Indian Journal of Experimental Biology Vol. 42, March 2004, pp. 235-243

Review Article

Transglutaminases, thioredoxins and protein disulphide isomerase: Diverse enzymes with a common goal of cross-linking proteins in lower organisms

Ramakrishna U Rao1 & Kapil Mehta2* IWashington University Sch~ol of Medicine, St. Louis, Missouri, USA

2The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA

Prokaryotes and various eukaryotes have remarkable ability to survive under adverse physiologic conditions and protect themselves from environmental stresses. An important mechanism by which they accomplish this is by synthesizing rigid and biochemically inert structures around them. In general, these structures are highly stable and resistant to mechanical and chemical insults. Biochemically, they are composed of complex carbohydrates, such as chitin and heavily crosslinked scaffold of proteins to form complex structures, such as sheath, cuticle, and epicuticle. Transglutaminases (TGases) are a family of enzymes that share catalytic function with thioredoxin and protein disulphide isomerases (POI) and catalyze protein crosslink reaction by establishing E-(y-glutamyl)lysine isopeptide bonds. The isopeptide bonds thus formed are of great physiologic significance because once formed, they cannot be hydorJysed by any known enzymes of the eukaryote system and exhibit high resistance to reducing agents, detergents, and chaotropic agents. Therefore, it is likely that protective structures viz., sheath, cuticle, epicuticle. and viral core proteins synthesized by microorganisms involve active participation of TGases. In this review, we briefly describe the current knowledge of non-mammalian TGases and their possible role in growth, development, and survival of small organisms. Special reference is made to filarial nematode and bacterial TGases since they are the most well-characterized and studied enzymes among non-mammalian TGases.

Keywords : Caellorhabditis eiegalls, Embryo, Filaria, Transglutaminase, Nematode, Protein cross-linking, Protein disulphide isomerase (POI). Thioredoxins

Transglutaminases (TGases; EC 2.3.2.13) are a family of intracellular and extracellular enzymes that catalyze Ca2+-dependent post-translational modification of proteins by establishing c(y-glutamyl) lysine isopeptide bonds and covalent conjugation of polyamines into proteins. During catalysis, acyl-transfer reaction occurs between carboxamide group of protein bound Glu- residue and a primary amine, including histamines and polyamines (Fig. la). If the primary amine donor is an epsilon-amino group of the protein­bound lysine residue, the product formed will be a covalently cross-linked proteins (Fig. Ib)l.2.

Thioredoxins are multifunctional enzymes that participate in many processes, including DNA synthesis and DNA repair. Published data also suggest that these enzymes playa role in cell division in animals. They are mainly localized at the site of DNA replication and transcription. Thioredoxin is required for meiosis in Drosophila and for development of their embryos. Moreover, thioredoxin is essential for generating sulphur via reduction of sulphate and plays a role in reducing methionine for

Correspondent author: Kapil Mehta, Department of Bioimmuno­therapy-Unit 422. The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA E-mail: krnehta@mdanderson.org

protein. repair of oxidized side chains. Methionine is also reduced by thioredoxin in the assembly of proteins3

. Thioredoxin systems involve two redox conditions of a dithiol/disulphide group. The protein contains an active site that involves cysteine residue. These -cys residues reverse from a dithiol (-SH HS-) group to a disulphide bridge( -S-S-). The oxidized protein is a disulphide with one bridge between two cysteines, whereas the reduced protein is a dithiol with two cysteines.

Similarly, protein disulphide isomerases (POI; EC 5.3:4.1) represent a family of yet another multifunctional endoplasmic reticulum (ER)-resident proteins that belong to the thioredoxin superfamily4. They can act as molecular chaperones, catalyze disulphide bond formation during protein folding5

. 6,

and can perform specialized functions as exemplified by ~-subunit of P4H7

• POI contains 2 redox active domains, near N- and C-termini of the protein that are similar to thioredoxins and both can play a role in disulphide isomerase activity. A number of ER proteins that differ from major POI isozyme contain 2 (e.g., ERp60, ERp55) or.3 (e.g., ERp72) thioredoxin domains but all of them exhibit POI activity. Several recent studies have revealed. that POI and thioredoxin enzymes can catalyze transamidation reaction which

236 INDIAN J EXP BIOL, MARCH 2004

can be linked to the presence of thioredoxin box present in these proteins. These observations suggest that POI, thioredoxins, and TGase enzymes may have a common biological role in regulating various cellular and tissue processes.

TGases are widely distributed enzymes and have been characterized from a diverse group of organisms including, vertebrates, invertebrates, mollusks, plants, and bacteria2

•8

. The evidence published in the literature suggests that these enzymes play an important role in various biologic processes that are beneficial to their hosts. In mammals, particularly in humans, at least nine TGases have been described which differ in their specificity toward substrate proteins8

• 9. They are encoded by separate genes and reveal a high degree of sequence homology. In general, TGases require Ca2

+ for catalytic activity (Fig. 1). However, in recent years, TGase-like activity independent of Ca2

+ has been reported in plants, eukaryotes, and bacteria (Table 1). With the advent of molecular biology tools, some of the TGases encoding genes in these organisms have been successfully cloned. Although, some TGases from these organisms have retained moderate to significant "'" homology to the known mammalian TGases, others show differences with little or no structural similarities to mammalian TGase. For example, TGases cloned from two bacteria (Bacillus subtilis and Streptoverticillium spp.) exhibit no homology to any of the known TGases or to each otherIO. Similarly, cytotoxic necrotizing factor 1 and 2 from Eschericia coli and dermonecrotic toxin from Bordetella pertussis both catalyze deamination of a specific glutamine residue in Rho GTPases. Both these proteins though retain the catalytic cysteine

Fig. 1 - TGase-catalyzed post-translational modification of proteins. (a) - TGases can catalyze covalent conjugation of amine (R-NH2) into the glutamine residue of the acceptor protein (black oval); and (b)-Covalent cross-linking of proteins by establishing NE(y-glutamyl)lysine isopeptide bond between the lysine donor residue of one protein (black oval, PI) and the receptor glutamine residue of another protein (gray rectangle. P2)·

(Cys) and histidine (His) residues and catalyze the transamidation reaction 11 , yet they exhibit no similarity to mammalian TGases. In contrast, mUltiple proteins (in silico) from Mycobacteria spp., the gene profiling data generated by PSI-BLAST program of sequenced genomes, contain sequences in three motifs that center around conserved Cys, His, and asparagine (Asp) residues that form the catalytic triad in the structurally characterized mammalian TGasesIO.

Table 1 - Transglutaminases and their homologues from the lower order organisms

Source

Straminopiles

Phytophthora sojae Algae Chlamydomonas reinhardtii

Fungus

Candida albicans

Plants

Soybean leaves Helianthus tuberosus Pisum sativum Malus domestica

Other eukaryotes & bacteria

Bacillus subtilis Bacillus circulans Physarum polycephalum Streptoverticillium spp. Streptomyces mobaraensis Eschericia coli

Protozoans

Giardia lamblia Plasmodium Jalciparum

Invertebrates

Ciona intestinalis (Sea squirt) Orconectes (Crayfish) Grasshopper

Limulus spp. (Horseshoe crab) Strongylocentrotus spp (Sea urchin)

Nematodes

Brugia malayi Dirofilaria immitis Caenorhabditis elegans

Other lower vertebrates

Pagellus bogaraveo

(Red sea bream)

Molecular weight, kDa

42

72

?

80 75.58 ? 80

28 45 96-101 38 ? 110

26,50,13 ?

80 86 97

86

55 57 55

78

Ca2+

requirement

Yes

Yes

?

No Yes/No Yes/No Yes/No

No Yes Yes No Yes No

Yes No

Yes Yes Catalytically inactive Yes Yes

Yes Yes Yes

Yes

RAO & MEHTA: TGases OF WWER ORGANISMS 237

The presence of multiple TGase homologues in Mycobacteria spp. may imply that these proteins play a role in the development of rigid membrane structures that have been observed in these bacteria. Moreover, the similarity of the catalytic triad and the reaction mechanism indicates that TGases share the core structural fold with thiol proteases. Therefore, it is likely that microbial homologues of TGase may act as proteases and that mammalian TGases might have been evolved from an ancestral protease.

TGase secreted from bacteria may play a role in cleaving the host proteins as a part of their intracellular life cycle, cell-to-cell migration or in induction of apoptosis. These proteins can thus serve as new therapeutic targets. Similarly, the proteins that share homology to mammalian TGase, but lack the active-site domain (e.g. annulin in the grasshopper) may perform structural and protein-binding functions similar to those of 4.2-band protein in red blood ceUs or alternatively may function as dominant-negative regulators of the active enzyme2

•

TGase in growth and development TGase-catalyzed post-translational modification of

proteins has been implicated in several biological processes2

,8. For example, in vertebrates TGase gene expression specifically characterizes cells undergoing apoptosis, the physiologic form of cell death that plays a critical role during reproduction and

developmentl2• In early 1990s, we have first reported

that in simple multicellular organisms, such as filarial nematodes, TGase-catalyzed protein cross-linking reactions are intimately involved in growth and differentiation frocess of early embryonic stages of these parasites I . The main function of fecund female worms is to produce millions of embryonic stages that undergo further differentiation into first-stage larvae (microfilariae). This in utero differentiation of early embryos to mature microfilariae is accompanied by substantial post-translational modifications of proteins and rearrangement of ceUular structures. The evidence that TGase is involved in the growth, development, and maturation of microfilariae and other larval stages is accumulating and is supported by the observations that the membranous structures, such as cuticle, epicutic1e, and sheath of these organisms are highly enriched with TGase-catalyzed E(y-glutamyl)lysine isopeptidesI4

