Stage-specific surface antigens during the morphogenesis of Trypanosoma cruzi: developmentally regulated expression of a glycosyl-phosphatidylinositol anchored glycoprotein of amastigotes

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

“…These data confirm that the hypothetical protein identified by mass spectrometry is Ssp-4, as previously identified by mAb2C2 ( Andrews et al, 1988 ). We sequenced nucleotides from the T. cruzi CL Brener, G and CL strains, and the protein alignment revealed that the Ssp-4 sequence is well conserved across all strains and among CL Brener haplotypes, clone Dm28c, and the Sylvio strain from the genome database ( Supplementary Figure S2A ).…”

“…In this set of results, it was observed that MAP2 reacts with mAb2C2 immunoprecipitates (mAb2C2-IPs) from less infectious EAs ( Figure 3D ). These data strongly suggest that the protein detected by mass spectrometry is the Ssp-4 that was previously described by Andrews et al (1988) . Immunostaining using the MAP2 antibody detected higher expression of Ssp-4 on non-infectious EAs, suggesting again that recognition of the carbohydrate epitope on Ssp-4 by mAb1D9 can hinder the interactions of the protein core epitopes ( Figure 4B ).…”

“…The first description of Ssp-4 revealed that mAb2C2 recognizes a surface GPI-anchored glycoprotein, which is cleaved and released into extracellular milieu in the presence of PI-PLC ( Andrews et al, 1988 ). Subsequently, Olivas-Rubio et al (2009) identified a partial sequence from the DGF-1 multigenic family, but identification of potential domains on the putative protein was challenging.…”

“…Additionally, Ssp-4 sequences exhibit homology with a hypothetical protein from Trypanosoma rangeli and Leishmania ssp., but not Trypanosoma brucei , which indicates that these proteins could be associated with the presence of an intracellular cycle defined by the amastigote form. In silico analysis of the amino acid sequences identified by MS led to the identification of Ssp-4 domains previously described by Andrews et al (1988) as potential sites to be targeted to the plasma membrane and secreted, i.e., the presence of N - and O -glycosylation sites and a possible GPI-anchor. Potential glycosylation sites observed explain the fact that hypothetical protein encountered by MS present 67 kDa, and by Western blotting molecular weight detect a band between 93 kDa.…”

“…EAs also express on their surfaces a major glycoprotein, stage-specific protein 4 (Ssp-4), initially described by Andrews et al (1987) . This protein is gradually released into extracellular milieu, which is mediated by an endogenous phosphatidylinositol-specific phospholipase C (PI-PLC) ( Andrews et al, 1988 ). We have demonstrated that Ssp-4 was also released in membrane trails when EAs were attached to poly-L-lysine-coated glass ( Barros et al, 1996 ).…”