The long-term goal of this project is to contribute to the understanding of the roles of modifications in N-linked oligosaccharides in the synthesis, routing, secretion, and development accumulation of lysosomal enzymes. Dictyostelium discoideum is bing used as a model system for this study because of the availability of a large variety of mutant strains which are altered in each of these physiologically important processes. We will conduct a detailed analysis of the structure and steps in the biosynthesis of the highly modified (3H)Mannose-labeled, N-linked oligosaccharides found on a single, well characterized lysosomal enzyme, Alpha-mannosidase. Mutant strains which are defective in the synthesis, routing, packaging and secretion of single or multiple lysosomal enzymes appear to have altered oligosaccharides. Therefore, similar structural analyses will be performed on this enzyme in the mutant strains to determine whether changes in oligosaccharide structure lead to or are correlated with mislocalization of the enzymes. Since mammalian cells target amny, but not all, of their lysosomal enzymes as a result of oligosaccharide modifications, this study may provide significant insight into these processes. Developmentally specific post-translational modifications occur in Dictyostelium. Certain mutant strains fail to both develop properly and to accumulate normal amounts of lysosomal enzymes, apparently due to alterations in these modifications. The structure and biosynthesis of the oligosaccharides of Alpha-mannosidase in these mutants will also be analyzed. An understanding of how oligosaccharide structures are modified and their cnsequences for development may contribute to our understanding such diverse processes as embryogenesis, turmoigenesis, and age-related alterations in protein modification.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032485-09
Application #
3281365
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1988-12-01
Project End
1992-12-31
Budget Start
1990-09-01
Budget End
1992-12-31
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
009214214
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Srikrishna, G; Wang, L; Freeze, H H (1998) Fucosebeta-1-P-Ser is a new type of glycosylation: using antibodies to identify a novel structure in Dictyostelium discoideum and study multiple types of fucosylation during growth and development. Glycobiology 8:799-811
Souza, G M; Mehta, D P; Lammertz, M et al. (1997) Dictyostelium lysosomal proteins with different sugar modifications sort to functionally distinct compartments. J Cell Sci 110 ( Pt 18):2239-48
Srikrishna, G; Varki, N M; Newell, P C et al. (1997) An IgG monoclonal antibody against Dictyostelium discoideum glycoproteins specifically recognizes Fucalpha1,6GlcNAcbeta in the core of N-linked glycans. Localized expression of core-fucosylated glycoconjugates in human tissues. J Biol Chem 272:25743-52
Freeze, H H; Lammertz, M; Iranfar, N et al. (1997) Consequences of disrupting the gene that encodes alpha-glucosidase II in the N-linked oligosaccharide biosynthesis pathway of Dictyostelium discoideum. Dev Genet 21:177-86
Chui, D; Oh-Eda, M; Liao, Y F et al. (1997) Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 90:157-67
Varki, A; Freeze, H H (1994) The major glycosylation pathways of mammalian membranes. A summary. Subcell Biochem 22:71-100
Freeze, H H; Bush, J M; Cardelli, J (1990) Biochemical and genetic analysis of an antigenic determinant found on N-linked oligosaccharides in Dictyostelium. Dev Genet 11:463-72
Freeze, H H; Koza-Taylor, P; Saunders, A et al. (1989) The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum. J Biol Chem 264:19278-86
Lacoste, C H; Freeze, H H; Jones, J A et al. (1989) Characteristics of the sulfation of N-linked oligosaccharides in vesicles from Dictyostelium discoideum: in vitro sulfation of lysosomal enzymes. Arch Biochem Biophys 273:505-15
Freeze, H H; Willies, L; Hamilton, S et al. (1989) Two mutants of Dictyostelium discoideum that lack a sulfated carbohydrate antigenic determinant synthesize a truncated lipid-linked precursor of N-linked oligosaccharides. J Biol Chem 264:5653-9

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