Bovine mammary gland UDP-GalNAc : GlcNAc beta-R beta 1 -> 4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-lactalbumin

We have identified a novel N-acetylgalactosaminyltransferase activity in la ctating bovine mammary gland membranes. Acceptor specificity studies and an alysis of products obtained in vitro by 400 MHz H-1-NMR spectroscopy reveal ed 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 for ming a GalNAc-beta 1-->4GlcNAc (N.N'-diacetyllactosediamine, lacdiNAc) unit . Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAc beta-R bet a 1-->4-N-acetylgalactosaminyltransferase (beta 4-GalNAcT), This enzyme res embles invertebrate beta 4-GalNAcT as well as mammalian beta 4-galactosyltr ansferase (beta P4GalT) in acceptor specificity. It can, however, be clearl y distinguished from the pituitary hormone-specific beta 4-GalNAcT by its i ncapability of acting with an elevated activity on a glycoprotein substrate carrying a hormone-specific peptide motif, Furthermore, the GalNAcT activi ty 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 compe tition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta 4-Ga lT polyclonal inhibiting antibody. Interestingly, under conditions where ma mmalian beta 4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose syn thase complex, the mammary gland beta 4-GalNAcT was similarly induced by al pha-LA to act on Glc with an increased efficiency yielding the lactose anal og GalNAc beta 1-->4Glc. This enzyme thus forms the second example of a mam malian glycosyltransferase the specificity of which can be modified by this milk protein. It is proposed that the mammary gland beta 4-GalNAcT functio ns in the synthesis of lacdiNAc-based, complex-type glycans frequently occu rring on bovine milk glycoproteins. The action of this enzyme is to be cons idered when aiming at the production of properly glycosylated protein bioph armaceuticals in the milk of transgenic dairy animals.