Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1051-1057
December 31, 1990
SIGNIFICANCE OF TRANSGLUTAMINASE-CATALYZED REACTIONS IN GROWTH AND DEVELOPMENT OF FILARIAL PARASITE, Brugiamalayi KAPIL MEHTA*, UNDARU R. RAO+, ANN C. VICKERY+ AND PAUL J. BIRCKBICHLER# * Department of Clinical Immunology and Biological Therapy, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 + Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa, FL 33612 # Biomedical Division, The Samuel Roberts Noble Foundation, Inc., Ardmore, OK73402 Received October 30, 1990
ABSTRACT: A novel form of transglutaminase enzyme [EC 2. 3.2. 13] was identified in adult worms of Brugia malayi. The molecular size of this enzyme was 22-kilodaltons as determined by Western blot and immunoprecipitation, using a monoclonal (CUB 7401) or polyclonal antibodies against guinea-pig liver tissue transglutaminase. The enzyme was present in female worms only; adult males contained no detectable levels of the enzyme peptide. Possible involvement of transglutaminase-catalyzed reactions in growth and survival of filari~l parasites was studied by using various enzyme-specific pseudosubstrates. Presence of these inhibitors resulted into a significant inhibition of microfilariae production and release by gravid female worms in a dose-dependent manner. These results suggest that transglutaminase-catalyzed reactions are essential for development of in utero growing embryos to mature microfilariae. ~ 1990~caaemioP..... ~nc.
Filarial nematodes cause chronic infections in nearly 120 million people worldwide and are a major cause of morbidity in endemic areas [1]. Infection {n humans is initiated by transmission of third-stage infective larvae (L3)by a blood sucking arthropod vector. These larvae develop into adult males or females over several months. After fertilization, mature females produce microfilariae that migrate into the blood stream or skin, where they are ingested by the appropriate vector and eventually develop into L3. Reduction or elimination of microfilariae could, therefore, interrupt transmission of infection in endemic areas and prevent microfilariae-associated pathogenesis [2]. Transglutaminases (EC 2. 3. 2. 13), a family of enzymes that catalyze the posttranslational modification of proteins through * To whom the correspondance should be addressed.
1051
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
exchange of primary amines for ammonia at the y-carboxamide group of pepfidebound glutamine residues, have been extensively studied in mammalian systems [36]. We report here the presence of a transglutaminase enzyme in female worms of
Brugia malayi and present evidence that participation of transglutaminase-catalyzed reactions is essential for differetiation of embryonic stages to mature microfilariae inside the female worms. MATERIALS AND METHODS
Parasites:.
Adult male and female worms of B. malayi were recovered from the peritoneal cavities of jirds 120 - 150 days post-infection with third-stage infective larvae (L3). Worms were washed twice with sterile saline and cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum (FCS), 10 mM HEPES, 100 ~tg mlq streptomycin and 100 U ml-1 penicillin (referred to as medium).
Effect of enzyme inhibition on mf release.
Adult female worms were rinsed thoroughly in sterile saline and incubated (two in each 1 ml of medium) at 370C in the presence or absence of the enzyme inhibitors. At the end of predetermined time intervals, the parasites were removed and 10 ~tl of the spent medium was used for the counts of released mf under the light microscope. Results shown for each time point and each inhibitor concentration are the average number of mf released by two female worms in at least two independent experiments performed in duplicate.
Detection of transglutaminase in parasite extracts:
Male and female worms were separated, rinsed three times in 20 mM Trisbuffered saline (pH 7.6), and lysed immediately in the cold by sonication in the same buffer containing 150 mM NaC1, 1 mM EDTA, 1.5 mM ~-mercaptoethanol (2-ME), 1 mM leupeptin, and 1ram phenylmethylsulfonyl fluoride. Worm extracts were centrifu,gecl (10,000 g for 1Omin); the supernatants were designated the 'soluble fraction. The pellet was resuspended in the same buffer containing 0.2% (v/v) zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPSJ and sonicated; it was desisnated the 'insoluble fraction. The soluble and insoluble fractions were mixed with SDS-sample buffer, boiled, and fractionated by electrophoresis on a 4 - 20 % polyacrylamiae-gradient minigel and then transferred onto nitrocellulose paper. The nitrocellulose paper was neutralized with 1% bovine serum albumin (BSA), and TGase was detected by a method described earlier [7], using mouse monoclonal antibody (CUB 7401) produced against guinea-pig liver tissue TGase [8] and alkaline phosphatase-conjugated goat antibody to mouse immunoglobulin G (Promega Corp., Madison, WI) as second antibody.
