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.
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