,ls. Moreover, inhibition of TGase activity by enzy~e-specific active-site or competitive inhibitors completely block the differentiation and production of microfilariae13

• Examination of uterine contents from TGase-inhibited Brugia worms reveal that the enzyme activity is absolutely critical for in utero development and maturation of embryos. Female worms when treated with TGase-specific inhibitors reveal deformed embryos that have been failed to differentiate into discrete stages (Fig. 2). Further studies with Brugia and other nematodes

Fig. 2-Effect of TGase inhibition on development and differentiation of embryos to first-stage larvae in filarial nematode. 8rugia malayi. (A) - Uterine contents of adult female worm show numerous embryos after 72 hr of incubation in medium alone or (B)­Medium containing TGase inhibitor (MOC. 100 ~M). (C) and (D)-Show the scanning electron micrograph of the embryos obtained from female worms after 72 hr incubation with medium alone; and medium containing MOC. respectively.

23~ INDIAN J EXP BIOL, MARCH 2004

clearly demonstrate that inhibition of TGase can completely block protein cross-linking reactions of the host and parasite proteins, the biochemical process that is essential for the assimilation of the new sheath and cuticle in developing embryos and microfilariae 14,16.

Significance of TGase-catalyzed protein cross­linking reactions in growth and development of larvae has been further supported by the observations that metabolically labelled proteins are effectively incorporated into the sheath and cuticle of live microfilariae only in the presence of enzymatically active TGasel4. Moreover, the adult worms of several nematodes showed high levels of TGase activity 17.18. TGase activity can be detected in both male and female worms, although the enzyme levels in female worms are generally much higher14.16-19. Immunohistochemical studies reveal that the enzyme protein in adult female worms of the canine heartworm nematode, D. immitis is predominantly localized in the hypodermis and muscle cells, the site of high metabolic activity19. The expression is also evident in the gut epithelium and in developing embryos. In male worms, the expression of

native TGase can be observed in somatic tissues. High levels of active TGase could be observed in developing embryos inside the female worms of human filarial nematode parasite (Brugia malayi) by probing in situ incorporation of the fluorescent enzyme substrate, monodanylcadaverine (MDC; Fig. 3). Morphological and biochemical evidence strongly support direct involvement of TGase-catalyzed cross-linking reactions in molting process of the third-stage infective larvae of filarial nematodes I9-21 .

Similarly, the free-living nematode Caenorhabditis elegans exhibits Ca2+-dependent TGase actIVIty associated with a 61-kDa protein22. Immunohisto­chemical studies reveal that the expression of enzyme protein is predominantly localized in the intestinal cells of adult worms. Higher expression of TGase protein has also been observed in Ll-stage larvae than in other larval stages. The increased TGase activity in the Ll stage was associated with apoptotic death of cells during this stage. Interestingly, TGase activity was lower in ced-3, ced-4, and ced-9 mutants when compared with the wild-type worms. It is noteworthy

Fig. 3 - Localization of catalytically active TGase in adult female worms of Brugia malayi. Adult worms were incubated in the presence of 200 11M of a fluorescent TGase substrate inhibitor, MDC. After 4 hr incubation, the worms were thoroughly washed in PBS, embedded in paraffin wax and 5 11M sections were cut and processed for visualization of covalently conjugated MDC following extensive washings in acidic alcohol to remove the free-unconjugated-MDC. (A)-Cross sections of female worms showing immature embryos; (B)­Microfilariae; (C)-Viewed under phase-contrast (Fig. A and B); and Fluorescent microscope (Fig. C and D).

RAO & MEHTA: TGases OF LOWER ORGANISMS 239

that mutations in ced-3, ced-4, and ced-9 genes confer survival phenotype to cells that otherwise die via apoptosis. In contrast, the ced-2 and ced-5 mutants in which the cells die normally but do not undergo phagocytosis, showed lower levels of the enzyme activity. The lower levels of enzyme activity in ced-3 mutant were not accompanied by a parallel decrease in E(y-glutamyl)lysine isopeptide bonds. In the ced-5 mutant and ced"5/ced-7 double mutants, in which the cells undergo normal apoptosis but are not phagocytosed, levels ' of isopeptide crosslinks were high and several immunopositive corps were detected ' in the head of the adult worms, where most neuronal cells normally die. These data suggest that TGase expression in C. elegans is either not linked with apoptotic events or other protein(s) with TGase activity may participate in these events.

