The natural mucin ligands for cell adhesion proteins (L- & P-selectin) and various tumor-associated core 2, 0-linked glycoproteins are known to contain the Galb1->4GlcNAcb1-6(Galb1-3)GalNAca-> structure. Relatively little is known of the precise substrate specificities of the glycosyltransferases that can modify this moiety. This proposal is centered on the study of selected glycosyltransferases which act on two galactose residues (Galb1-4 and Galb1-3) of this branched tetrasaccharide. Our prime interest is to study 2,3-sialyltransferases. We have also included the study of two a1,2-fucosyltransferases and a1,4GlcNAc-transferases. One unique and common characteristic of these enzymes is that they prefer this core 2, branched structure for their activities over the simple Galbl-4GlcNAcb and Galbl 3GalNAca disaccharides. We plan to procure a series of core 2 branched structures and other structures which occur as a part of glycoproteins and glycolipids for investigating the specificity of these enzymes. We will examine the specificity of different a2,3-sialyltransferases and ST3Gal I - IV. We also plan to purify these enzymes from selected sources and examine their specificity with a spectrum of compounds. Our rationale for the study of these enzymes is that the combined knowledge of their actions will enable us to elucidate the biosynthetic pathways of; e.g. Fuca1 -2Galbl->GlcNAcbl-6(NeuAca2-3Galb1-3)GalNAca1-, as found in 0-linked glycoproteins. We also hypothesize that the combined knowledge of these enzymes from a given tumor tissue or cell line source will provide us information on the structures of oligosaccharide moieties which can occur as part of tumor-associated antigens. Thus, as a long term objective, the proposed biochemical studies can lead to novel carbohydrate epitopes for future use. This program also allows us to design new approaches for the chemical synthesis of our target structures. NMR studies play an important role in determining the structure of our synthetic compounds. This program has been the prime source of support for our synthetic efforts which are focused to fulfill the following objectives: a) To obtain compounds that are important for specificity studies. b) To synthesize specific modified acceptors which can be used for a single enzyme activity. c) To acquire highly specific ligands for purification of our selected glycosyltransferases. d) Specificity studies will provide us with valuable information for designing modified analogs as inhibitors of these enzymes. As a long term objective, the combination of both inhibitors of critical enzyme for generating human tumor associated antigen and antigens as vaccine may prove beneficial in the treatment of cancer.
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