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

Abstract

We found a novel human glycosyltransferase gene carrying a hypothetical ␤1,4-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 TM 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 ␤-benzyl. The product was deduced to be GalNAc␤1-4GlcNAc␤-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme ␤1,4-N-acetylgalactosaminyltransferase-III (␤4GalNAc-T3). ␤4GalNAc-T3 effectively synthesized N,N -diacetylgalactosediamine, GalNAc␤1-4GlcNAc, 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 ␤4GalNAc-T3 to synthesize N,N -diacetylgalactosediamine structures in N-glycans on a model protein.When fetal calf fetuin treated with neuraminidase and ␤1,4-galactosidase was utilized as an acceptor protein, ␤4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that ␤4GalNAc-T3 could transfer GalNAc residues, producing N,N -diacetylgalactosediamine structures at least in Nglycans and probably in both N-and O-glycans.

B4GALNT3 Expression Predicts a Favorable Prognosis and Suppresses Cell Migration and Invasion via β1 Integrin Signaling in Neuroblastoma

“…B4GALNT3 modifies N-and O-glycans decorated with GlcNAc in vitro. 7 In the present study, we found that B4GALNT3 adds LacdiNAc to ␤ 1 integrin and, thereby, suppresses its downstream signaling. Glycolipids on the cell surface may interact with functional membrane proteins, such as integrins, growth factor receptors, and Src family kinases to form glycosynaptic microdomains that control cell adhesion, growth, and motility.…”

“…7 This enzyme can transfer GalNAc to any nonreducing terminal GlcNAc-␤ in vitro, resulting in synthesis of GalNAc␤1, 4GlcNAc (LacdiNAc or LDN). The terminal ␤1,4 -linked GalNAc of LDN can be recognized by a lectin, Wisteria floribunda agglutinin (WFA).…”

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…By evaluating alkaline phosphatase activity in glycan-related gene knockdown mESCs, LacdiNAc, which is synthesized by b4GalNAc-T3 [35], was identified as a candidate cell surface glycan required for self-renewal of mESCs. We designed two constructs that expressed different shRNAs targeting b4GalNAc-T3 (b4GalNAc-T3-1 and b4GalNAc-T3-2), as described previously [40], and additionally constructs that expressed shRNAs targeting EGFP as negative controls.…”

“…By evaluation of alkaline phosphatase activity in RNAi-mediated knockdown mESCs, we identified glycans essential for selfrenewal. One of the glycan sequences of interest is the LacdiNAc (GalNAcb1-4GlcNAc) motif, which is synthesized by b4GalNAc-T3 [35]. LacdiNAc is frequently present on glycoproteins and glycolipids in invertebrates but is only present on a limited number of glycoproteins and glycolipids, such as glycoprotein hormones, in vertebrates [36,37].…”

β1,4-N-Acetylgalactosaminyltransferase III Enhances Malignant Phenotypes of Colon Cancer Cells

“…b4GalNAc-T3 has been shown to be significantly expressed in the gastrointestinal tract (14). In addition, the recombinant enzyme can transfer GalNAc to GlcNAc and form N,N ¶-diacetyllactosediamine (GalNAch1,4GlcNAc) in both N-and O-glycans by an in vitro enzyme activity assay.…”