A beta-1,3-N-acetylglucosaminyltransferase with poly-N-acetyllactosamine synthase activity is structurally related to beta-1,3-galactosyltransferases

Abstract

Human and mouse cDNAs encoding a new beta-1, 3-N-acetylglucosaminyltransferase (beta3GnT) have been isolated from fetal and newborn brain libraries. The human and mouse cDNAs included ORFs coding for predicted type II transmembrane polypeptides of 329 and 325 aa, respectively. The human and mouse beta3GnT homologues shared 90% similarity. The beta3GnT gene was widely expressed in human and mouse tissues, although differences in the transcript levels were visible, thus indicating possible tissue-specific regulation mechanisms. The beta3GnT enzyme showed a marked preference for Gal(beta1-4)Glc(NAc)-based acceptors, whereas no activity was detected on type 1 Gal(beta1-3)GlcNAc and O-glycan core 1 Gal(beta1-3)GalNAc acceptors. The new beta3GnT enzyme was capable of both initiating and elongating poly-N-acetyllactosamine chains, which demonstrated its identity with the poly-N-acetyllactosamine synthase enzyme (E.C. 2.4.1.149), showed no similarity with the i antigen beta3GnT enzyme described recently, and, strikingly, included several amino acid motifs in its protein that have been recently identified in beta-1,3-galactosyltransferase enzymes. The comparison between the new UDP-GlcNAc:betaGal beta3GnT and the three UDP-Gal:betaGlcNAc beta-1,3-galactosyltransferases-I, -II, and -III reveals glycosyltransferases that share conserved sequence motifs though exhibiting inverted donor and acceptor specificities. This suggests that the conserved amino acid motifs likely represent residues required for the catalysis of the glycosidic (beta1-3) linkage.

Figure 1

Primary structure and deduced amino acid sequence of the human and mouse β3GnT cDNA. Amino acids identical in both species are in boldface. The predicted transmembrane region is shaded and the single potential N-glycosylation site (N-X-[S/T]) is underlined.

Figure 2

clustalw alignment of mouse β3GalT-I, -II, -III, and -IV and mouse β3GnT proteins. Conserved residues are shaded. The white arrows mark the positions of the cysteine residues conserved among β3GalT proteins. The black arrow shows the position of the cysteines conserved in the five proteins.

Figure 3

Expression pattern of the β3GnT gene in adult human and mouse tissues as determined by using Northern blot analysis. Each lane represents about 2 μg of poly(A)⁺ RNA. At the left, the size of the RNA markers is indicated in kilobases.

Figure 4

Flow cytometry analysis of HeLa cells. HeLa cells transfected with an empty pcDNA3.1 vector (mock) or with the β3GnT expression vector (β3GnT) were stained with tomato lectin (Left) or anti-i antiserum (Right).

Figure 5

600-MHz one-dimensional (a) and two-dimensional (b) ROESY (200 ms) ¹H NMR spectra of the isolated trisaccharide GlcNAc(β1–3)Gal(β1–4)Glc(β1-OBn) (C-B-A), recorded at 300 K.