The long range objective of the proposed program is to understand molecular mechanisms that regulate the biosynthesis of N-linked glycoproteins during growth and differentiation. The model to be used is the mammary gland during its ontogeny. N-linked glycoproteins constitute the largest class of glycoproteins and participate in myriads of cellular reactions and other phenomena that are fundamental to biological recognition. Alterations in glycoprotein metabolism are associated with a variety of pathological and grave consequences for the host. The enzyme UDP-GlcNAc:Dol-P GlcNAc-l-P-transferase initiates the assembly of the oligosaccharide precursor Glc3Man9GlcNAc2-PP-dolichol for the biosynthesis of the carbohydrate component of these proteins. Following a transfer en bloc of the oligosaccharide to the nascent polypeptide, the enzymes glucosidase I and II trigger the processing of these proteins. Additional processing and post-translational modifications give rise to different subclasses, viz., high mannose, hybrid and complex glycoproteins. Throughout the reproductive life of the mammalian female, the mammary gland goes through highly ordered, cyclic changes under the influence of a variety of hormones that control its growth, differentiation, and regression. Our preliminary studies have shown that several of the key enzymes for the biosynthesis of N-linked glycoproteins in this tissue are developmentally controlled and prolactin appears to participate in this regulation. The enzymes GlcNAc-l-P-transferase and Glucosidases I and II being the early enzymes in biosynthesis and processing, appear to be excellent candidates for the regulation of N- linked glycoproteins in the mammary gland. We have purified these enzymes from the bovine gland; monospecific polyclonal antibodies raised against these enzymes show excellent tissue and species crossreactivity. In the proposed program, the genes for these enzymes will be cloned in the expression vector lambda gtll. These would then be used to select clones from a mouse mammary library in lambda gt10. This will be followed by a study of regulation of these enzymes, both in vivo and in vitro during gland development. Finally, using the antibodies, the biosynthesis and turnover of these enzymes would be investigated.
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