No abstract

No abstract

A 1.3-kilobase cDNA clone (7A) coding for bovine galactosyltransferase (glycoprotein 4-3-galactosyltransferase, EC 2.4.1.38) was isolated from a Xgtll expression library by immunological screening with monospecific polyclonal antisera to the affinity-purified bovine enzyme. The nucleotide sequence of this clone predicts an open reading frame that starts at the 5' end of the insert and codes for a polypeptide of 334 amino acids with Mr 37,645. Based on a Mr of 57,000 for the membrane-bound enzyme this clone accounts for approximately 61% of the coding sequence. Portions of the predicted amino acid sequence matched the six tryptic peptides isolated from affinity-purified bovine galactosyltransferase. Clone 7A hybridizes to a 4.8-kilobase bovine mRNA and identifies multiple EcoRI restriction fragments in bovine, murine, and human DNA.UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-p-D-galactosyltransferase (glycoprotein 4-/-galactosyltransferase; EC 2.4.1.38) is a Golgi membrane-bound enzyme that participates in the biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids (1, 2). Galactosyltransferase catalyzes the following reaction of UDPgalactose (UDP-Gal) and N-acetylglucosamine (GlcNAc): MnI2 UDP-Gal + GlcNAc Mn Gal(/31-+4)GlcNAc + UDP where the acceptor sugar, N-acetylglucosamine, may be either the free monosaccharide or the nonreducing terminal monosaccharide of a carbohydrate side chain of a glycoprotein or glycolipid (3). Galactosyltransferase can also interact with the regulatory protein a-lactalbumin to form the heterodimer, lactose synthetase (EC 2.4.1.22). This complex catalyzes the transfer of galactose from UDPgalactose to glucose, forming lactose (4). The net result of the specific interaction of galactosyltransferase with a-lactalbumin is to lower the Km for glucose so that lactose synthesis can take place at physiological concentrations of glucose.Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical (5-9) and biochemical procedures [comprehensively reviewed in Pierce et al. (10) and Shur (11)]. This cell surface distribution has led to the postulate that, in addition to its biosynthetic role, galactosyltransferase has a functional role in intercellular recognition and/or adhesion (12). A detailed analysis of the cell surface galactosyltransferase localized to the plasma membrane overlying the intact acrosome of mouse sperm supports a functional role in sperm-egg recognition (7, 13). There is also evidence to suggest that galactosyltransferase might be functionally involved in the T/t complex of the mouse, a region in which mutations lead to abnormal embryonic development and sperm production (14).Because of the multiple functions of this membrane-bound enzyme and our long range goal of determining the relationship between the cell surface and intracellular form of galactosyltransferase, we have taken advantage of molecular cloning techniques to obtain structural information on this enzy...

No abstract

No abstract