoplasmic reticulum and nuclear envelope (Reed, 2000). Till now, about 20 proteins of this family have been identified. Many of the Bcl-2 family of proteins are crucial as they function as regulators of the fate of the cells. We can categorize them into two different types, based on their role in apoptosis. One group is called as pro apoptotic, that is they initiate the apoptotic process. The other group is anti apoptotic, they potentiate the inhibition of apoptosis. Mitochondrial dependent apoptotic pathways are generally due to the regulated action of these proteins.

Based on the predicted or experimentally determined three-dimensional structures, Bcl-2 family proteins can be broadly divided into two groups (Reed, 2000). One

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subset of these proteins are similar to the structure of pore forming domains of bacterial toxins such as colicins and bacterial toxins (Reed, 2000). These include anti apoptotic proteins such as, Bcl-2, Bcl-XL, Mcl-1, Bif-1 Bcl-W and Boo and pro apoptotic proteins such as Bax, Bak, Bok and Bid. Other subsets of Bcl-2 family proteins are those who have only the presence of BH-3 domains. They include Bad, Bik, Bim, Hrk, Bcl-Gs, p193 and APR (Reed, 2000). All these proteins are pro apoptotic in their function and the cell death inducing activity depends on their ability to form dimers with the anti apoptotic Bcl-2 members.

There are reports that Bcl-2 family members regulate the release of cytochrome c from mitochondria, hence regulating the apoptotic process (Reed, 1997). One good example is Bcl-2. This protein prevents the release of cytochrome c from mitochondria, and in turn this prevents the cells from undergoing apoptosis.

Conversely, BID, which is a pro-apoptotic member; mediates the release of cytochrome c from mitochondria, which in turn forms the apoptosome by interacting with Apaf-1 and caspase 9. This leads to the apoptotic cell demise. BID is also capable of interacting with both pro apoptotic and anti apoptotic Bcl-2 proteins. As predicted from their structure, Bcl-2, Bcl-XL and Bax can form ion channels when they added to the synthetic membranes. Deletion of pore forming domains could abolish the channel formation by Bcl-2 and Bax in the synthetic membranes (Schendel et al., 1997). It is interesting to note that, apart from inhibiting caspase dependent apoptosis, in some of the cases Bcl-2 can also prevent hypoxia induced necrosis (Jurgensmeier et al., 1998).

.

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2. Aim

L-amino acid oxidase (LAAO) is a classical flavoprotein. Because of its ease of purification and stability, it has attracted the attention of researchers over the past and present years. In the case of Malayan pit viper (Calloselasma rhodostoma), 30% of its venom constitutes LAAO. LAAO from Malayan pit viper is a homodimeric, glycosylated protein containing non covalently bound FAD. Asp 172 and Asp 361 are the glycosylation sites. The crystal structure of this protein was solved and its glycan moiety has been completely characterized. Hydrogen peroxide produced by its enzymatic activity was shown to be necessary for all its physiological properties.

However, the role of the glycan moieties in the physiological effects has not been studied in detail. The three-dimensional structure of LAAO shows that there is a long funnel that leads to the active center, which allows for substrate entry, and the same funnel is used for the release of the products hydrogen peroxide and ammonia.

Interestingly, there is a glycan moiety near the active center. The terminus of the glycan moiety contains sialic acids, which are cell recognition molecules and bind to the cells with the help of specific receptors called sialic acid binding lectins (Sig-lecs).

Based on this, it has been hypothesized that LAAO might bind to the cells with the help of sialic acids and thereby generate high local concentrations of hydrogen peroxide. This might be an important factor in the course of cell death.

The aim of this work was to study a) the role of LAAO from Malayan pit viper in inducing cell death, b) to understand the molecular mechanisms involved in it and c) to elucidate the possible roles of glycan moiety in the induction of cell death.

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3. Antibacterial activity of L-amino acid oxidase

Sudharsana Rao Ande, Sandro Ghisla, Kai-Uwe Fröhlich and Peter Macheroux

(Manuscript in preparation)

The work presented in this chapter was as a part of collaboration between Prof. Dr.

Sandro Ghisla, Prof. Dr. Peter Macheroux and Prof. Dr. Kai-Uwe Fröhlich (PM and KUF are at the University of Graz). The entire work presented in this chapter was done by myself at the Institute of Microbiology, Biochemistry and Molecular biology University of Graz, Graz, Austria.

3.1. Introduction

L-amino acid oxidase is a flavoenzyme that utilizes L-amino acids and converts them into corresponding keto acids, there by producing hydrogen peroxide and ammonia.

L-amino acid oxidases are cosmopolitan in distribution. They are present in diverse sources ranging from prokaryotic bacteria to the higher order organisms, mammals.

These LAAO’s are highly stereo specific but have different selective substrates and differ in their properties. LAAO has been discovered by Zeller in snake venom around 60 years ago (Zeller, 1977). From that time, it has grabbed the attention of the researchers for its various biological properties such as apoptosis inducing activity (Ali et al., 2000), inhibition (Nathan et al., 1982) or activation of platelet aggregation (Li et al., 1994), anti bacterial activity , anti parasitic activity (Tempone et al., 2001), anti HIV activity (Zhang et al., 2003) and oedema inducing properties (Ali et al., 2000).

Antibacterial activity of L-amino acid oxidase was first discovered by Skarnes in 1970. Later on several reports of proteins that have L-amino acid oxidase activity, have been shown to induce antibacterial activity (Stabeli et al., 2004; Stiles et al., 1991). Hydrogen peroxide produced was shown to be necessary for the antibacterial activity of LAAO. Interestingly D-amino acid oxidase (DAAO) which produced similar products upon utilization of D-amino acids could not elicit antibacterial activity (Skarnes, 1970). These findings suggested that there could be different