The S. sanguis Platelet Aggregation-associated Protein (PAAP) has been isolated as a precursor N-formylmethionyl glycoprotein of 115 kDa. While other prokaryotic glycoproteins have been suggested in the literature, this is the first protein from a Gram-positive organism shown by direct experimental evidence to have covalently associated carbohydrate. Preliminary studies also suggest that the carbohydrate polymers are not present in the wall-associated form of PAAP. Therefore, by studying PAAP biosynthesis and export, S. sanguis provides a unique model system to understand the novel biosynthetic pathway of prokaryotic glycoproteins. The applicant hypothesizes that PAAP provides a mechanism for incorporation of carbohydrate polymers onto the cell wall of S. sanguis. Acting as a co-transporter, PAAP may translocate the carbohydrate through the membrane in covalent association with the exported protein. Once PAAP is transported, the carbohydrate oligosaccharides may be cleaved from the protein backbone and transferred to other cell wall components, such as lipid or peptidoglycan. To test this hypothesis, the specific aims of this research project are to: 1) identify and characterize the cell wall component which contains the PAAP oligosaccharides, 2) isolate the S. sanguis oligosaccharyltransferase responsible for the intracellular transfer of oligosaccharide to the PAAP protein backbone, 3) isolate the glycosidase/transferase(s) responsible for removal and transfer of oligosaccharides from the exported PAAP protein to another site in the cell wall, 4) biochemically characterize the isolated proteins/enzymes associated with intracellular and wall-associated transfer of the oligosaccharides, and 5) screen for these proteins/ enzymes in other streptococci. To accomplish these aims, immunologic, biochemical and molecular biological techniques will be employed. The elucidation of this biosynthetic pathway for streptococcal glycoproteins may impact the understanding of other biosynthetic pathways, such as eukaryotic glycoprotein biosynthesis and prokaryotic cell wall polysaccharide synthesis. The cell walls of oral streptococci are rich in polysaccharides, and, if these structures are cotransported to the cell surface as part of the PAAP glycoprotein, identification of the glycosylation pathway may suggest strategies to alter the function of these polysaccharides.
