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Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae
S Lustigman1, B Brotman, T Huima
1Department of Virology and Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021, USA.
Abstract:
Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.
Insights
Transglutaminase (TGase) enzyme activity is crucial for filarial worm development. Inhibiting TGase prevents molting from L3 to L4 larvae by disrupting cuticle separation, essential for parasite survival.
Area of Science:
- Parasitology
- Biochemistry
- Molecular Biology
Background:
- Filarial parasites, like Onchocerca volvulus, possess highly insoluble proteins in their cuticle and epicuticle.
- These proteins are likely cross-linked, suggesting enzymatic involvement in cuticle formation and development.
Purpose of the Study:
- To investigate the role of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4).
- To assess the impact of TGase inhibitors on the survival and molting of third-stage larvae (L3) to L4 in vitro.
Main Methods:
- Culturing Onchocerca volvulus L3 larvae in vitro with specific TGase inhibitors (monodansylcadaverine, cystamine, N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-alanine benzylamide).
- Assessing larval viability and molting rates.
- Performing ultrastructural examination of inhibited larvae.
- Localizing TGase reaction products using a monoclonal antibody against epsilon-(gamma-glutamyl)lysine.
Main Results:
- TGase inhibitors did not affect the viability of L3 or L4 larvae.
- Inhibitors significantly reduced L3 to L4 molting in a time- and dose-dependent manner, with complete inhibition at 100-200 microM.
- Ultrastructural analysis revealed synthesized L4 cuticle but incomplete separation from the L3 cuticle in treated larvae.
- TGase products were localized to cuticle regions involved in separation and in L3 amphids.
Conclusions:
- TGase-catalyzed reactions are essential for the successful molting of Onchocerca volvulus from L3 to L4.
- Inhibition of TGase disrupts the cuticle separation process during molting, impacting parasite development.