The biosynthesis of N-linked glycoproteins involves a step wise, dolichol- linked assembly of a tetrasaccharide precursor, Glc3Man9GlcNAc2 and its en bloc transfer to the nascent peptide followed by co- and post-translational modifications of the oligosaccharyl moiety. The donor for the glycosyl residues in the precursor is Glc-P-Dol; its biosynthesis is catalyzed by UDP-Glc:Dol-P glucosyltransferase. It has been shown that glucosylation of the precursor signals its transfer to the acceptor polypeptides. One may hypothesize that the action of glucosyltransferase represents a key step for the overall regulation of N-glycosylation. The mammary gland possesses some unique features for studying the biosynthesis and regulation of glycoproteins. It is under intense hormonal modulation for its growth and development as it repeatedly cycles between dormancy -> gestation -> lactation -> regression -> dormancy throughout the reproductive life of the female. Further, the lactating gland synthesizes and secretes massive amounts of only a limited number of well characterized glycoproteins into milk. Previous studies from our laboratory have elucidated the outlines of the dolichol cycle and the stereochemical structures of the intermediates of the cycle in the mammary gland. The first enzyme, i.e., UDP-GlcNAc:Dol- P GlcNAc-1-P-transferase and processing-specific glucosidases I and II have been purified. The proposed program will purify, characterize and clone the glucosyltransferase. Innovative methodologies, based on the synthesis of novel analogs of the enzyme substrate will be incorporated in the purification protocol. The enzyme cDNA will be cloned in the expression vector lambdaZAP-II. IN studies outside the scope of this work, molecular probes for glucosyltransferase will be employed to investigate the gland. The recent pioneering studies on the expression of glycosylated and functional glycoproteins tPA, alpha-antitrypsin and blood clotting factor IX in the milk of transgenic animals illustrate the power of the mammary gland as a bioreactor of immense potential for biomedical and agrochemical purposes. Glycoproteins participate in a wide spectrum of biological functions and recognition phenomena. Pathological states including malignancy, atherosclerosis and many autosomal recessive disorders are accompanied with marked alterations in cellular glycoprotein metabolism. A glycoprotein-glycoprotein interaction between gp120 of HIV and CD4 receptor on the lymphocyte provides the entry port for the introduction of viral genetic material into the host leading to AIDS.