Gene sequence analysis of TGases Genes coding for various members of TGase

family have been characterized from vertebrates2•8 and

invertebrates including ascidians, grasshopper and Limului3

-28

, Cloning of bacterial TGase has been reported from Bacillus subtilii9 and Streptoverticillium Spp.30, but their protein sequences do ,not match with each other or to other members of the ' TGase family. The crystal structure of Streptoverticillium TGase has been recently described31 and reveals some interesting features. The overall structure of bacterial TGase is completely different from the mammalian TGases, Nevertheless, CYS64, Asp255, and His274 of the bacterial TGase perfectly superimpose with Cys, Asp, His catalytic triad of the mammalian TGases, which is sufficient to confer it the transamidation activity . On the other hand, TGase gene has been recently cloned from Physarum spp. demonstrate remarkable similarity to mammalian TGases, including the conserved Cys, His, Asp catalytic triad32

. Moreover, similar to the mammalian tissue-type TGase, a GTP-binding domain has been observed in the sequence of Physarum TGase and the enzyme protein can catalyze GTP-hydrolyzing activity and GTP can inhibit the transamidation activity of the bacterial TGase in a manner, similar to the tissue-type TGase32.

Though, the TGase activity has been described in c.elegani2

, no gene with significant homology to . vertebrate or bacterial TGases has been identified in

its (nearly) complete genome sequence database. This suggests that a TGase homologue is among the gene products that are not yet sequenced or that C. elegans nematode has a completely different TGase that has

no resemblance to other members of TGase superfamily. In fact, the evidence obtained so far in other nematodes strongly supports the latter contention. PDI and other thioredoxin-motif containing proteins have been shown to catalyze the transamidation reaction in a calcium-dependent manner, similar to the mammalian TGase. It is, therefore, likely that in C. elegans these proteins could catalyze the protein cross-linking functions, Indeed, evidence is accumulating that proteins with thioredoxin-motifs, such as Erp6033, PDI_334, and Erp5735 from C. elegans can catalyze transamidation reaction and effectively cross-link proteins. Conversely, TGases of the mammalian origin (e.g. tissue-type TGase) can also catalyze PDI activiti6

,

In our laboratory, we have first purified TGase protein to homogeneity from a filarial nematode, Brugia malayi based on its ability to catalyze calcium-dependent transamidation reaction3

?, The N­terminal amino acid sequence of the purified TGase shows no homology with any of the known TGases or other protein sequences in the GenBank database. An antibody raised against the synthetic N-terminal peptide of Brugia malayi TGase, has recognized a 56-kDa protein in immunoblots of crude extracts from Brugia spp. and other nematooe parasites38. By using this anti-TGase peptide antibody, the cDNA libraries from a closely related dog heartworm nematode (Dirofilaria immitis) have been screened and a positive cDNA clone encoding full-length TGase protein has been obtained39

• The nucleotide sequence or the deduced amino acid sequence of this cDNA clone shows no homology to any of the known TGases. The protein expressed in E. coli, however, is fully functional and like other TGases, recombinant D. immitis protein requires Ca2

+ for catalytic activity. In addition, its activity is inhibited by TGase-specific inhibitors, ammonia, primary amines, EDT A, and -SH group blocking reagents37.39. Interestingly, the nematode TGase shows significant homology to a PDI-related endoplasmic reticulum (ER) protein, ERp60. Another interesting aspect of nematode TGase is that it contains two distinct regions that are identical to the active-site sequences of PDVthioredoxin family of proteins39. More importantly, TGase from filarial nematodes exhibits the catalytic triad composed of Cys, His and Asp residues suggesting that PDI and PDI-like proteins (such as Erp60) play an important role in catalyzing transamidation and other posttranslational modifications during growth and development of these parasites._ Preliminary studies suggest that

240 INDIAN J EXP mOL, MARCH 2004

individuals living in areas, endemic for filarial infections (endemic normals) harbor high titers of anti-TGase antibodies (P Kaliraj and K Mehta, unpublished observations) implicating a possible role for TGase in mounting protective immune response in the human host against filarial parasites. These initial observations walTant further evaluation of TGase as a target for developing effective vaccine and/or chemotherapeutic agents for controlling infections caused by these nematode parasites.

