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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD016942-04
Application #
3314069
Study Section
Biochemistry Study Section (BIO)
Project Start
1983-07-01
Project End
1990-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Eunice Kennedy Shriver Center Mtl Retardatn
Department
Type
DUNS #
City
Waltham
State
MA
Country
United States
Zip Code
02254
Daniel, P F; Evans, J E; De Gasperi, R et al. (1992) A human lysosomal alpha(1----6)-mannosidase active on the branched trimannosyl core of complex glycans. Glycobiology 2:327-36
al Daher, S; De Gasperi, R; Daniel, P et al. (1992) Substrate specificity of human liver neutral alpha-mannosidase. Biochem J 286 ( Pt 1):47-53
De Gasperi, R; Daniel, P F; Warren, C D (1992) A human lysosomal alpha-mannosidase specific for the core of complex glycans. J Biol Chem 267:9706-12
al Daher, S; de Gasperi, R; Daniel, P et al. (1991) The substrate-specificity of human lysosomal alpha-D-mannosidase in relation to genetic alpha-mannosidosis. Biochem J 277 ( Pt 3):743-51
DeGasperi, R; al Daher, S; Daniel, P F et al. (1991) The substrate specificity of bovine and feline lysosomal alpha-D-mannosidases in relation to alpha-mannosidosis. J Biol Chem 266:16556-63
DeGasperi, R; Thomas, L J; Sugiyama, E et al. (1990) Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene. Science 250:988-91
Daniel, P F; Newburg, D S; O'Neil, N E et al. (1989) Effects of the alpha-mannosidase inhibitors, 1,4-dideoxy-1,4-imino-D-mannitol and swainsonine, on glycoprotein catabolism in cultured macrophages. Glycoconj J 6:229-40
Warren, C D; Azaroff, L S; Bugge, B et al. (1988) The accumulation of oligosaccharides in tissues and body fluids of cats with alpha-mannosidosis. Carbohydr Res 180:325-38
Warren, C D; Daniel, P F; Bugge, B et al. (1988) The structures of oligosaccharides excreted by sheep with swainsonine toxicosis. J Biol Chem 263:15041-9
Daniel, P F (1987) Separation of benzoylated oligosaccharides by reversed-phase high-pressure liquid chromatography: application to high-mannose type oligosaccharides. Methods Enzymol 138:94-116

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