Metabolic Labeling and Immunoprecipitation of transglutaminase:
Male and female worms were separated and cultured in methionine/cysteinefree medium (GIBCO Labs., Grand Island, NY) containing 200 ~tCi ml-1 of Tran-[35S] label ( sp. act. 1,197 Ci/mmol; ICN Labs., Costa Mesa, CA). After a 45 min pulse at 370C, ttie parasites were removed and washed twice with phosphate-buffered saline (PBS) and. resuspended in Tris-HCl.(20 mM; pH 7.5, con.taming protease inhibitors).
were sedimented by adding 3IY0, I R I P A buffer (10 mM :Yris-iiIC1, pH 7.4, 15~ w~n~th NaC1, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) and 20 ~tl of a 10% suspension of fixed Staphylococcus aureus cells (Pansorbin; Calbiochem, San Diego, CA) [9]. The precipitated complexes were washed with RIPA and the proteins were solubilized in an SDS-containmg electrophoresis buffer. The samples were fractionated by SDS-gel electrophoresis on a 4 - 20% continuous gradient slab gel, 1052
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the radiolabeled proteins were located by fluorography under conditions described in detail previously [10].
Indirect immunofluorescence staining: Freshly isolated female worms were cut into small pieces with a surgical blade and smeared on clean glass slide. After air drying, the smears were fixed with methanol at -200 C for 15 rain. After extensive washing with PBS, they were incubated with either undiluted CUB 7401 monoclona] antibody supernatant or with supernatant from a control hybridoma (CUB 11) [8]. The second antibody in each case was a 1:1,000 dilution offluorescein-conjugated goat anti-mouse immunoglobulin G (y-chain specific; Sigma, St. Louis, MO). The stained slides were examined under a Nikon fluorescence microscope using transmitted dark field illumination and a 40 x objective. Pictures were taken on Kodak X-pan film using an exposure time of 2 rain. RESULTS AND DISCUSSION We began this study after we observed that the sheath of the microfilariae and embryos in the filarial parasites, when viewed under the phase-contrast microscope, looks very much like the cornified envelopes formed by keratinocyte transglutaminase in epidermis [11]. In the first series of experiments to determine the significance of transglutaminase-catalyzed reactions in filarial parasites, we studied the effect of monodansylcadaverine (DC), a known high affinity pseudosubstrate for transglutaminase [12]. Interestingly, micromolar concentration of DC blocked the production and release of microfilariae by B. malayi female worms in a dose-dependent manner. Significant inhibition (75%) was detected with 50 ~M DC, and a 200 I~M concentration of DC completely inhibited microfilariae release. Concentrations of DC (,
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1051-1057
December 31, 1990
SIGNIFICANCE OF TRANSGLUTAMINASE-CATALYZED REACTIONS IN GROWTH AND DEVELOPMENT OF FILARIAL PARASITE, Brugiamalayi KAPIL MEHTA*, UNDARU R. RAO+, ANN C. VICKERY+ AND PAUL J. BIRCKBICHLER# * Department of Clinical Immunology and Biological Therapy, University of Texas M.D. Anderson Cancer Center, Houston, TX 77030 + Department of Environmental and Occupational Health, College of Public Health, University of South Florida, Tampa, FL 33612 # Biomedical Division, The Samuel Roberts Noble Foundation, Inc., Ardmore, OK73402 Received October 30, 1990
ABSTRACT: A novel form of transglutaminase enzyme [EC 2. 3.2. 13] was identified in adult worms of Brugia malayi. The molecular size of this enzyme was 22-kilodaltons as determined by Western blot and immunoprecipitation, using a monoclonal (CUB 7401) or polyclonal antibodies against guinea-pig liver tissue transglutaminase. The enzyme was present in female worms only; adult males contained no detectable levels of the enzyme peptide. Possible involvement of transglutaminase-catalyzed reactions in growth and survival of filari~l parasites was studied by using various enzyme-specific pseudosubstrates. Presence of these inhibitors resulted into a significant inhibition of microfilariae production and release by gravid female worms in a dose-dependent manner. These results suggest that transglutaminase-catalyzed reactions are essential for development of in utero growing embryos to mature microfilariae. ~ 1990~caaemioP..... ~nc.
Filarial nematodes cause chronic infections in nearly 120 million people worldwide and are a major cause of morbidity in endemic areas [1]. Infection {n humans is initiated by transmission of third-stage infective larvae (L3)by a blood sucking arthropod vector. These larvae develop into adult males or females over several months. After fertilization, mature females produce microfilariae that migrate into the blood stream or skin, where they are ingested by the appropriate vector and eventually develop into L3. Reduction or elimination of microfilariae could, therefore, interrupt transmission of infection in endemic areas and prevent microfilariae-associated pathogenesis [2]. Transglutaminases (EC 2. 3. 2. 13), a family of enzymes that catalyze the posttranslational modification of proteins through * To whom the correspondance should be addressed.
1051
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
exchange of primary amines for ammonia at the y-carboxamide group of pepfidebound glutamine residues, have been extensively studied in mammalian systems [36]. We report here the presence of a transglutaminase enzyme in female worms of
Brugia malayi and present evidence that participation of transglutaminase-catalyzed reactions is essential for differetiation of embryonic stages to mature microfilariae inside the female worms. MATERIALS AND METHODS
Parasites:.
