The structural diversity of complex carbohydrates provides ample information for mediating cell recognition events characterizing the morphogenetic assembly of cells into functional tissues during early mammalian embryogenesis. The diversity of these macromolecules directly results from the expression of the enzymes responsible for the biosynthesis of their oligosaccharide chains. In the proposed study, the developmental expression of the enzymes in the pathway for synthesizing asparagine-linked oligosaccharides will be investigated in mouse embryos, focusing on the question of whether the biosynthesis of N-glycosylated proteins is 1) altered during the normal developmental program from unfertilized egg to the early post-implantation stage, 2) different in the cells of the trophectoderm and the inner-cell mass, and the tissues derived from each and 3) affected by perturbing development during the compaction and attachment/outgrowth stages. These studies will utilize an in vitro culture system in which embryos develop through the early post-implantation stages free of maternal systemic influence. The idea that asparagine-linked oligosaccharide synthesis is developmentally regulated during embryogenesis will be tested 1) by measuring the in vitro activities of key enzymes involved both in the assembly and processing of these oligosaccharide chains and 2) by characterizing the structures of glyconjugates newly synthesized in vivo to see if they correspond with the capabilities predicted from in vitro enzyme assays. These goals will be met by using quantitative microtechniques for enzymatic analysis which, in preliminary experiments, were shown to accurately measure the activity of Man-P-Dol synthase in single mouse embryos. The activities of the enzymes involved in the synthesis of oligosaccharide chains of N-linked glycoproteins coupled with structural and compositional analyses of the oligosaccharide chains synthesized in vivo should prove important not only for elucidating the control of oligosaccharide synthesis but also in the study of cell-surface oligosaccharides and their functions in cell-cell interactions during development.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Unknown (R23)
Project #
5R23HD021326-03
Application #
3448323
Study Section
Human Embryology and Development Subcommittee 2 (HED)
Project Start
1985-06-01
Project End
1988-07-31
Budget Start
1987-04-01
Budget End
1988-07-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
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Ram, P A; Fung, M; Millette, C F et al. (1989) Thin-layer chromatographic method for the determination of glycosyltransferase activity. Anal Biochem 178:421-6
Millette, C F; Cardullo, R A; Armant, D R et al. (1987) Fucosylation events during mammalian spermatogenesis. Ann N Y Acad Sci 513:58-73
Cardullo, R A; Armant, D R; Millette, C F (1987) Quantitation of macromolecular binding using size exclusion filters: application to a fucosyltransferase assay. Anal Biochem 161:57-63
Armant, D R; Kaplan, H A; Mover, H et al. (1986) The effect of hexapeptides on attachment and outgrowth of mouse blastocysts cultured in vitro: evidence for the involvement of the cell recognition tripeptide Arg-Gly-Asp. Proc Natl Acad Sci U S A 83:6751-5