TRANSGLUTAMINASE AS A TARGET FOR NOVEL ANTI-FILARIAL AGENTS

KAPIL MEHTA

Division of Medicine, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030

ABSTRACT

A rational approach for developing effective fi laricides awaits greater knowledge on the biochemical pathways operating in filarial parasites. For example, meta- bolic pathways or key enzymes that are so pivotal to the parasite that their interruption or inhibit ion causes spontaneous death, could be exploited to develop effective chemotherapeutic agents. Similarly, developing a safe and nontoxic f i laricidal will require precise understanding of whether or not the potential molecular target in the parasite is unique or at least different enough from the host system. Recently, we identified a novel transglutaminase enzyme (EC2.3.2.13; TGase) in filarial parasites that plays an important role dur ing their normal growth and development. Based on these principles, TGase may serve as a new target for the development of effective chemotherapeutic agent and vaccine for controlling and preventing the infections caused by these parasites.

KEY WORDS : Filariasis, transglutaminase, chemotherapy, cuticle, sheath

INTRODUCTION

Of the filarial genera that infect humans, the most pathogenic and prevalent are Wuchereria, Brugia, and Onchocerca. The first two are termed lymphatic filariae because the lymphatic system is the main site of pathology. They are respon- sible for over 90 million worldwide cases of lym- phatic filariasis and another 905 million people live in endemic areas and are at risk. Onchocerca volvulus that causes blindness and skin disease, is endemic in several parts of Africa, Latin America and the Eastern Mediterranean. At present, 17.6 million people are infected and about 336,000 are blind due to onchocerciasis. From these data it is clear that filadasis continues to be a serious health

Address for Correspondence Dr. Kapil Mehta, Division of Bioimmunotherapy- 60, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030 (USA) E-mail : kmehta @ mdacc.tmc.edu

problem in many parts of the world. There is at present no safe and reliable chemotherapeutic agent active against infective larvae and adults of the filarial species. This problem is further compounded by the acquired resistance seen in vector populations to conventional insecticides. Therefore, identification of pathways that are parasite-specific and vital for their growth or survival, may offer new opportunities for devel- oping effective chemotherapeutic agents. In the following section, I summarize our findings on ident i f icat ion of one such key enzyme, transglutaminase (EC2.3.2.13; TGase) and its role during developmental stages.

Significance of TGase-catalyzed reactions in filarial parasites

TGases are a family of enzymes that cata- lyze calcium-dependent crosslinking of cellular proteins by establishing ~:-(~,-glutamyl) lysine isopeptide bonds (Chart 1). These covalent isopeptide cross-links are of great physiological significance since they are highly resistant to Droteolysis, denat~at~ls, and reducing ao~nts.

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Mehta TG ase reactions in filarial nematodes

NH 2 ---- Lys--.-- P2

I P1 ..-Glu - + Lys TGase + Ca 2+ P1 -.Glu + NH 3 )

Chart 1 : TGase-catalyzes a calcium-dependent-acyl transfer-reaction between the carboxamide group of a peptide (P1)-bound glutamine residue and ~-amine group of peptide (P2)-bound lysine residue.

Out interest in studying the role for TGase in filarial parasites started following the observa- tion that "egg shell" or sheath of developing embryos and microfilariae, when viewed under a phase-contrast or scanning electron microscope, closely resembled the other transglutaminase- catalyzed structures of the mammalian system. Using a highly-sensitive microwell plate assay, we demonstrated that adult worms of Brugia malayi, B. pahangi, Dirofilaria immitis, and O. volvulus contained high levels of TGase activity. The enzyme activity could be detected in both male and female worms as well as in third- (L3) and fourth (L4)-stage infective larvae. The direct proof for the presence of biologically active TGase in filarial parasites came from the observation that adult worms as well as microfilariae contained large amounts of~-(~,-glutamyl)lysine isopeptides, the product of TGas-catalyzed reaction. Further- more, in situ labeling of the fmale worms with monodansylcadaverine (MDC), a fluorescent enzyme substrate revealed that embryos in early stages of development are particulady enriched in TGase. Interestingly, the enzyme levels as determined by fluourescence intensity was much higher at peripheral sites in developing embryos. Female worms also showed evidence for the presence of TGase at specific sites in the cuticle.

To determine the role of TGase-catalyzed reactions in filarial parasites, we studied the effect of MDC, a known high affinity substrate inhibitor of TGase. Incubation of B. malayi female worms in the presence of micromolar concentrations of MDC blocked the production and release of mi- crofilariae in a dose-dependent manner. Signifi- cant inhibition (75%) was detected with 50pM MDC, and a 1001JM concentration of MDC com- pletely inhibited the microfilariae release by fe- male worms. Concentrations of MDC ~ 1001JM) that inhibited microfilariae production had no gross toxic effect on adult worms or fully developed

microfilariae. For example, incubation of adult worms in the presence of MDC at a 100pM con- centration did not affect their motility even after 3 days of culture. However, at concentrations > 200pM, the adult worms became sluggish with time and after 24 h of culture they were immobile and eventually died. The inhibitory effect of MDC on microfilariae production was evident as early as 60 min after the incubation and continued as long as the drug was present in the incubation mixture. These initial observations in Brugia were confirmed in several other filarial parasites. Simi- larly, other structurally unrelated inhibitors of TGase also inhibited the Mf production and output by adult famale worms.

