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

We found a novel human glycosyltransferase gene carrying a hypothetical β1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5′-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank™ accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc β-benzyl. The product was deduced to be GalNAcβ1–4GlcNAcβ-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme β1,4- N -acetylgalactosaminyltransferase-III (β4GalNAc-T3). β4GalNAc-T3 effectively synthesized N,N ′-diacetylgalactosediamine, GalNAcβ1–4GlcNAc, at non-reducing termini of various acceptors derived not only from N -glycans but also from O -glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N ′-diacetylgalactosediamine structures in their N -glycans, we examined the ability of β4GalNAc-T3 to synthesize N,N ′-diacetylgalactosediamine structures in N -glycans on a model protein. When fetal calf fetuin treated with neuraminidase and β1,4-galactosidase was utilized as an acceptor protein, β4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that β4GalNAc-T3 could transfer GalNAc residues, producing N,N ′-diacetylgalactosediamine structures at least in N -glycans and probably in both N - and O -glycans.