Two closely related forms of UDP-GlcNAc: alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II occur in the clawed frog Xenopus laevis.
UDP-GlcNAc:alpha6-D-mannoside beta1,2-N-acetylglucosaminyltransferase II (GnT II; EC 2.4.1.143) is a medial-Golgi resident enzyme that catalyses an essential step in the biosynthetic pathway leading from high mannose to complex N-linked oligosaccharides. Screening a cDNA library from Xenopus laevis ovary with a human GnT II DNA probe resulted in the isolation of two cDNA clones encoding two closely related GnT II isoenzymes, GnT II-A and GnT II-B. Analysis of the corresponding genomic DNAs revealed that the open reading frame of both X. laevis GnT II genes resides within a single exon. The GnT II-A gene was found to be transcriptionally active in all X. laevis tissues tested. In contrast, expression of the GnT II-B gene was detected only in a limited number of tissues. Both GnT II-A and GnT II-B exhibit a type II transmembrane protein topology with a putative N-terminal cytoplasmic tail of 9 amino acids followed by a transmembrane domain of 18 residues, and a C-terminal luminal domain of 405 residues. The two proteins differ at 28 amino acid positions within their luminal regions. Heterologous expression of soluble forms of the enzymes in insect cells showed that GnT II-A and GnT II-B are both catalytically active and exhibit similar specific activities. Both recombinant proteins are modified with N-linked oligosaccharides. N-terminal deletion studies demonstrated that the first 49 amino acid residues are not essential for proper folding and enzymatic activity of X. laevis GnT II.
Mucha
Jan
J
Zentrum für Angewandte Genetik, Universität für Bodenkultur Wien, Muthgasse 18, A-1190 Wien, Austria.
Svoboda
Barbara
B
Kappel
Sonja
S
Strasser
Richard
R
Bencur
Peter
P
Fröhwein
Ulrike
U
Schachter
Harry
H
Mach
Lukas
L
Glössl
Josef
J
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005260:Female; D015723:Gene Library; D005822:Genetic Vectors; D016678:Genome; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis* / Q000737:chemistry*; D009844:Oligosaccharides / Q000737:chemistry; D010053:Ovary / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000737:chemistry*; D014982:Xenopus laevis
D018076:DNA, Complementary; D009844:Oligosaccharides; D011134:Polysaccharides; D020033:Protein Isoforms; D012333:RNA, Messenger; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase