Transglutaminase-catalyzed incorporation of host proteins in Brugia malayi microfilariae

Molecular and Biochemical Parasitology 76 (1996) 105- 114

Transglutaminase-catalyzed incorporation of host proteins in Brugia malayi microfilariae

Kapil Mehta”,*, Ramaswamy Chandrashekarlb, Undaru R. Rae”

“Department of Bioimmunotherap.v, The Universitv of Texas M.D. Anderson Cancer Center,Houston, TX. USA bDepartment of Medicine and Molecular Microbiology, Washington University School qf’ MedicineSt. Louis, MO. USA

‘School of Veterinary Medicine, Louisiana State University,Baton Rouge, LA, USA

Received 11 September 1995; accepted 30 November 1995

Abstract

Recently, we have characterized and purified a novel transglutaminase (pTGase) from adults of the filarial worms Brugia malayi. pTGase-catalyzed reactions seem to play an essential role during in utero growth and development of microfilariae. The results presented here demonstrate that exudates from the peritoneal cavity of jirds, the site where adult worms of B. malayi reside and produce microfilariae, contain several host proteins that can serve as substrates in pTGase-catalyzed reactions. The peritoneal exudate proteins are avidly taken up by adult female worms in vitro and incorporated into the developing microfilariae. Among the several host proteins that were crosslinked, a 6%kDa molecular weight protein (~68) was found to be the major protein taken up by the parasites. Following uptake by the parasites, the peritoneal exudate proteins are crosslinked to form high molecular weight aggregates, that are subsequently incorporated into in utero developing embryos and microfilariae. The cross-linking of host proteins was, however, inhibited by monodansylcadaverine (MDC), a competitive inhibitor of pTGase. Antibodies raised against the jird peritoneal exudate proteins strongly immunoreacted with a 68-kDa protein in adult worms and microfilariae extracts but not with infective-stage larvae (L,) of B. malayi. These results suggest that pTGase is involved in covalent incorporation of host proteins (such as ~68) into developing embryos and microfilariae of B. malayi.

Keywords: Transglutaminase; Filariasis; Cross-linked proteins; Sheath; Cuticle

Abbreviations: BPA, 5-(biotinamido) pentylamine; GPT- Gase, transglutaminase from guinea-pig liver; L,, third-stage larvae; MDC, monodansylcadaverine; mf, microfilariae; pTGase, parasite-transglutaminase

* Corresponding author: Department of Bioimmunother- apy, Box 60, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Fax: + 1 713 7961731; E-mail: [email protected].

’ Present address: HESKA Inc., 1825, Sharp Point Dr., Fort

1. Introduction

Filarial nematodes are ovoviviparous parasites

that give birth to first-stage larvae called microfi-

lariae (mf). Many species of filarial parasites (e.g.,

Brugia and Wuchereria) release mf that are encap-

sulated within a highly-resistant structure, called a

Collins, CO 80525, USA. sheath. A clear correlation between the disappear-

0166-6851/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved

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106 K. Mehta et al. / Molecular and Biochemical Parasitology 76 (1996) 105-114

ante of rnf from the circulation and the appear- ance of antisheath antibodies has been reported [l-3]. Moreover, the sheath represents the major area of interaction between mf and the immuno- logical components of the host [4]. Ultrastructural studies indicate that the sheath of mf is derived from the embryonic egg shell during in utero growth and development [5,6]. Detailed knowl- edge of the biochemical nature of the sheath and cuticle of filarial parasites may offer a rational approach to understanding the protective role of sheath.

Unfortunately, little information is available on the biochemical composition of the sheath except that it contains protein, carbohydrate, sulfated proteoglycans and inorganic components such as phosphate, Nat, and K+ [4,7,8]. Sheaths are highly resistant structures and can withstand strong treatments such as boiling in presence of sodium dodecyl sulfate (SDS) [9]. Based on this property as well as their physical resemblance to other transglutaminase-catalyzed structures, such as cornified envelopes [lo] and apoptotic bodies [l 11, the microfilarial sheath was postulated to be the product of transglutaminase (TGase)-catalyzed reactions [9]. TGases (EC 2.3.2.13) are a family of enzymes that catalyze a posttranslational modification of proteins by introducing an isopeptide bond between internal glutamine residues and an amine donor [12,13]. Often the amine donor is a peptide-bound lysine, and the resultant product is two proteins covalently crosslinked by an l -(y-glutamyl)lysine isopeptide bond [ 12- 151. TGase-catalyzed cross-linking reactions are of great physiological interest, because the isopeptide cross-links introduced into the proteins are exceptionally stable and can be broken only by the total degradation of the two peptide chains.

We have demonstrated the presence of a TGase (pTGase) in adult worms of Brugia malayi [16]. More recently, the pTGase was purified from adult B. malayi [17]. Extracts of filarial parasites contained significant levels of l -(y-glutamyl)lysine isopeptide bonds [9], the product of the TGase- catalyzed reaction, suggesting that pTGase is bio- logically active in these nematodes. More recently, Tracsa et al. [18] confirmed these observations

and reported that purified microfilarial sheaths from a rat filarial parasite, Litomosoides carinii, contain high levels of isopeptide bonds. Inhibition of pTGase activity by known TGase inhibitors led to a complete arrest of in utero growth and maturation of embryos to mf and of their release by the female worms [18], suggesting that pT- Gase-catalyzed reactions may play a critical role in growth and development of the larval stages of the filarial parasites.

In this paper we demonstrate that certain host- proteins can serve as effective substrates for pT- Gase and are incorporated into in utero developing mf.

2. Materials and methods

2.1. Reagents

Partially purified TGase from guinea pig liver (GPTGase; sp. act., 1.9 U/mg protein); a competitive TGase inhibitor, monodansylcadaverine (MDC); NJ-dimethyl casein; streptavidin alkaline phosphatase, and p-nitrophenyl phosphate were purchased from Sigma Chemical Co. (St. Louis, MO). The amine donor substrate S’(biotinamido) pentylamine (BPA) was purchased from Pierce Laboratories (Rockford, IL). The radioisotopes [2,3-3H(N)]putrescine (sp. act., 28.8 Ci/mmol) and carrier-free [1251]NaI were obtained from NEN Research Products (Boston, MA). RPM1 1640 medium (Gibco Laboratories, Grand Island, NY) was supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 2 mM glutamine, 10 mM Hepes buffer, penicillin (I 00 U/ml), and streptomycin (100 pg/ ml).

2.2. Parasites

Adult female worms and mf of B. malayi were recovered from the peritoneal cavities of jirds (Meriones unguiculatus) 4-6 months after infection with third-stage infective larvae (L3). The mf collected from the peritoneal cavity lavage were purified by density gradient centrifugation over Percoll (Sigma), as described previously [19]. L,

K. Mehta et al. I Molecular and Biochemical Parasitology 76 (1996) 105-114 107

were recovered from Aedes aegypti mosquitoes, 12

days after they were allowed to feed on jirds with patent infection. The parasites were thoroughly washed in sterile saline containing high concentra- tions of antibiotics (200 pg streptomycin/ml, 200 U penicillin/ml, and 0.25 ,ug fungizone/ml) and were cultured in RPM1 1640 under appropriate conditions.

2.3. Determinution of the enzyme activity

Enzyme activity in partially purified adult

worm extracts (pTGase) or purified guinea pig

liver transglutaminase (GPTGase) was determined according to a recently described microtiter plate assay [20] with slight modification as described [17]. In brief, the microtiter plates were coated with 0.2% dimethyl casein or peritoneal exudate

proteins at 37°C for 1 h. Uncoated sites were blocked with 0.5% nonfat dry milk. The reaction mixture contained in a total of 200 pl volume, 100 mM Tris-HCl (pH 8.5) 10 mM CaCl, or EDTA, 10 mM DTT, 1 mM BPA, and partially purified pTGase or GPTGase. The reaction was carried

out at 37°C for 1 h. Enzyme-catalyzed conjuga- tion of BPA into dimethyl casein was determined by streptavidin-alkaline phosphatase and p-nitrophenyl phosphate as a reporter system. The reac-

tion was stopped by adding 100 pl of 2 mM sodium bicarbonate solution, and absorbance was determined at 405 nm using a Vmax kinetic microtiter plate reader (Molecular Devices, Palo

Alto, CA).

2.4. Iodination of peritoneal exudate proteins

Peritoneal exudates from uninfected jirds were

lavaged in a minimum volume (0.5 ml) of phosphate-buffered saline (PBS, pH 7.4) and cen- trifuged at 10 000 x g for 10 min to remove contaminating cells. The cell-free exudates were then labeled with Iodogen, using 50 pug of Iodo- gen reagent (Pierce, Rockford, IL) and 0.5 mCi of Na”‘1. This resulted in iodinated proteins with a specific activity of N 2.2 x lo6 cpm/pg protein. To study the uptake and fate of host proteins by B. mala_vi adult female worms, radiolabeled peritoneal exudate proteins (2-4 x lo7 cpm) were in-

cubated in the presence of female worms under

appropriate conditions. At the end of the incubation period, adult worms were removed, washed extensively in PBS and processed for further analysis.

2.5. Detection of host protein in adult purusite

Mice were immunized by injecting at multiple sites cell-free exudates from normal jirds (5.0 c(g protein) mixed in Freund’s complete adjuvant.

Primary immunization was followed by two boosters with the same antigen but in incomplete

adjuvant. Two weeks after the last immunization, mice were bled from the retro-orbital plexus; serum was isolated and used at a 1: 100 to 1:500 dilution in an immunoblot assay and at 1: 100 dilution in an immunofluorescence staining assay

to detect host proteins in the parasites.

2.6. Western blotting unalysis

Extracts from adult female worms, mf, and

third-stage infective larvae (L,) were prepared by sonication in 100 mM TrissHCl buffer (pH 7.4) containing 150 mM NaCl, 1 mM PMSF, and 1

mM EDTA. The proteins were solubilized by boiling in SDS-sample buffer and fractionated by 5525% polyacrylamide gel electrophoresis according to the method of Laemmli [21]. Western blots were probed with mouse anti-peritoneal exudate protein antiserum (1: 100 and 1:500 dilution). The antigen-antibody reaction was detected with per- oxidase-labeled secondary antibody to mouse immunoglobulin G ( 1: 1000 dilution).

2.7. Localizution oj “51-lubeled host proteins in embryonic stages

Adult female worms were incubated in the presence of l’SI-labeled peritoneal exudate proteins as described above. After a 4-h pulse, the parasites were extensively rinsed in sterile PBS and incubated in RPM1 1640 for an additional 18 h. After the incubation, adult female worms were placed on glass slides in 20 ~1 PBS and dissected. Dam- aged worms were allowed to release embryos by incubating at 37°C for 5 min. The contents were

108 K. Mehta et al. 1 Molecular and Biochemical Parasitology 76 (1996) 105-114

smeared on glass slides, air-dried, fixed in ethanol/ acetic acid (30: 1, v/v) at - 20°C and coated with bulk emulsion (NBT-2, Eastman Kodak, Rochester, NY) diluted 1: 1 with water. The glass slides bearing parasites were processed for autoradiography according to the manufacturer’s in- structions.

3. Results

We employed a highly sensitive microwell plate assay to determine whether pTGase from B. malayi adult worms could utilize host peritoneal exudate proteins as substrates [ 17,201. Results shown in Fig. 1 demonstrate that pTGase effec-

1.4 n +5 mM CaCl 2

q +O.SmMM)C

1.2-

r l.O-

g

z 0.8- 8 3 e 0.6-

8 P 4 0.4-

0.2 -

0 +5 mMEDTA

exudate

Fig. 1. The ability of peritoneal exudate protein(s) and casein to serve as substrate for pTGase. Microtiter plates were coated with 0.2% (w/v) each of dimethyl casein or cell-free peritoneal exudate proteins. Five pg of partially purified pTGase were added to the wells and tested for its ability to conjugate an amine donor substrate 5-(biotinamido)-pentylamine (BPA), to the substrate proteins. pTGase-catalyzed incorporation of BPA into substrate proteins was then determined by a calori- metric method as described in Materials and methods. The results are expressed as mean absorbance k SD from replicate wells (n = 6).

tively catalyzed incorporation of amine donor substrate BPA into peritoneal exudate proteins. The reaction was absolutely Ca*+-dependent, an important feature of transglutaminase-catalyzed reactions, and could be inhibited in the presence of the competitive inhibitor MDC. Dimethyl casein in which the ~-amine group of lysine residues are blocked to prevent intra- molecular cross- links, was a better substrate than peritoneal exudate proteins for pTGase. Similar results were obtained with GPTGase; both the jird-peritoneal exudate proteins as well1 as dimethyl casein served as effective substrates (data not shown).

To determine whether peritoneal exudate proteins could be utilized in vivo as substrates by pTGase, the uptake and fate of ‘251-labeled exudate proteins by live adult worms was studied. Freshly isolated parasites were incubated for 4 h in the presence of 1251-labeled peritoneal exudate proteins. After the incubation, adult worms were washed and chased in RPM1 1640 for 8 h. The incorporation of host proteins in adult female worms at various time intervals was analyzed by polyacrylamide gel electrophoresis and autoradiography. Immediately following the incubation with radiolabled host proteins (0 h), the parasite extracts revealed the presence of three major radioactive bands at; 35-, 6%, and a smear of protein bands at 2 12%kDa (Fig. 2, lane 1). One, 2, 4 and 8 h after incubation (Fig. 2, lanes 2, 3, 4 and, 5 respectively), a rapid and time-dependent decrease in radioactive bands was observed, de- spite loading of equal cpms in each lane. These results suggest that the host proteins taken up by adult female worms may undergo heavy crosslinking reactions to form high molecular weight aggregates that fail to enter the gel. Since pTGase could effectively use these proteins as substrate (Fig. I), it is likely that such post-translational modification of the host proteins was catalyzed by this enzyme. To test this contention, we directly quantitated the radioactivity associated with high molecular weight aggregates following incubation of adult female worms in the presence or absence of active pTGase. Incubation of the adult worms in the presence of ‘251-labeled peritoneal exudate proteins resulted in uptake of host proteins by the parasites (Table 1). The presence of MDC, a

K. Mehta et al. I Molecular and Biochemical Parasitology 76 (1996) 105-114 109

116

12 3 4 5

Fig. 2. Post-translation modification of host proteins in adult B. malayi female worms. Adult female worms were incubated with radiolabeled-peritoneal exudate proteins (15 fig/ml, 2.8 x 10’ cpm) for 4 h at 37°C. After incubation, the worms were thoroughly rinsed in PBS and re-cultured in RPMI- medium at 37°C for 0 h (lane l), 1 h (lane 2), 2 h (lane 3), 4 h (lane 4) or 8 h (lane 5). At each time interval three female worms were removed, washed and lysed by sonication. The proteins (50 pg/lane) were separated by polyacrylamide gel electrophoresis and the radioactive bands were visualized by fluorography.

competitive inhibitor of pTGase, had no significant effect on this phenomenon. Incubation of labeled parasites for an additional 24 h in the phase of active endogenous pTGase resulted in about 76% recovery of radioactivity in the high moIecular weight aggregates that failed to pass through 0.4 PM pore size membranes (Table 1).

However, inhibition of endogenous pTGase by MDC under similar conditions resulted in only 22% recovery of the radioactivity in the high molecular weight fraction. These results suggest that in the presence of active pTGase, host proteins taken up by the parasites are post-trans- lationally modified, resulting in high molecular weight aggregates.

To localize the host proteins after their uptake by the parasites, freshly obtained gravid female worms were incubated for 4 h in the presence of radiolabeled peritoneal exudate proteins. Subse- quently, the parasites were washed and further incubated in RPM1 medium for an additional 18 h. At the end of the incubation period, uterine contents from the worms were collected and processed for autoradiography as described in Mate- rials and methods. The labeled host proteins predominantly localized in the embryonic membranes of cuticle/sheath of in utero developing eggs (Fig. 3A) and embryos (Fig. 3B).The radiolabeled host proteins in general accumulated more towards the periphery of in utero developing embryos (Fig. 3C).

We also tested the presence of host proteins in adult worms by using antibodies raised against normal jird- peritoneal exudate proteins in mice. Western blot analysis using this antibody revealed the presence of a strong immunoreactive band of 68 kDa in adult worm extracts (Fig. 4; lanes 1 and 2). A similar immunoreactive band of 68 kDa was detected in mf extracts (Fig. 4). However, the antiserum did not label any protein either in the extracts prepared from L, of B. malayi (Fig. 4) or from adult worms of the dog filarial parasite Dirojilaria immitis (data not shown). We also used this antiserum to localize host proteins in B. malayi adult worm sections. Cross-sections of adult female worms were treated with a 1:lOO dilution of the antiserum, and the antigen-antibody reaction was detected with FITC-conjugated anti-mouse immunoglobulin and examined under a fluorescence microscope. There was a strong immunoreactivity of antiserum raised against the host proteins (normal peritoneal exudate) with adult worm components (Fig. 5B). Interestingly, the developing embryos inside the uteri of adult female worms also showed a strong immu-

