Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans

Abstract

The core 3 structure of the O-glycan, GlcNAc␤1-3Gal-NAc␣1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide ␤1,3-Nacetylglucosaminyltransferase (␤3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human ␤3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it ␤3Gn-T6. The putative amino acid sequence of ␤3Gn-T6 retains the ␤3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of ␤3Gn-T6 expressed in insect cells showed ␤3Gn-T activity toward GalNAc␣-p-nitrophenyl and GalNAc␣1-serine/threonine. The ␤1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. ␤3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the ␤3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that ␤3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.

Aberrant O‐glycosylation contributes to tumorigenesis in human colorectal cancer

“…It is known that mature O‐glycosylation depends specifically on T‐synthase activity and its single chaperone‐Cosmc 31. Besides, in colonic tissues, defective core 3 O‐glycosylation could also lead to the exposure of Tn antigen 21, 32. Here we found that the expression of C3GnT that controls core 3 O‐glycosylation in cancer tissues expressing Tn antigen (Tn‐positive) was comparable to that in Tn‐negative cancerous tissues, thereby suggesting that there were only possible defects in T‐synthase and/or Cosmc (Figure 2A).…”

“…Knockout of either T‐synthase or Cosmc in mice causes Tn expression in vivo 18, 19, 20. Besides, in gastrointestinal tract, Tn antigen can also be elongated into core 3 O‐glycans by specific glycosyltransferase‐C3GnT 21. Overall, T‐synthase, Cosmc and C3GnT are essential for complete O‐glycosylation in colonic tissues.…”

A novel β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T8), which synthesizes poly‐N‐acetyllactosamine, is dramatically upregulated in colon cancer

“…Three b3-glycosyltransferase (b3-GT) motifs, XIRX(S/T)W(G/L/M), (F/Y)-XXXXDXD and (E/D)DVXXGX, which we found in a previous study, are commonly encoded in b3-GTs that combine two sugars with a b1,3-linkage [1][2][3][4]. These specific motifs are shared by b3Gal-Ts, b3GalNAc-Ts and b3Gn-Ts.…”

“…In the past, we succeeded in the molecular cloning of a series of b3-GT genes. Thirteen human b3-GTs, i.e., five b3Gal-Ts, two b3GalNAc-Ts and six b3Gn-Ts, have been identified and reported [1][2][3][4][5][6][7][8].…”

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

“…Quantitative Analysis of ␤4GalNAc-T3 Transcripts in Human Tissues by Real Time PCR-For the quantification of human ␤4GalNAc-T3 transcripts, we employed the real time PCR method, as described in detail previously (40,41). Total RNA of various human tissues was purchased from Clontech.…”