CGC Bibliography Paper 5471

Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAcB-R B1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans.

Kawar ZS, Van Die I, Cummings RD

Medline:

12167666

Citation:

Journal of Biological Chemistry 277: 34924-34932 2002

Type:

ARTICLE

Genes:

sqv-3

Abstract:

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Galbeta4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAcbeta4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAcbeta-R beta1,4-N-acetylgalactosaminyltransferase (Cebeta4GalNAcT) that synthesizes the LDN structure. Cebeta4GalNAcT is a member of the beta4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Cebeta4GalNAcT expressed in CHO-Lec8 cells was active using UDP-GalNAc, but not UDPGal, as a donor toward a variety of acceptor substrates containing terminal beta-linked GlcNAc in both N- and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and H-1 NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Cebeta4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic beta4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.