Our long term goals are to develop a complete understanding of the F. meningosepticum oligosaccharide-cleaving enzymes, peptide-N4-(N-acetyl- beta-glucosaminyl) asparagine amidase (PNGase F) and endo-beta-N- acetylglucosaminidase F (Endo F), including a comprehensive analysis of their substrate specificity, mechanism of action, amino acid sequence and protein structure, active-center, and of their utility in deglycosylation of normal and pathological Asn-linked glycans. The discovery and characterization of related enzymes with new and unique specificities in prokaryotic and eukaryotic systems represents a continual extension of this broad objective, with important implications for oligosaccharide analysis and structure/function studies of glycoproteins into the next decade. The research is divided by specific aims into separate but related fields including molecular cloning and expression of PNGase F and Endo F to complement the proposed active-center studies. The experiments will draw heavily on the chemical synthesis of modified glycopeptide substrates to (1), understand the mechanism of action of PNGase F, and 2) to explore the role of this prototypical enzyme in the normal catabolism of Asn-linked glycans and in the pathological lysosomal storage diseases.
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