Adult male and female worms of B. malayi were recovered from the peritoneal cavities of jirds 120 - 150 days post-infection with third-stage infective larvae (L3). Worms were washed twice with sterile saline and cultured in RPMI 1640 medium, supplemented with 10% fetal calf serum (FCS), 10 mM HEPES, 100 ~tg mlq streptomycin and 100 U ml-1 penicillin (referred to as medium).
Effect of enzyme inhibition on mf release.
Adult female worms were rinsed thoroughly in sterile saline and incubated (two in each 1 ml of medium) at 370C in the presence or absence of the enzyme inhibitors. At the end of predetermined time intervals, the parasites were removed and 10 ~tl of the spent medium was used for the counts of released mf under the light microscope. Results shown for each time point and each inhibitor concentration are the average number of mf released by two female worms in at least two independent experiments performed in duplicate.
Detection of transglutaminase in parasite extracts:
Male and female worms were separated, rinsed three times in 20 mM Trisbuffered saline (pH 7.6), and lysed immediately in the cold by sonication in the same buffer containing 150 mM NaC1, 1 mM EDTA, 1.5 mM ~-mercaptoethanol (2-ME), 1 mM leupeptin, and 1ram phenylmethylsulfonyl fluoride. Worm extracts were centrifu,gecl (10,000 g for 1Omin); the supernatants were designated the 'soluble fraction. The pellet was resuspended in the same buffer containing 0.2% (v/v) zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPSJ and sonicated; it was desisnated the 'insoluble fraction. The soluble and insoluble fractions were mixed with SDS-sample buffer, boiled, and fractionated by electrophoresis on a 4 - 20 % polyacrylamiae-gradient minigel and then transferred onto nitrocellulose paper. The nitrocellulose paper was neutralized with 1% bovine serum albumin (BSA), and TGase was detected by a method described earlier [7], using mouse monoclonal antibody (CUB 7401) produced against guinea-pig liver tissue TGase [8] and alkaline phosphatase-conjugated goat antibody to mouse immunoglobulin G (Promega Corp., Madison, WI) as second antibody.
Metabolic Labeling and Immunoprecipitation of transglutaminase:
Male and female worms were separated and cultured in methionine/cysteinefree medium (GIBCO Labs., Grand Island, NY) containing 200 ~tCi ml-1 of Tran-[35S] label ( sp. act. 1,197 Ci/mmol; ICN Labs., Costa Mesa, CA). After a 45 min pulse at 370C, ttie parasites were removed and washed twice with phosphate-buffered saline (PBS) and. resuspended in Tris-HCl.(20 mM; pH 7.5, con.taming protease inhibitors).
were sedimented by adding 3IY0, I R I P A buffer (10 mM :Yris-iiIC1, pH 7.4, 15~ w~n~th NaC1, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) and 20 ~tl of a 10% suspension of fixed Staphylococcus aureus cells (Pansorbin; Calbiochem, San Diego, CA) [9]. The precipitated complexes were washed with RIPA and the proteins were solubilized in an SDS-containmg electrophoresis buffer. The samples were fractionated by SDS-gel electrophoresis on a 4 - 20% continuous gradient slab gel, 1052
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and the radiolabeled proteins were located by fluorography under conditions described in detail previously [10].
Indirect immunofluorescence staining: Freshly isolated female worms were cut into small pieces with a surgical blade and smeared on clean glass slide. After air drying, the smears were fixed with methanol at -200 C for 15 rain. After extensive washing with PBS, they were incubated with either undiluted CUB 7401 monoclona] antibody supernatant or with supernatant from a control hybridoma (CUB 11) [8]. The second antibody in each case was a 1:1,000 dilution offluorescein-conjugated goat anti-mouse immunoglobulin G (y-chain specific; Sigma, St. Louis, MO). The stained slides were examined under a Nikon fluorescence microscope using transmitted dark field illumination and a 40 x objective. Pictures were taken on Kodak X-pan film using an exposure time of 2 rain. RESULTS AND DISCUSSION We began this study after we observed that the sheath of the microfilariae and embryos in the filarial parasites, when viewed under the phase-contrast microscope, looks very much like the cornified envelopes formed by keratinocyte transglutaminase in epidermis [11]. In the first series of experiments to determine the significance of transglutaminase-catalyzed reactions in filarial parasites, we studied the effect of monodansylcadaverine (DC), a known high affinity pseudosubstrate for transglutaminase [12]. Interestingly, micromolar concentration of DC blocked the production and release of microfilariae by B. malayi female worms in a dose-dependent manner. Significant inhibition (75%) was detected with 50 ~M DC, and a 200 I~M concentration of DC completely inhibited microfilariae release. Concentrations of DC (,
