The ultimate objective of our program is to delineate the molecular basis for gene expression during animal growth and development. The focus of the investigation is on the hormonal regulation of protein N- glycosylation in the mammary gland. Asparagine-linked glycoproteins comprise the largest class of glycoproteins and are involved in a myriad of phenomena that are fundamental to biological recognition. Alterations in glycoprotein metabolism are associated with a variety of pathologies, e.g., malignancy, atherosclerosis, numerous genetic disorders and the initial host-parasite interaction leading to AIDS. A concert of seventeen glycosyltransferases and two glycosidases, viz., glucosidase I and II are minimally required for the assembly of all N-linked glycoproteins. Among these, UDP-GlcNAc:Dolichol-P GlcNAc-1-P-transferase (GPT) and glucosidase I are critically positioned at the first committed step for the biosynthesis and the initial reaction for processing, respectively, in the multienzyme pathway of assembly. These enzymes, therefore, control the flux of carbohydrate and would appear to be excellent candidates for the overall regulation of protein N- glycosylation. The proposed investigation will focus on examining the hormonal modulation of these enzymes using molecular-biological approaches with HC11 cells, an epithelial cell line derived from the mouse mammary gland. Additionally, biochemical experiments are proposed that will exploit the potential of a novel methodology developed in our laboratory to map and delineate the active site of glucosidase I. A preliminary analysis by sequentially deleting various domains for the molecular basis for the ER-retention of glucosidase I is also proposed. these studies represent an extension of our ongoing studies and the work that has been accomplished in the previous funding period. The mammary gland offers a unique model to the investigator for studying glycoprotein biosynthesis and regulation at the biochemical and molecular-biological level. This tissue is intensely modulated by hormones for its growth and differentiation throughout the reproductive life of the female. It can potentially serve as an excellent bioreactor in transgenic animals to harvest large quantities of biomedically significant glycoproteins in its secretion, i.e., milk. Preliminary successes with the secretion of alpha-antitrypism, tissue plasminogen activator, and blood clotting factors appear very promising.
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