110

Table 1

K. Mehta et al. / Molecular and Biochemical Parasitology 76 (1996) 105-114

pTGase-catalyzed post-translational modification of peritoneal exudate proteins by adult female worms of B. malayi

Incubation time” (h) Incubation condition Uptake/incorporation of the host proteinb (cpm/O.l mg protein)

0

24

Untreated 4438 * 300 +0.2 mM MDC 3450* 110 Untreated 3381& 93 f0.2 mM MDC 770 k 56

aFive female worms each were incubated in presence or absence of MDC (0.2 mM) and 1251-labeled peritoneal exudate proteins (3.6 x 10’ cpm) from uninfected jirds. After 4 h incubation, the worms were rinsed thoroughly in PBS and were either lysed immediately (0 h) by sonication or were incubated in RPM1 1640 with or without MDC for an additional 24 h and were lysed by repeated cycles of boiling in 10% SDS containing 50 mM DTT. bThe O-h samples were TCA precipitated to determine the uptake of radiolabeled-exudate protein(s) by the parasites, whereas 24-h samples were passed through 0.4 ,uM pore-size Nucleopore membranes. The membranes were washed, 3 x 5 ml each, with lo”/ SDS containing 50 mM DTT and the counts retained on membranes were determined which reflected the amount of radioactivity incorporated into high M,,, protein aggregates.

nofluorescence with the antiserum, confirming the presence of host proteins in these developmental stages of the parasite. The presence of human serum albumin did not inhibit the interaction of this antibody with antigenic components of the parasite (Fig. 5C). Similarly, preimmune serum did not exhibit any significant fluorescence in adult worms or the embryos (Fig. 5A).

4. Discussion

The host proteins present in the peritoneal cavities of jirds (the site where adult worms of B. malayi reside and produce mf) were shown to be actively taken up by the adult female worms and incorporated covalently onto the surface (sheath/ cuticle) of in utero developing mf. The host proteins served as suitable substrates for the parasite protein cross-linking enzyme, pTGase. Trans- glutaminases (TGase) are a family of enzymes that catalyze posttranslational modification of proteins by introducing an irreversible isopeptide bond between internal glutamine residues and an amine donor, such as the e-amine group of protein-bound lysine residues [ 12- 151.

It is believed that live mf are relatively inert to the immune reactions and provoke minimal infl- ammatory reactions in their infected hosts. Mi- crofilaremic patients can bear well over 1000 mf per ml of peripheral blood for over 10 years without any adverse effects from the presence of

this stage of the parasite [22]. The pathology caused by mf is predominantly due to the inflam- matory reactions evoked by the dead or dying parasites. The mechanism of ‘host antigen’ dis- guise has been suggested by several investigators to explain these observations. Convincing evi- dence for this belief has come from studies with Schistosoma mansoni, where the host’s blood group antigens were shown to be acquired by the parasites and conferred protection from host immunity [23,24]. The similar presence of blood group antigens on mf of Wuchereria bancrofti and Loa Zoa parasites has been reported [25]. An association of immunoglobulin to the surface of B. pahangi mf was suggested to confer resistance to the hosts’ immune attack by this stage of parasite [26]. Furthermore, the presence of host serum albumin on the surface of filarial nematodes has been reported by several investigators. Rodent serum albumin was detected on the cuticle of adult male and female worms of L. carinii [27]; human serum albumin on W. bancrofti microfilariae [28,29]; and bovine serum albumin on 0. gibsoni mf [30].

Interestingly, the major protein (~68) from jird’s peritoneal cavity that we observed in this study to be incorporated onto the surface of developing embryos, resembled closely to albumin (67 kDa) in terms of its Mol. wt. We, therefore, decided to determine whether ~68 was indeed an albumin. Due to the lack of appropriate probes available against jird proteins, we were unable to

K. Mehta et al. ! Molecular and Biochemical Parasitology 76 (1996) 105.-114 III

Fig. 3. pTGase-catalyzed incorporation of jird-proteins in in utero developing embryos of B. malayi. Adult female worms were

incubated with radiolabeled peritoneal exudate proteins (I 5 pg), followed by 18 h incubation in plain medium. After incubation, the

uterine contents were dissected out and processed for autoradiography as described in Materials and methods. (A) An egg stage of

microfilaria; (B) an early embryonic stage; and (C) cross-section of female worms showing several eggs in the uterus.

completely rule out that ~68 is not an albumin.

