Further characterization of negative regulatory element involved in the hormonal regulation of GlcNAc-1-P transferase gene in mouse mammary gland.

The gene encoding UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase (GPT), the enzyme that initiates the pathway for the biosynthesis of asparagine-linked glycoproteins, is ubiquitously expressed in eukaryotic cells. However, its expression in the mammary gland is developmentally and hormonally regulated; transcription of the mouse mammary GPT gene is stimulated by the lactogenic hormones, insulin, glucocorticoid, and prolactin. Earlier, we demonstrated that a distal negative regulatory element in mouse GPT (mGPT) promoter plays an important role in developmental and hormonal control of mGPT gene expression in mammary gland (Ma J, Saito H, Oka T and Vijay IK (1996) J Biol Chem, in press). In this report, a tissue distribution of the repressor that binds the negative regulatory element was examined; a comparison of the negative regulatory element to other consensus sequences for known transcription factors is discussed.

Conserved structural features in glycoprotein processing glucosidase I from several tissues and species.

Glucosidase I initiates the processing of the oligosaccharide, Glc3Man9GlcNAc2, in newly assembled glycoproteins by excising the distal alpha 1,2-linked glucosyl residue in the oligosaccharide. Earlier, the enzyme purified from the ER of rat and bovine mammary gland has been found to have M(r) of 85 kDa, as examined by SDS-PAGE along with a domain structure in which a 39 kDa lumenally-oriented region is anchored to the ER through a transmembrane segment and a short cytoplasmic tail. These studies were further extended to include the enzyme from several different tissues of the rat, mouse, guinea pig and bovine mammary glands, sheep liver and pig kidney. Using anti-rat glucosidase I antibody as a probe and several biochemical parameters such as SDS-PAGE analysis, trypsin-catalyzed digestion, ConA-binding, endo H susceptibility and peptide mapping analysis by cleavage of the tryptophanyl peptide linkages within the enzyme, it was found that glucosidase I in all of the tissue sources examined has an M(r) of 85 kDa and is cross-reactive to anti-rat glucosidase antibody. The enzyme is a high mannose glycoprotein, and has domain features in its structure; the enzyme from mouse, rat, guinea pig and bovine mammary glands and sheep liver is sequentially cleaved by trypsin to generate fragments of 69, 55 and 39 kDa. The rate of release of the different fragments differs for different sources, indicating some evolutionary changes in its primary structure. The trypsin-released fragments from pig kidney enzyme are 69, 45 and 29 kDa in size, identical to the same observed earlier for pig liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on hormonal modulation of asparagine-linked glycoprotein biosynthesis in explant cultures of rat mammary gland.

Glucosidase I has been purified to homogeneity and polyclonal antibodies against the enzyme have been prepared. The anti-glucosidase I antibodies recognized a single band of 85 kDa on western blot at a dilution as high as 1:2000 and also inhibited the enzyme activity, suggesting the specificity of the antibodies. Con A-Sepharose binding experiment indicates that this enzyme itself is a high mannose type N-linked glycoprotein. The increase in the electrophoretic mobility of 85 kDa band following digestion with endoglycosidase H and F strengthened this observation. The presence of any O-linked sugar attached covalently to glucosidase I could not be detected by binding assays with O-linkage specific biotinylated lectins. The studies on developmental regulation suggest that the synthesis of glucosidase I is modulated with the ontogeny of the gland. Lactogenic hormones, viz. insulin, hydrocortisone and prolactin, appeared to regulate the synthesis of glucosidase I. The possible role of these hormones in the overall regulation of protein N-glycosylation has been discussed.