Characterization Of The Subcellular Localization And Secretion Of The Glycoprotein GP900 In Cryptosporidium Parvum

Cryptosporidium parvum is a zoonotic apicomplexan parasite possessing a set of unique secretory organelles(e.g.,micronemes,rhoptries and dense granules).These organelles release their contents during the parasite invasion.GP900 protein,as a mucin-like glycoprotein with a single transmembrane domain,was previously reported as a microneme protein,and was transferred from the microneme to the sporozoite surface and released to form a gliding trajectory during secretion.However,the biological processes involved in the transfer and release of this protein to the sporozoite surface remain unclear.The main objective of this study was to determine whether the GP900 protein is anchored to the sporozoite surface plasma membrane as a transmembrane protein,and whether it is cleaved before the sporozoite membrane is released.In this study,two antibodies against GP900 were raised: one was a mouse monoclonal antibody against the N-terminus of the transmembrane region(i.e.,antiGP900-N m Ab),and the other was a rabbit polyclonal antibody against the C-terminus of the transmembrane region(i.e.,anti-GP900-C p Ab)to differentiate intact and cleaved proteins.The localizations of the GP900 protein in the sporozoites and different intracellular developmental stages,as well as the secretion and cleavage of GP900,were verified by indirect immunofluorescence microscopic assay(IFA)and immunogold electron microscopy(IEM),and the secretion of GP900 protein was detected by enzyme-linked immunosorbent assay.The micronemal localization of GP900 was confirmed by IFA and IEM,in which both anti-GP900 antibodies labeled the apical region of unexcysted and excysted sporozoites.However,only anti-GP900-N m Ab,but not anti-GP900-C p Ab,labeled the membrane of excysted sporozoites and the trails of gliding sporozoites.IFA using both anti-GP900-N and anti-GP900-C antibodies also labeled mature and immature meronts in the intracellular stages,but not macrogametes and microgametes.Additionally,only anti-GP900-N m Ab,but not anti-GP900-C p Ab,detected GP900 in the mediums secreted by C.parvum sporozoites and intracellular parasite.In conclusion,GP900 protein is present on the microneme of sporozoites and intracellular merozoites,but not in the sexual stage.During secretion from sporozoites,the protein is cleaved to release its intracellular portion,and the cleavage is complete before GP900 protein is transferred to the outside the parasite cell.Based on the molecular features of GP900,including heavy glycosylation with simple N-and Clinked glycans and the lack of adhesive domains,together with the fact that GP900 is secreted as a cleaved protein unanchored to the parasite plasma membrane,we speculate that GP900 plays a lubricating role during sporozoite gliding,invasion and intracellular development.