Mannosidosis is an inherited storage disorder in which a deficiency of lysosomal Alpha-mannosidase leads to the accumulation of mannose-rich oligosaccharides in many organs including brain. During the initial funding period, we investigated bovine and feline animal models of the disease and noted significant species differences in the structures of the most abundant oligosaccharides in respect to both the number of Alpha-Man residues and their linkage and also in the number of G1cNAc residues present. We hypothesize that the apparent substrate specificity of Alpha-mannosidase in different species in determined by whether the oligosaccharide chain exists preferentially in an extended or folded back conformation. Hydrogen bonding between the core G1cNAc-G1cNac-Asn and Man residues on the 6-arm is postulated to stabilize the folded back conformation, which would alter the relative accessibility of certain Man residues to Alpha-mannosidase. In humans, a lysosomal endoglucosaminidase is responsible for cleaving glycopeptides prior to exo-glycosidase degradation, thus eliminating the possibility of hydrogen bond formation. The absence of such a lysosomal endoglucosaminidase in bovine and feline mannosidosis may explain both the preponderance of chitobiosyl oligosaccharides and also the differences in mannosyl linkages as compared to oligosaccharides isolated from human mannosidosis. The structures of oligosaccharides containing a single GlcNAc, that are abundant in bovine mannosidosis pancreas, suggest that they are synthesized by the alternate glycosylation pathway and are cleaved by an endoglucosaminidase with novel specificity. The proposed studies will investigate the substrate specificity of endoglucosaminidases from bovine, feline, human and rat tissues using a wide range of purified oligosaccharide and glycopeptide substrates, in the presence of exo-glycosidase inhibitors. The reaction products will be characterized by a combination of reversed phase HPLC and FAB-MS. We will also compare the abundance and structure of accumulated oligosaccharides in both genetic and swainsonine-induced mannosidosis in calves and kittens, and in transformed human fibroblasts. A lysosomal Alpha-mannosidase that is unaffected in human mannosidosis will be isolated from transformed fibroblasts and its substrate specificity studied. The availability of tissues and urines from animals with natural and swainsonine-induced mannosidosis, coupled with out proven abilities to purify individual oligosaccharides by HPLC and to perform structural studies on the, make us uniquely qualified to perform such a study.
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