Molecular Cloning and Enzymatic Characterization of a UDP-GalNAc:GlcNAcβ-R β1,4-N-Acetylgalactosaminyltransferase fromCaenorhabditis elegans

Abstract

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal␤4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc␤4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding ␤4GalTs responsible for LN synthesis have been reported, the ␤4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc␤-R ␤1,4-N-acetylgalactosaminyltransferase (Ce␤4GalNAcT) that synthesizes the LDN structure. Ce␤4GalNAcT is a member of the ␤4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce␤4GalNAcT expressed in CHOLec8 cells was active using UDP-GalNAc, but not UDPGal, as a donor toward a variety of acceptor substrates containing terminal ␤-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 1 H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce␤4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic ␤4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.

Resistance to a Bacterial Toxin Is Mediated by Removal of a Conserved Glycosylation Pathway Required for Toxin-Host Interactions

“…The 150-kilobase region between these boundary single nucleotide polymorphisms contains 20 predicted ORFs. Sequencing of cDNA isolated from the bre-4 alleles revealed changes in the enzymatically characterized glycosyltransferase Y73E7A.7 (8). cDNA rescue of a bre-4 mutant confirmed the identity of bre-4.…”

“…1B). BRE-4 has been previously characterized biochemically as a UDPGalNAc:GlcNAc ␤1,4-N-acetylgalactosaminyltransferase (Ce␤4 GalNAcT) (8). BRE-4/Ce␤4GalNAcT specifically synthesizes the GalNAc␤1,4GlcNAc sequence commonly found on glycoproteins and glycolipids.…”

“…Our identification of two additional genes, bre-2 and bre-4, extends this pathway. The bre-4 gene product has been previously demonstrated to have glycosyltransferase activity (8); the bre-2 gene product has not, and thus, BRE-2 should be regarded as a putative glycosyltransferase. A likely bre-4 homologue in Drosophila is noted above.…”

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

“…Its ortholog was also found in mouse. Kawar et al (23) has reported the cloning of a nematode LacdiNAc synthase, Ce␤4GalNAc-T, which is probably an ortholog of human ␤4Gal-T1. ␤4GalNAc-T3 in the present study is a novel glycosyltransferase, and this is the first report of the molecular cloning and characterization of mammal LacdiNAc synthases, ␤4GalNAc-T3 and m␤4GalNAc-T3.…”

“…(N,NЈ-diacetyllactosediamine, LacdiNAc), has been cloned and named Ce␤4GalNAcT (23). Ce␤4GalNAcT has Ile at the same position as Tyr-289 of ␤4Gal-T1, and transfers GalNAc to a variety of acceptor substrates with terminal ␤-linked GlcNAc, resulting in the synthesis of the LacdiNAc structure.…”