A Lymnaea stagnalis gene, with sequence similarity to that of mammalian beta 1-->4-galactosyltransferases, encodes a novel UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyltransferase.
A cDNA encoding a novel glycosyltransferase, that may be involved in a variant pathway for the synthesis of complex type oligosaccharide chains, was cloned from the pond snail Lymnaea stagnalis. By heterologous hybridization, using bovine beta 1-->4-galactosyltransferase cDNA as probe, a genomic clone from a snail library was isolated. This genomic clone was subsequently used to clone the corresponding cDNA from a prostate gland library. The isolated cDNA encodes a polypeptide of 490 amino acids with a type II membrane protein topology typical for glycosyltransferases. The carboxyl-terminal part, encoding the putative catalytic domain, reveals considerable sequence similarity with the corresponding region of mammalian beta 1-->4-galactosyltransferases, suggesting an evolutionary relationship. Expression of this cDNA in COS cells and insect cells revealed that the encoded enzyme transfers GlcNAc, rather than Gal or GalNAc, from the corresponding nucleotide sugars to several beta-N-acetylglucosaminides. Structural characterization by 1H NMR spectroscopy of products formed in vitro demonstrated that the enzyme can be identified as a UDP-GlcNAc:GlcNAc beta-R beta 1-->4-N-acetylglucosaminyl-transferase. A new family of glycosyltransferases has hereby been discovered, consisting of enzymes that act on acceptor substrates with a terminal beta-linked GlcNAc residue and establish a beta 1-->4-linkage, but have a different nucleotide sugar requirement.
Bakker
H
H
Department of Medical Chemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Agterberg
M
M
Van Tetering
A
A
Koeleman
C A
CA
Van den Eijnden
D H
DH
Van Die
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D015698:Genomic Library; D007313:Insecta; D008195:Lymnaea / Q000201:enzymology* / Q000235:genetics*; D008322:Mammals; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000235:genetics*; D009237:N-Acetyllactosamine Synthase / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D017931:DNA Primers; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012313:RNA; D017351:N-Acetylglucosaminyltransferases; C090677:UDP-GlcNAc-GlcNAc-R 1-4-N-acetylglucosaminyltransferase; D009237:N-Acetyllactosamine Synthase