Browsing by Author "Daya R. Pokharel"

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Setaria cervi collagenase: IgG cleavage and inhibition by Wuchereria bancrofti infected sera
(2004) Yashodhara Srivastava; Sarika Gupta; Daya R. Pokharel; Sushma Rathaur
Significant protease activity has been detected in somatic extract of adults and microfilarial stage of Setaria cervi, using general protease substrates and collagen. The pH optima studies of the somatic extract of adult females showed two peaks at 7.0 and 5.0 for collagenase activity. Both forms were purified using sequential DEAE-sepharose and Sephadex G-100 column chromatography. The purified enzymes had the molecular masses of 175 and 20 kDa and pH optima at 7.0 and 5.0, respectively. The 175 kDa collagenase was more sensitive to metal chelators and serine protease inhibitors. However, 20 kDa collagenase was sensitive to cysteine protease inhibitors. The IgG antibodies from W. bancrofti infected human sera inhibited both enzymes. Further the purified collagenases were used to digest total human IgG at their respective pH and for different lengths of time. The 175 kDa protein was capable of cleaving human IgG. The digestion appeared to be restricted to a single cleavage point of H-chain within the hinge region of IgG molecule and produced fragments of similar molecular mass (27 kDa) indicating cleavage to Fab and Fc fragments. The degree of digestion was found to be proportional to the incubation time at 37°C. No further digestion of either fragments were observed. The L-chains were apparently resistant to collagenase digestion in all cases. Thus, the results suggest that S. cervi collagenase might be involved in the defense mechanisms of the parasite against the immune response of the host.
Tissue localization of collagenase and leucine aminopeptidase in the bovine filarial parasite Setaria cervi
(2006) Daya R. Pokharel; Reeta Rai; Pankaj Kumar; C.M. Chaturvedi; Sushma Rathaur
Background: Like other helminth proteases, filarial proteases have also been shown to require for parasite survival inside the host and mediate various physiologic processes such as tissue invasion, feeding, embryogenesis and host immune evasion. Many of these proteases have shown potential for vaccines and chemotherapeutic agents against active filarial infections. Setaria cervi is a bovine filarial parasite and serves as a good parasite model for the studies in lymphatic filariasis. Recently, a 175 kDa collagenase and leucine aminopeptidase (LAP) have been purified and characterized from the bovine filarial parasite S. cervi and shown to be potential vaccine candidate and diagnostic marker, respectively for human lymphatic filariasis. However, their tissue localizations and putative roles in the parasite biology have not yet been examined and thus remain unclear. Therefore, the current study attempts to localize and explore the putative roles of these two enzymes in S. cervi. Methods: The tissue distributions of 175 kDa collagenase and leucine aminopeptidase in S. cervi were examined by immunohistochemical and histochemical methods, respectively. Immune sera obtained from the jirds immunized with collagenase served as primary antibody, rabbit anti-mouse IgG-HRP conjugate as secondary antibody and DAB as the substrate for the immunostaining of collagenase. Leu-βNA was used as the substrate for the histochemical staining of LAP. Results: Both the collagenase and LAP were present in the body wall; however, they differ in their distribution pattern in different layers of body wall. Collagenase was mainly localized in epicuticle, cuticle, syncytial hypodermis and the nerve cord region whereas LAP was more concentrated in epicuticle, longitudinal muscle layers and almost absent or very faintly stained in syncytial hypodermis and nerve cord region. Both collagenase and LAP showed their common distributions in intestine, uterus and mature eggs, growing embryos and mf. Very strong immunostaining of collagenase in the outer body surface of the parasite indicates its major role in host-parasite relationship whereas the presence of LAP in muscular region suggests its role in tissue remodeling. The common presences of collagenase and LAP in the S. cervi intestine, ovary, uterus, eggs and mf suggest that they also have collaborative roles in molting, nutrition and embryogenesis. The data obtained on their immunological characterizations and their presence in important parasite organs give strong indication that they are critical for the survival of filarial parasite and thus can be good vaccine candidates and/or diagnostic markers for human lymphatic filariasis. Conclusion: The manuscript reports for the first time the tissue distribution of collagenase and LAP in the bovine filarial parasite S. cervi and discuss their putative roles in vivo. Our findings also open the avenue to examine the roles of these two proteases in vivo, which will require further experiments like using their natural substrates and/ or specific inhibitors in each tissues. © 2006 Pokharel et al; licensee BioMed Central Ltd.