The focus of this proposal is to determine if specific structural changes occur in the cell surface glycoprotein Asn- and/or in Ser/Thr-linked oligosaccharides upon differentiation in vitro of mouse embryonal carcinoma F9 cells into endoderm cells and to define chemically these structures. Additionally, a major aspect of this proposal is to define the changes in the activities of certain glycosyltransferases which may be responsible for the changes in oligosaccharide structure. Several studies suggest that the average sizes of cell surface oligosaccharides are reduced during differentiation of F9 cells; however, detailed structural analyses comparing these cell types have not been reported. This proposal will examine the structures in the two cell types by metabolically radiolabeling cell surface glycoproteins with ?3?H-sugar precursors and then fractionating the glycopeptides by serial lectin affinity. This is a newly developed technique for separating oligosaccharides on the basis of specific structural features and not size alone. Oligosaccharides will be separated from the partly purified glycopeptides by either hydrazinolysis or NaOH/ NaBH?4? treatments and the released oligosaccharides will be separated further by ion exchange column chromatography and high performance liquid chromatography. Structural analyses of the isolated oligosaccharides will be performed using methylation and specific exo- and endoglycosidases. The structures determined for oligosaccharides from these cell types should suggest which glycosyltransferase activities may be altered during differentiation. Newly developed assays allowing for greatly increased sensitivity and specificity will be used to assess the activity of a number of glycosyltransferases in both F9 and endodermal cell-free extracts. Concurrently with these studies two additional lectins will be purified from both Solanum tuberosum (potato) and Lycopersicon esculentum (tomato) and their high affinity carbohydrate binding specificities will be investigaed by immobilizing the lectins and analyzing the interactions of purified glycopeptides with the lectins in column chromatography. These lectins may be useful in the expansion of the technique of serial lectin affinity and should greatly facilitate the analyses of cell derived oligosaccharides in this proposal. In this proposal embryonic carcinoma cells serve as a model system for the early events in embryo development. The long term goal of this research is to understand the molecular events at the cell surface accompanying cellular differentiation and embryogenesis. (A)
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