The long term goal of this work is to study the function of oligosaccharides in the soil amoeba Dictyostelium discoideum and then to extrapolate these results to higher organisms. The focus of this work is on the structure, biosynthesis and function of two novel types of glycosylation found in different populations of vesicle associated proteins in this amoeba. One of these is the specific methylation of mannose-6-phosphate (Man6P) residues by a phosphate methyl transferase. In mammalian cells, Man6P is used to target newly made lysosomal enzymes to the lysosome, but the role of methylation for lysosomal enzyme targeting or other functions in Dictyostelium is unknown. Partial purification and biochemical characterization of this transferase will be done. This will be followed by creation mutants that specifically disrupt the methyltransferase gene to assess its function. The other type of glycosylation is called serine phosphoglycosylation, and it is initiated by the transfer of N-acetylglucosamine-l-phosphate (GlcNAc-alpha-l-P) to serine residues. The modification occurs on some lysosomal proteins in vegetative cells and later in development, in spore cell-specific organelles called prespore vesicles. This modification may also be involved in targeting. Other sugars may be linked to GIcNAc-l-P, and this study will examine the structural diversity of the these chains, how they are made and what structural features of the acceptor proteins induce or permit phosphoglycosylation. A novel mutagenesis procedure will be used to generate mutants that cannot add these chains because the GlcNAc-alpha-1-P transferase gene is disrupted. The consequences of its loss will be examined for intracellular protein movement, targeting and development. The availability of an antibody against GlcNAc-alpha-1-P, and a specific enzyme assay will be used to search for phosphoglycosylation in higher organisms. Preliminary studies that show its presence in the cell adhesion molecule T-cadherin will be expanded to include further work on its biosynthesis and structure. If either of these modifications is used for lysosomal enzyme targeting in Dictyostelium, this may have implications for other novel ways of targeting proteins in mammalian cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032485-11
Application #
2176602
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1988-12-01
Project End
1997-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
11
Fiscal Year
1994
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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