However, based on several other criteria we feel

that ~68 may not be the serum albumin. For

example, serum albumin from various sources is

known to be a poor substrate for TGase-catalyzed

reactions [31]. In contrast, the ~68 from peritoneal

exudates served as an excellent substrate for both,

the mammalian TGase (data not shown) as well

as for pTGase (Fig. 1). In addition, the presence

of excess cold serum albumin failed to compete

out the uptake of iodinated-p68 by adult female

worms (data not shown). In general, the presence

of albumin on the surface of mf has been sug-

gested to be an acquired phenomenon as the

protein could be detected only on mature mf after

they are released by the female worms [27-301.

Immature mf and embryos obtained directly from

the uteri did not possess albumin on their surface.

Contrary to this, labeled-p68 could be effectively

taken up by the female worms and could be seen

112 K. Mehta et al. 1 Molecular and Biochemical Parasitology 76 (1996) 105-l 14

Mr kDa

1;s

66

45

31

14

123 123 123 Adults Mt

Fig. 4. Western blot analysis of B. malayi adults, microfilariae (mf), and third-stage infective larvae (Ls). Lane 1, mouse antibody to jird peritoneal exudate proteins (1: 100 dilution): lane 2, same as lane 1 except for the serum dilution (1:500); lane 3, pre-immune serum (1: 100). Five /(g of total protein was loaded on each lane.

incorporated onto the surface of all the in utero developing stages (Fig. 3). This contention was further supported by previous observation that anti-albumin antibody failed to react in an immunofluorescence assay [30] and fixation of mf with ethanol removed albumin from the surface [28]. However, we were able to demonstrate a strong immunoreactivity of anti-p68 antisera (raised against cell-free peritoneal exudates from uninfected jirds) by immunofluorescent assay using ethanol-fixed adult worm cross-sections (Fig. 5B).

Fig. 5. Localization of host-protein(s) by immunofluorescence in B. malayi female worm cross-sections by pre-immune (A) and anti-peritoneal exudate protein anti-serum (B). Human serum albumin failed to inhibit anitbody binding to the parasite antigenic components (C).

These observations suggested that p68 from peritoneal exudate may represent a distinct host protein that is covalently conjugated onto the surface of in utero growing mf. The work that is currently in progress in our laboratory to further characterize p68 should unequivocally address this issue.

K. Mehta et al. / Molecular and Biochemical Parasitology 76 (1996) 105-114 113

Whether other host tissues, such as lymphatics for which the adult parasites of B. malayi have tropism, contain ~68 remains to be seen. Covalent incorporation of host protein(s) onto the surface of mf during early developmental stages, as demonstrated in the present study, is an interest- ing possibility that may explain prolonged sur- vival of the parasites in host’s hostile environments. Interestingly, the TGase-catalyzed crosslinking of host fibrinogen onto the surface of highly metastatic melanoma tumor cells (B16/ FlO) has also been recently observed. The fibrinogen coating on the surface of these tumor cells was shown to provide protection against the lytic effect of autologous lymphokine-activated killer cells [32]. Assimilation of host proteins in developing embryos (Fig. 4) may suggest that pTGase- catalyzed crosslinking of the host protein(s) may be an essential step during in utero development of mf. Several mechanisms, including pTGase-mediated post-translational modifications of proteins in microfilarial sheaths/cuticle have been pro- posed [8]. In this context, B. muluyi adult worms [9] and purified sheaths from Litomosoides carinii mf [18] have been shown to contain high levels of TGase-catalyzed products, c-(y-glutamyl)lysine isopeptides. Inhibition of pTGase activity in adult female worms by enzyme-specific inhibitors, completely blocked the in utero maturation and release of mf from the grdvid female worms [9,16,331, suggesting that pTGase-mediated crosslinking reactions are critical during growth of mf. In order to further understand the significance of pTGase-catalyzed reactions in growth and development of filarial nematodes, we recently purified the pTGase from B. tnalayi to homogeneity [ 171. Delineation of host and parasite proteins that could serve as substrates for pTGase will be extremely useful in providing the insight into the role of this enzyme in growth, development and maturation of filarial parasites.

Acknowledgements

The authors would like to thank Mr. Walter Page1 for editorial review of the manuscript. This work was supported by Onchocerciassis Control

Programme in West Africa-United Nations De- velopment Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases.

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Transglutaminase-catalyzed incorporation of host proteins in Brugia malayi microfilariae