Bovine mammary gland UDP-GalNAc:GlcNAcβ-Rβ1→4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with α-lactalbumin

We have identified a novel N-acetylgalactosaminyltransferase activity in lactating bovine mammary gland membranes. Acceptor specificity studies and analysis of products obtained in vitro by 400 MHz H-1-NMR spectroscopy revealed that the enzyme catalyses the transfer of N-acetylgalactosamine (GalNAc) from UDP-GalNAc to acceptor substrates carrying a terminal, beta-linked N-acetylglucosamine (GlcNAc) residue and establishes a beta 1-->4-linkage forming a GalNAc-beta 1-->4GlcNAc (N.N'-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase (beta 4-GalNAcT), This enzyme resembles invertebrate beta 4-GalNAcT as well as mammalian beta 4-galactosyltransferase (beta P4GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta 4-GalNAcT by its incapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif, Furthermore, the GalNAcT activity appeared not to be due to a promiscuous action of a beta 4-GalT as could he demonstrated by comparing the beta 4-GalNAcT and beta 4-GalT activities of the mammary gland, bovine colostrum, and purified beta 4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta 4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta 4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta 4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAc beta 1-->4Glc. This enzyme thus forms the second example of a mammalian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta 4-GalNAcT functions in the synthesis of lacdiNAc-based, complex-type glycans frequently occurring on bovine milk glycoproteins. The action of this enzyme is to be considered when aiming at the production of properly glycosylated protein biopharmaceuticals in the milk of transgenic dairy animals.