Three isoforms of PDI have been recently isolated from another eukaryote, Giardia lamblia, exhibited Ca2+-dependent transamidation activitlO In addition to TGase activity, these proteins can also catalyze PDI reaction as determined by their ability to reconstitute the scrambled RNase into its native form. Unlike TGase activity, these proteins do not require calcium for catalyzing disulphide isomerase activity. Thus, identification of these novel homologues of TGase with 'thioredoxin-active sites' suggests an interesting aspect of protein post-translational modification in primitive forms of living organisms. It is possible that TGases require PDI activity to promote catalysis of cross-linking reactions in these organisms and vice­versa. Similar TGase homologues have been described in other filarial nematodes as well as from C. elegans and they demonstrate high level of structural and functional similarity among themselves but not with mammalian TGases27

.

Protein substrates of TGase TGase-mediated cross-linking of proteins confer

stability as well as resistance to mechanical disruption and chemical attacks . Therefore, it is likely that TGase-catalyzed reactions play a role in protecting the free-living microorganisms from environmental · stresses and parasitic microorganisms from their hosts' immune attack. To understand the physiologic functions of TGase, it is necessary to identify the nature of endogenous proteins that serve as substrates for these enzymes. For example, the plasmodial stage of the lower eukaryote Physarum spp. upon alcohol­induced stress activates transglutaminase with accumulation of cross-linked proteins. During this process, LA V 1-2 protein and actin seem to act as internal substrates41

. In addition, TGases have been shown to alter Candida albicans surface proteins, envelope proteins and aspartyl-proteinase from HIV and the hepatitis-C-virus core protein by which they

. f h . 28 I alter the pathogeneclty 0 t ese orgal11sms . n our laboratory, we have tested the ability of endogenous TGase to utilize nematode proteins as substrates by

studying MDC incorporation by the live worms. Incubation of adult Brugia male and female worms with MDC lead to the fluorescent labeling of several proteins ranging from 10 kDa to 200 kDa'4 . The labeling is much more prominent in female worms than in male worms suggesting high TGase activity . levels in female worms. A 22-kDa proline-rich and glutamine-rich protein (p22) that has been cloned from filarial nematode, B. malayi, serves as an excellent substrate in TGase-catalyzed reactions (unpublished results). Similar to bacteria and viruses, many parasites can evade hosts' immune attack by opsonizing themselves with hosts ' proteins. Recently, one such protein (p68) from gerbil peritoneal fluid (gerbils are naturally susceptible to Brugia infections) has been found covalently incorporated in a TGase­dependent manner onto the surface of young microfilariae l6

• Similarly, in C. elegans, a 50 kDa protein, in addition to several other proteins that could serve as substrates for the enzyme, is found predominantly cross-linked in adult worm extracts by

22 I . the endogenous TGase . More recent y, two protellls (enolase and mitochondrial A TP synthase) have been isolated from C. elegans and shown to serve as glutamine donor substrates for endogenous TGase42

.

These observations suggest that TGase-catalyzed protein cross-linking reactions may not only play a role during growth and development of these organisms but also contribute toward their longevity and pathogenicity.

Amino acid ,residues involved in TGase activity of thioredoxins and PDls

PDls are members of the thioredoxin family of enzymes. They catalyze the formation, isomerization, and reduction of disulphide bonds in the endoplasmiC reticulum, and display chaperone activity. PDIs have a characteristic a-b-b' -a'- c domains, where domains a and a' show sequence homology to thioredoxins, including the reactive CXXC thioredoxin motif and catalyze the isomerase activity . The c domain contains a putativ~ calcium-binding site.

The Ca2+ -dependent TGases have a cysteine residue in the active site and crosslink proteins using a Cys-His-Asp catalytic triad in an acyl transfer reaction where y-carboxamide group of a protein bound glutamine residue is the acyl donor and c­amino group of a lysine residue or a primary amine serves as acyl acceptor. As discussed in previous sections, D. immitis protein with no sequence similarity to any of the known TGases displays TGase

RAO & MEHTA: TGases OF LOWER ORGANISMS 241

activity. However, it shows significant homology to a POI-related protein, Erp60, and the recombinant protein exhibits both PDI and TGase activities 19,20.39. Similarly, three isofonns of PDI that have been isolated from the protozoan Giardia lamblia4o, C. eLegans PDI_l ,2,333-35, and a mammalian PDI39 are also active in a TGase assay.

