Identification and Characterization of Three Novel β1,3-N-Acetylglucosaminyltransferases Structurally Related to the β1,3-Galactosyltransferase Family

Abstract

We have isolated three types of cDNAs encoding novel ␤1,3-N-acetylglucosaminyltransferases (designated ␤3Gn-T2, -T3, and -T4) from human gastric mucosa and the neuroblastoma cell line SK-N-MC. These enzymes are predicted to be type 2 transmembrane proteins of 397, 372, and 378 amino acids, respectively. They share motifs conserved among members of the ␤1,3-galactosyltransferase family and a ␤1,3-N-acetylglucosaminyltransferase (designated ␤3Gn-T1), but show no structural similarity to another type of ␤1,3-N-acetylglucosaminyltransferase (iGnT). Each of the enzymes expressed by insect cells as a secreted protein fused to the FLAG peptide showed ␤1,3-N-acetylglucosaminyltransferase activity for type 2 oligosaccharides but not ␤1,3-galactosyltransferase activity. These enzymes exhibited different substrate specificity. Transfection of Namalwa KJM-1 cells with ␤3Gn-T2, -T3, or -T4 cDNA led to an increase in poly-N-acetyllactosamines recognized by an anti-i-antigen antibody or specific lectins. The expression profiles of these ␤3Gn-Ts were different among 35 human tissues. ␤3Gn-T2 was ubiquitously expressed, whereas expression of ␤3Gn-T3 and -T4 was relatively restricted. ␤3Gn-T3 was expressed in colon, jejunum, stomach, esophagus, placenta, and trachea. ␤3Gn-T4 was mainly expressed in brain. These results have revealed that several ␤1,3-Nacetylglucosaminyltransferases form a family with structural similarity to the ␤1,3-galactosyltransferase family. Considering the differences in substrate specificity and distribution, each ␤1,3-N-acetylglucosaminyltransferase may play different roles.A family of human ␤1,3-galactosyltransferases (␤3Gal-Ts) 1 consisting of five members (␤3Gal-T1, -T2, -T3, -T4, and -T5) was recently identified (1-4). The first ␤1,3-galactosyltransferase (␤3Gal-T1), which catalyzes the formation of type 1 oligosaccharides, was isolated by us using an expression cloning approach (1). Expression patterns of ␤3Gal-T1 and type 1 oligosaccharides strongly suggested the existence of ␤3Gal-T1 homologs. For instance, type 1-derived oligosaccharides such as sialyl-Le a were known to be expressed in colon and pancreatic cancer cell lines, whereas expression of ␤3Gal-T1 was detected in brain, but not in cancer cells. Our early approach using Southern hybridization failed to detect the existence of ␤3Gal-T1 homologous genes. However, recent accumulation of nucleotide sequence information on human cDNAs and genes such as expressed sequence tags (ESTs) enabled us to search homologous genes that do not have high similarity as detected by hybridization, but show significant similarity. A homology search based on the nucleotide or amino acid sequence of ␤3Gal-T1 led to the isolation of ␤3Gal-T2, -T3, and -T4, indicating that ␤3Gal-Ts form a family (1-3).␤3Gal-T2 catalyzed a similar reaction, but showed different substrate specificity compared with ␤3Gal-T1. The activity of ␤3Gal-T3 has not been detected, whereas the corresponding mouse enzyme exhibits weak ␤3Gal-T activity for both GlcNAc and GalNAc (5). On the other...

Biosynthesis of the Linkage Region of Glycosaminoglycans

“…These enzymes in fact contain a motif in common with other ␤1,3-glycosyltransferases. One of the enzymes, ␤3GalT3, has been shown to also transfer GalNAc in ␤1,3-linkage to globosides (42), and four structurally related ␤1,3-N-acetylglucosaminyltransferases have been recently cloned based on sequence homology to ␤3GalT1 (43)(44)(45). In contrast to these enzymes, GAG GalTII (␤3GalT6) preferentially acts on Gal␤1,4Xyl, which is found in the linkage region of glycosaminoglycans.…”

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

“…Construction and purification of human b3Gn-T8 proteins fused with FLAG peptide We performed a BLAST search of the EST databases and identified a cDNA (AW444713), homologous in amino acid sequence to the open reading frame (ORF) of b3Gn-T3 [3]. On searching the human genomic DNA database, we found a single contig (AC011462) containing the EST sequence.…”

“…elongation of polylactosamine chains, however they exhibit a different substrate specificity dependent on the length of the polylactosamine chain [1,3]. Our in vitro analysis previously demonstrated that b3Gn-T2 is most active toward polylactosamine substrates having 2-5 LacNAc repeats [1].…”

“…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].…”