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We recently reported the cDNA cloning and functional characterization of a novel transglutaminase (TGase) from the dog filarial parasite Dirofilaria immitis. D. immitis TGase (DiTG) has no sequence similarity with any other known TGase, but has significant similarity to protein disulfide isomerase (PDI)-related endoplasmic reticulum protein ERp60. In the present study. we further characterized the recombinant DiTG (rDiTG) and studied its role in the molting process of third-stage larvae. The enzymatic activity of rDiTG requires Ca2+, and the maximum activity was observed at a calcium concentration of 4 mM. Interestingly, the rDiTG was highly thermostable, with optimal activity observed at 55 degrees C, similar to that seen with the native enzyme. Dithiothreitol (DTT) was not essential for enzyme activity. In fact, rDiTG was more active in the absence of DTT. The known inhibitors of TGase, such as monodansylcadaverine (MDC), cystamine and iodoacetamide, inhibited the TGase activity, but not the PDI activity, of rDiTG, demonstrating the dual activity of rDiTG. The TGase-specific pseudosubstrate, MDC, completely inhibited the molting of D. immitis L3 to L4 if present during the first 24-48 h of the molting process. Electron microscopic studies revealed that MDC-treated infective larvae failed to show separation between the L3 and L4 cuticles. The L4 cuticle and accompanying hypodermis were much thinner in MDC-treated worms than in controls. Using anti-rDiTG antiserum, the native DiTG antigen was localized in the hypodermis, afibrillar muscle cells and gut epithelium in adult male and female worms as well as developing embryos in the females.

Prior studies have shown that filarial nematodes can effectively metabolize hydrogen peroxide in excess of that generated by activated host cells. However, the mechanisms of H2O2 removal by the filarial parasites are unclear. Herein we report the results of studies carried out on the biochemical activity and on immunolocalization of a recombinant peroxiredoxin (Prx) enzyme from the dog filarial parasite Dirofilaria immitis. A full-length cDNA encoding a 1-Cys Prx enzyme from the dog heartworm D. immitis was expressed in Escherichia coli as a recombinant polyhistidine fusion protein (rDiPrx-1). rDiPrx-1 was capable of reducing H2O2 in the presence of dithiothreitol. The apparent kinetic constants determined for DiPrx-1 using H2O2 as a substrate were a Michaelis constant (Km) of 16.28 mM and a maximal velocity (Vmax) of 16 micromol/min(-1). Consistent with the enzyme activity, D. immitis adult worms could detoxify exogenously added H2O2 in vitro. Antibodies to rDiPrx-1 identified a 27-kDa native antigen in parasite extracts and larval and adult excretory-secretory products. The antibodies were used to localize the native antigen to the lateral hypodermal chords of both male and female worms by immunohistochemistry. In addition, labeling was seen in the afibrillar muscle cells in male worms and in some areas of the uterine wall in female worms. Thus, DiPrx-1 is the first parasite Prx to be shown to detoxify exogenously added H2O2 in an in vitro system.