Mutagenesis studies reveal that the second Cys in CXXC motif of the thioredoxin domain of C. elegans POI-3 and His adjacent to it are critical for TGase activity of this enzyme33, and these studies conclude that the crucial catalytic residues for transglutamylation are found in the thioredoxin-like domains of POls. This result raises the possibility that thioredoxins themselves are able to accomplish TGase type catalytic reactions. Indeed, when tested, both E. coli and human thioredoxins have been found to display Ca2+-dependent TGase activity34. Analysis of known PDIs and thioredoxin sequences .has revealed the presence of conserved Cys, His and Asp residues required for TGases to catalyze the incorporation of primary amines into protein-bound glutamine residues. The available 3D structures of PDIs and thioredoxins show that these residues are in close proximity to achieve transglutamylation of substrate proteins. Evidence for this also comes from the fact that filarial worm thioredoxin sequences contain conserved Cys, His, Asp residues. Interestingly, some of the known human and bacterial thioredoxins, containing catalytic triad, show TGase activity and can be inhibited by blocking His residues31 . Recent findings suggest that PDIs of both higher and lower organisms can function like TGase cross-linking enzymes by catalyzing the formation, reduction and isomerization of disulphide bonds. In addition, evidence is accumulating that thioredoxins, like their functionally related proteins PDIs, can also serve as polypeptide-binding proteins and participate in protein-folding mechanism43. Dual functional activity has also been observed with PDI-3 of Giardia lambLia4o. Recently, several PDI orthologues have been discovered and Erp57, Erp60, Erp72 are a few of them that are widely distributed and have also been reported in several nematodes like Trichinella, Strongyloides, Haemonchus and Ostertagia Spp33. PDI-protein sequence alignments with nematode Erp60 clearly show conserved catalytic triad of Cys, His and Asp residues. Both PDI and TGase are known to use Cys as an essential residue in the active site for transglutamination functions. The protein crystallography on C. elegans POI and E. coli thioredoxins reveal that His, Cys, and Asp residues in

these structures closely resemble the His, Cys, Asp catalytic triad of other transglutaminases34. Taken together, these observations suggest that the shared enzymatic activities among various members of the PDI, thioredoxin and trans glutaminase enzyme families may have important biological implications, especially in small organisms whose main function is to reproduce and maintain the progeny.

Perspective Identification of novel TGases and other proteins,

such as POls and thioredoxins with ability to catalyze TGase reactions in lower organisms, has started to provide important leads in the areas of therapeutic development and industrial applications in food processing. For example, TGases produced by microorganisms are being applied extensively to replace meat products with vegetable proteins without compromising the taste and texture44

,45. Similarly, the realization that TGase-catalyzed reactions play a pivotal role in the growth, development, and survival of nematode parasites may offer excellent biochemical targets to control debilitating conditions caused by these organisms in humans and animals. It is estimated that filarial nematodes alone afflict about 190 million people worldwide of which 22.5 million cases are in Indian subcontinent and continues to be a major cause of morbidity and suffering to the mankind. Furthermore, with the insurgence of HIV­infections and tuberculosis, dual infections with filarial worms in some of these individuals may increase the clinical outcome of the disease pathogenicity. Since TGase-mediated post-transla­tional modification of viral and bacterial proteins has been well documented, these could be used as suitable targets for drug development against these potentially fatal diseases. From the data described above, it is clear that protein cross-linking reactions catalyzed by multiple enzymes (TGaseIPDIlErp60) play a critical role in the synthesis and assimilation of new cuticle in developing larvae . . Moreover, the nematode TGase shows no homology to their hosts' counterparts, making it an attractive target to selectively inhibit the enzyme activity in parasites. Similarly, TGase­mediated reactions playa role in the development and survival of Plasmodium46 parasite which is responsible for resurgence of malaria and in Giardia40

that causes severe water-borne diarrhoeal infections in tropics. Therefore, it is tempting to speculate that inhibition of TGase-catalyzed reactions may offer pathogen-selective and therefore, less toxic agents to

242 INDIAN J EXP BIOL, MARCH 2004

control and eradicate the debilitating and life­threatening infections caused by these parasites.

Acknowledgement The authors would like to thank Drs R

Chandrashekar, R Singh and E Devarajan for their contributions to this project. Due to the space limitation, we regret our inability to cite the work of several col\eagues in the field.

References I Folk J E & Finlayson J S, The epsilon-(gamma-glutamyl)

lysine crosslink and the catalytic role of transglutaminases, Adv Protein Chem, 3 (1977) I.

2 Lorand L & Graham R M, Transglutaminases: Cross-linking enzymes with pleiotropic functions, Nat Rev Mol Cell Bioi, (2003) 140.

3 Buchanan B B, Scheurmann P, Decottignies P & Lozano R M B. Thioredoxin : A multifunctional regulatory protein with a bright future in technology and medicille, Arch Biachem Biophys, 314 (1994) 257.

4 Clissold PM & Bicknell R, The thioredoxin-like fold : Hidden domains in protein disulphide isomerases and other chaperone proteins, Bioassays, 25 (2003) 603.