Next we determined the significance of TGase-catalyzed crosslinking reactions in devel- oping larval stages of O. volvulus and D. immitis. In agreement with their in vitro inhibitory effect, both MDC and cystamine caused a significant inhibition in molting of L3 larvae to L4-stage. Tranmission electron microscopy revealed that the cuticle in MDC-treated larvae failed to sepa- rate from the newly synthesized underlying L4 cuticle. The cuticle is a elastic extracellular matrix secreted by the underlying hypodermis and is an exceptionally stable and insoluble structure that is composed of highly cross-linked proteins. They are mainly collagenous proteins, crosslinked by covalent disulfide bonds and localized in the basal and inner cortical layers. The nonreducible interchain covalent bonds are of two types; ~-(~,- glutamyl) lysine isopeptide bonds and bonds between tyrosine side chains. Indeed, the pres- ence of ~-(~,-glutamyl) lysine isopeptide bonds has been confirmed in the developing embryos and microfilarial sheath of Litomosoides carinfi and B. malayi and in the infective larvae of O. volvulus. It is, therefore, conceivable that inhibition of TGase-catalyzed crosslinking reactions by en- zyme-specific inhibitors prevent the assimilation

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Mehta TG ase reactions in filarial nematodes

of new cuticle that is needed during molting of L3 to L4 stage.

In an attempt to further define the molecular nature of TGase-catalyzed reactions in the as- similation of new cuticle, we purified TGase from adult worms of B. malayi. The six-step purification protocol that we used gave a 20% yield with a 2200-fold purification. Biochemical characteriza- tion of the purified TGase revealed that it is a 56- kDa protein and shares some characteristics to its mammalian enzyme. Most intriguing was the ob- servation that N-terminal amino acid sequence of the purified TGase protein upto 20 amino acid residues revealed no homology with any of the known transglutaminases or other protein se- quences when searched in GenBank TM data base. The isoelectric point of the pure enzyme was determined to be 7.2. The enzyme activity was Ca2§ and was inhibited by ammonia, primary amines, EDTA, and -SH group blocking reagents. The enzyme activity was moderately inhibited at micromolar concentrations of GTP. The TGase was highly thermostable (stable at 60~ for several hours) with optimum activity ob- served at 55~ The enzyme was active in the basic pH range with an optimum activity observed at pH 8.5. The TGase activity was affected by the

presence of high salt concentration (NaCI, KCI), detergents, ampholines, metal ions, and organic solvents. However, SDS-PAGE or other physical parameters such as freezing and thawing, had no appreciable effect on catalytic functions of TGase. Based on unique N-terminal sequence and other physico-chemical properties, TGase appeared to be a novel member of the TGase enzyme family. We recently confirmed this by cloning a cDNA, encoding full length TGase from dog heartworm, D immitis. The derived amino acid sequence of this cDNA clone predicted a protein of 57.2 kDa size, similar to purified B. malayi TGase. The encoded protein showed no sequence similarity with any of the known TGases but has several features similar to the purified TGase.

Thus, our finding of a novel tranglutaminase from filarial nematodes suggests an interesting aspect of protein post-translational modifications in developing nematode larvae. Intervention of these pathways in turn may provide useful leads for successful elimination of nematode parasites and treatment of diseases caused by them.

Acknowledgements : The work in author's laboratory was supported in part by a grant from WHO's Special Program in Tropical Disesases.

REFERENCES

1. Mehta, K., Rao, U.R., Vickery, A.C. and Birckbichler, P.J. (1990) Significance oftransglutaminase- catalyzed reactions in growth and development of filarial parasite, B. malayi. Bicochem. Biophys. Res. Commun. 225, 625-634.

2. Mehta, K., Rao, U.R., Vickery, A.C. and Fesus, L. (1992) Identification of a novel transglutaminase from the filarial parasite B. malayi and its role in growth and development. Mol. Biochem. Parasitol. 74, 105-114.

3. Singh, R.N. and Mehta, K. (1994) Purification and characterization of a novel transglutaminase from filarial nematode B. malayi, Eur. J. Biochem, 225, 625-634.

4. Sing, R.N., Chandrashekar, R. and Mehta, K. (1995) Purification and partial characterization of transglutaminase from dog filarial parasite, Dirofilaria immitis. Int. J. Bicochem. Cell Biol. 27, 1285-1291.

5. Mehta, K., Chandrashekar, R. and Rao, U.R. (1996) Transglutaminase-catalyzed incorporation of host proteins in Brugia malayi microfilariae. Mol. Biochem. Parasitol. 76, 105-114.

6. Lustigman, S., Brotman, B., Huima, T., Castelhano, A.L., Singh, R.N., Mehta, K. and Prince, A.M. (1995) Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae. Antimicrob. Agents Chemother. 39, 1913-1919.

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