Molecular cloning and characterization of a Schistosoma mansoni gynecophoral canal protein

This dissertation describes the molecular cloning and biochemical characterization of SmGCP, an 86-kDa glycoprotein localized to the gynecophoral canal of adult male Schistosoma mansoni. A cDNA encoding SmGCP was identified by immunoscreening an adult S. mansoni expression library with polyclonal sera and a monoclonal antibody reactive with SmGCP. The 2.4-kb cDNA encoded a 688-aa protein with limited sequence similarity to Drosophila fasciclin I, a developmentally-regulated, GPI-anchored glycoprotein involved in cell-cell adhesion.;Native SmGCP was detected strongly and consistently in acetone-fixed cercariae, schistosomula and juvenile liver forms of S. mansoni. Mild fixation procedures failed to expose SmGCP, suggesting that the protein was not associated with the parasite's outer membrane. Analysis of SmGCP transcript abundance by RT-PCR suggested that the level of mRNA encoding SmGCP was increased in unisexually-reared male worms and in bisexually-reared male worms which had been separated from their mating partners for five days. These findings were consistent with mating-partner-dependent transcriptional regulation of SmGCP in male worms. No evidence of pairing-dependent transcriptional regulation of SmGCP was observed in female worms.;Mice injected intramuscularly with an expression plasmid encoding SmGCP generated antibodies which reproduced gynecophoral canal staining by indirect immunofluorescence. These mice were not significantly protected against subsequent challenge; no effects on worm burden, maturation, or pairing of adult worms were observed.;Enzymatic deglycosylation of native SmGCP demonstrated that N-linked glycans account for 7kDa of the protein's molecular mass. Multiple predicted phosphorylation sites identified in the SmGCP deduced sequence were found not to be utilized in the native protein. Native SmGCP partitioned to the aqueous phase of detergent extracts, suggesting that the protein lacks a lipid anchor. Recombinant SmGCP expressed in eukaryotic cells was detected by immunoblot as a double band in the culture supernatant. Native SmGCP was found to bind heparin-Sepharose with moderate affinity, suggesting that SmGCP could functionally associate with heparin-sulfated schistosome proteins. However, coimmunoprecipitation experiments demonstrated no proteins physically associated with native SmGCP in bisexually-reared adult male worms.