5 Rietsch A & Beckwith J, The genetics of disulphide bond metabolism, AIlIlU Rev Genet, 32 (1998) 163.

6 Ferrari D M & Soling H D, The protein disulphide-isomerase family: Unraveling a string offolds, Biochem J, 339 (1999) I.

7 Winter A D & Page A P, Prolyl 4-hydroxylase is an essential procollagen-modifying enzyme required for exoskeleton formation and the maintenance of body shape in the nematode Caellorhabditis eiegans, Mol Cell Bioi, 20 (2000) 4084.

8 Chen J S & Mehta K, Tissue transglutaminase: An enzyme with a split personality, lilt J Biochem Cell Bioi, 31 (1999) 817.

9 Grenard P, Bates M K & Aeschlimann D, Evolution of transglutaminase genes: Identification of a transglutaminase gene cluster on human chromosome 15q 15 . Structure of the gene encoding transglutaminase X and a novel gene family member, transglutaminase Z , J Bioi Chem, 276 (2001) 33066.

10 Makarova K S, Aravind L & Koonin E V, A superfamily of archaeal , bacterial, and eukaryotic proteins homologous to animal transglutami nases, Protein Sci, 8 (1999) 1714.

II Schmidt G, Selzer J, Lerm M & Aktories K, The Rho­deamidating cytotoxic necrotizing factor I f;om Escherichia coli possesses transglutaminase activity. Cysteine 866 and histidine 881 are essential for enzyme activity, J Bioi Chelll , 273 (1998) 13669.

12 Tomei L D, Apoptosis: A program for death or survival? In: Apoptosis: The molecular basis of cell death, edited by L D Tomei and F 0 Cope (Cold Spring Harbor Press, New York) 1991,279.

13 Mehta K, Rao U R, Vickery A C, Birckbichler P J, Significance of transglutaminase-catalyzed reactions in growth and development of filarial parasite, Brugia malayi. Biochem Biophys Res Commull, 173 (1990) 1051.

14 Mehta K, Rao U R, Vickery A C & Fesus, Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development, Mol Biochem Parasitol, 53 (1992) 1.

15 Tarcsa E et al. , Epsilon-(gamma-glutamyl)lysine crosslinks in Litol1los'oides carinii microfilarial sheaths, Parasitol. Res. 78 (1992) 623.

16 Mehta K, Chandrashekar R & Rao U R. Transglutaminase­catalyzed incorporation of host proteins in Brllgia malayi microfilariae, Mol Biochem Parasitol, 76 (1996) 105.

17 Rao U R, Chapman M R, Singh R N, Mehta K & Klei T R. Transglutaminase activity in equine strongyles and its potential role in growth and development, Parasite, 6 (1999) 131.

18 Rao U R, Mehta K, Subrahmanyam D & Vickery A C, Bnigia malayi and AcalJthocheilonema viteae: antifilarial activity o f transglutaminase inhibitors in vitro, Amimicrob AgelJts Chemother, 35 (1991) 2219.

19 Chandrashekar R & Mehta K, Transglutaminase-catalyzed reactions in the growth, maturation and development of parasitic nematodes. Parasitol Today, 16 (2000) II.

20 Chandrashekar R, Devarajan E & Mehta K, Dirofilaria illlmitis: Further characterization of the transglutaminase enzyme and its role in larval molting, Parasitol Res, 88 (2002) 185.

21 Lustigman S, Brotman B, Huima T , Castelhano A L, Singh R N, Mehta K & Alfred M P, Transg lutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae, Antimicrob Agents Chemother, 39 ( 1995) 1913.

22 Madi A, Punyiczki M, Rao D, Piacentini M & Fesus L, Biochemical characterization and localization ' of transglutaminase in wild-type and cell-death mutants of the nematode CaelJorhabditis elegans. Eur J Biochelll, 253 (1998) 583.

23 Cariello L, Ristoratore F & Zanetti L, A new transglutaminase-like from the ascidian Ciona ' intestinalis , FEBS Lett, 408 (1997) 171.

24 Wang R, Liang Z, Hal M & Soderhall K A, A transglutaminase involved in the coagulation system of the freshwater crayfish, Pacifastacus ieniuscllius. Tissue localisation and cDNA cloning, Fish Shellfish Immunol, II (2001) 623.

25 Singer M A, Hortsch M, Goodman C S & Bentley D, Annulin, a protein expressed at limb segment boundaries. in the grasshoppe r embryo, is homologous to protein cross­linking transglutaminases, Del' Bioi, 154 (1992) 143.

26 Tokunaga F, Muta T, Iwanaga S, Ichinose A, Davie E W, Kuma K-I & Miyata T , Limulus hemocyte transglutaminase. cDNA cloning, amino acid sequence, and tissue localization, J Bioi Chem, 268 (1993) 262.

