Differential galactosylation of neuronal and haematopoietic signal regulatory protein-alpha determines its cellular binding-specificity.

Signal regulatory protein-alpha (SIRP alpha) is a member of the Ig superfamily selectively expressed by neuronal and myeloid cells. The molecule mediates functional interactions with CD47/integrin-associated protein. Here we provide evidence for the tissue-specific glycosylation of neuronal and haematopoietic SIRP alpha. We demonstrate a major difference in the galactosylation of N-linked glycans isolated from neuronal (i.e. brain-derived) SIRP alpha as compared to myeloid (i.e. spleen-derived) SIRP alpha, with neuronal SIRP alpha almost completely lacking galactose. beta 4-galactosyltransferase assays demonstrated that this is most likely due to a low galactosylation capacity of the brain. In order to investigate the role of galactosylation of SIRP alpha in cellular interactions, soluble recombinant SIRP alpha glycoforms containing galactose (SIRP alpha-Fc) or lacking galactose (SIRP alpha(Delta Gal)-Fc) were produced. Binding studies demonstrated superior binding of SIRP alpha(Delta Gal)-Fc to cerebellar neurons and isolated lymphocytes. In contrast, SIRP alpha-Fc bound relatively strong to macrophages. These data show that the galactosylation of SIRP alpha determines its cellular binding specificity.

Recombinant glycodelin carrying the same type of glycan structures as contraceptive glycodelin-A can be produced in human kidney 293 cells but not in chinese hamster ovary cells.

We have produced human recombinant glycodelin in human kidney 293 cells and in Chinese hamster ovary (CHO) cells. Structural analyses by lectin immunoassays and fast atom bombardment mass spectrometry showed that recombinant human glycodelin produced in CHO cells contains only typical CHO-type glycans and is devoid of any of the N, N'-diacetyllactosediamine (lacdiNAc)-based chains previously identified in glycodelin-A (GdA). By contrast, human kidney 293 cells produced recombinant glycodelin with the same type of carbohydrate structures as GdA. The presence of a beta1-->4-N-acetylgalactosaminyltransferase functioning in the synthesis of lacdiNAc-based glycans in human kidney 293 cells is concluded to be the cause of the occurrence of lacdiNAc-based glycans on glycodelin produced in these cells. Furthermore, human kidney 293 cells were found to be particularly suited for the production of recombinant glycodelin when they were cultured in high glucose media. Lowering the glucose concentration and the addition of glucosamine resulted in higher relative amounts of oligomannosidic-type glycans and complex glycans with truncated antennae. Human glycodelin is an attractive candidate for the development of a contraceptive agent, and this study gives valuable information for selecting the proper expression system and cell culture conditions for the production of a correctly glycosylated recombinant form.

The lactose analog GalNAcbeta1-->4Glc is present in bovine colostrum. Enzymatic basis for its occurrence.

We have isolated from bovine colostrum the lactose analog GalNAcbeta1-->4Glc. The enzymatic basis for its occurrence was studied by assaying the activities of GlcNAcbeta-R beta4-N-acetylgalactosaminyltransferase (beta4-GalNAcT) and GlcNAcbeta-R beta4-galactosyltransferase (beta4-GalT) in primary milk and several lactating bovine mammary gland fractions. As the beta4-GalNAcT, which appears to be tightly membrane bound, is induced by the milk protein alpha-lactalbumin (alpha-LA) to act on Glc, it is concluded that beta4-GalNAcT is responsible for the synthesis of GalNAcbeta1-->4Glc in the gland. The comparatively low level (15-20 mg/l) at which this disaccharide is produced may be due to the relatively poor interaction of beta4-GalNAcT with alpha-LA as well as to the fact that alpha-LA does not inhibit the action of the enzyme on N-acetylglucosaminides.

Bovine mammary gland UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase is glycoprotein hormone nonspecific and shows interaction with alpha-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 MHz1H-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 beta1-->4-linkage forming a GalNAcbeta1-->4GlcNAc ( N, N '-diacetyllactosediamine, lacdiNAc) unit. Therefore, the enzyme can be identified as a UDP-GalNAc:GlcNAcbeta-R beta1-->4-N-acetylgalactosaminyltransferase (beta4-GalNAcT). This enzyme resembles invertebrate beta4-GalNAcT as well as mammalian beta4-galactosyltransferase (beta4-GalT) in acceptor specificity. It can, however, be clearly distinguished from the pituitary hormone-specific beta4-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 beta4-GalT as could be demonstrated by comparing the beta4-GalNAcT and beta4-GalT activities of the mammary gland, bovine colostrum, and purified beta4-GalT, by competition studies with UDP-GalNAc and UDP-Gal, and by use of an anti-beta4-GalT polyclonal inhibiting antibody. Interestingly, under conditions where mammalian beta4-GalT forms with alpha-lactalbumin (alpha-LA) the lactose synthase complex, the mammary gland beta4-GalNAcT was similarly induced by alpha-LA to act on Glc with an increased efficiency yielding the lactose analog GalNAcbeta1-->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 beta4-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.