Our primary objective is to identify and characterize new types of glycosyltransferases responsible for biosynthetic pathways which are different or reverse to the currently accepted pathways. We plan to identify alpha(1,2)-L-fucosyltransferase which can incorporate fucose at the C-2 position of galactose in the X-determinant (Le(x)) structure, Galbeta1 yields 4 (Fucalpha1 yields 3) GlcNAc, to give the Le(y) sequence, Fucalpha1 yields 2 Galbeta1 yields 4 (Fucalpha1 yields 3) GlcNAcbeta1 yields OR. Similarly, we plan to study the alpha (2,3)- sialyltransferases which can use Le(x) and Le(a) structures as acceptors. Recently, we have isolated and partially characterized beta(1-3)-galactosyltransferase which incorporates galactose to the alpha-linked GalNAc residue in the R-GlcNAcbeta1 yields 6 GalNAcalpha1 yields OB sequence to give R-GlcNAcbeta1 yields 6 (Galbeta1 yields 3) GalNAcalpha1 yields OR (Core II structure). We are interested in utilizing different tumor cell lines and both normal and cancerous human tissues as source material for our investigations. We will also attempt to purification of some of these new enzyme activities, such as beta(1,3)-galactosyltransferase and alpha(1,3)-L-fucosyltransferase present in human lung carcinoma cell lines. Affinity chromatography will be employed as a key step in these purifications. Both nucleotide and specific-acceptor ligands will be made available for this technique. Our chemical synthetic approach will be primarily used for the procurement of the variety of well-defined carbohydrate structures required for the study of these enzymes, including acceptors and ligands. Certain unique and highly specific acceptor moieties can be provided to enable us to examine the activity of a single glycosyltransferase activity in spite of the presence of other glycosyltransferases present in the same source. We also intend to evaluate some of our modified analogs as inhibitors of certain glycosyltransferase activities. The identification of new types of glycosyltransferase activities will shed more light on the biosynthetic pathways of glycoconjugates, especially fucosylated glycoconjugates containing Le(x), Le(a) and sialyl (Le(x) and Le(a)) determinants. We intend through this research to fill the various gaps that exist in our knowledge of the specificity of those enzymes proposed for study. A successful outcome of the proposed program will open various avenues toward other long-term objectives. For example, the discovery and documentation of glycosyl- transferases of novel specificities can enable the development of unique enzymatic and immunological probes to detect these enzymes.
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