27 Mehta K, Rao U R & Chandrashekar R. Transglutaminases of the lower organisms, Minerva Biotechnoi, 14 (2002) .129.

28 Ruiz-Herrera J , Iranzo M, Elorza M V, Sentandreu R, Mormeneo S, Involvement of transglutaminase in the formation of covalent cross-links in the cell wall o f Candida albicalls, Arch Microbial, 164 (1995) 186.

29 Kobayashi K, Hashiguchi K, Yokozekin K & Yamanaka S, Molecular cloning of the transglutaminase gene from Bacillus subtilis and its expression in Escherichia coli , Biosci Biotechnol Biochem, 62 (1998) 1109.

30 Duran R, Junqua M, Schmitter J M, Gancet C & Goulas P, Purification, characterisation and gene cloning of transglutaminase from Streptoverticilliunl cinnamolleulIl CBS 683.68, Biochimie, 80 (1998) 313.

31 Kashiwagi T, Yokoyama K, Ishikawa K, Ono K, Ejima D. Matsui H & Suzuki E. Crystal structure of microbial

RAO & MEHTA: TGases OF LOWER ORGANISMS 243

transglutaminase from Streptoverticillium mobaraense. J Bioi Chem. 277 (2002) 44252.

32 Wada F, Nakamura A, Masutani T, Ikura K, Maki M & Hitomi K, Identification of mammalian-type transglutaminase in Physarum polycephalum. Evidence from the cDNA sequence and involvement of GTP in the regulation of transamidating activity, Eur J Biochem, 269 (2002) 3451.

33 Eschenlauer S C & Page A P, The Caenorhabditis elegans ERp60 homolog protein disulphide isomerase-3 has disulphide isomerase and transglutaminase-like cross-linking activity and is involved in the maintenance of body morphology, J Bioi Chem, 278 (2003) 4227.

34 Blasko B, Madi A & Fesus L, Thioredoxin motif of Caenorhabditis elegans PDI-3 provides Cys and His catalytic residues for transglutaminase activity, Biochem Biophys Res Commun, 303 (2003) 1142.

35 Natsuka S, Takubo R, Seki R & Ikura K, Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity, J Biochem, 130 (2001) 731.

36 Hasegawa G, Suwa M, Ichikawa Y, Ohtsuka T, Kumagai S, Kikuchi M, Sato Y, Saito Y, A novel function of tissue-type transglutamiriase: Protein disulphide isomerase, Biochem J, 373 (2003) 793.

37 Singh R N & Mehta K, Purification and characterization of a novel transglutaminase from filarial nematode Brugia malayi. Eur J Biochem, 225 (1994) 625.

38 Singh R N, Chandrashekar R & Mehta K, Purification and partial characterization of a transglutaminase from dog filarial parasite, Dirofilaria immitis, Int J Biochem Cell Bioi, 27 (1995) 1285.

39 Chandrashekar R, Tsuji N, Morales T, Ozols V & Mehta K, An ERp60-like protein from the filarial parasite Dirofilaria immitis has both transglutaminase and protein disulphide isomerase activity, Proc Natl Acad Sci . USA, 95 (1998) 531.

40 Knodler L A, Noiva R, Mehta K, McCaffery J M, Aley S B. Svard S G. Nystul T G, Reiner D S, Silberman J D & Gillin F D, Novel protein-disulphide isomerases from the early­diverging protist Giardia lamblia, J BioI Chem, 274 (1999) 29805.

41 Mottahedeh J & Marsh R, Characterization of 101-kDa transglutaminase from Physarnm polycephalum and identification of LA V 1-2 as substrate, J Bioi Chem, 273 (1998) 29888.

42 Madi A, Kele Z, Janaky T, Punyicki M & Fesus L, Identification of protein substrates for transglutaminase in Caenorhabditis elegans, Biochem Biophys Res Commun, 283 (2001) 964.

43 Freedman R B, Hirst T R & Tuite M F, Protein disulphide isomerase: Building bridges in protein folding, Trends Biochem Sci. 19 (1994) 33.

44 Zhu Y, Rinzema A & Tramper J, Microbial transglutaminase - A review of its production and application in food processing, Appl Microbiol Biotechnol, 44 (1995) 277.

45 Coilighan R, Cortez J & Griffin M, The biotechnological applications of transglutaminases, Minerva Biotechnol, 14 (2002) 143.

46 Adini A, Krugliak M, Ginsburg H, Li L, Lavie L & Warburg A, Transglutaminase in Plasmodium parasites: Activity and putative role in oocysts and blood stages, Mol Biochem Parasitol, 117 (2001) 161.