Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I.
UDP-GlcNAc:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) catalyzes an essential first step in the conversion of high-mannose N-glycans to hybrid and complex N-glycans. Cloning of the gene encoding this enzyme was carried out by mixed oligonucleotide-primed polymerase chain reaction amplification of rabbit liver single-stranded cDNA using sense and antisense 20- to 24-base-pair (bp) primers. A rabbit liver library in phage lambda gt10 yielded a 2.5-kilobase (kb) cDNA with a 447-amino acid coding sequence. None of the nine asparagine residues were in an Asn-Xaa-(Ser or Thr) sequence, indicating that the protein is not N-glycosylated. There is no sequence homology to other previously cloned glycosyltransferases, but GnT I appears to have a domain structure typical of these enzymes--i.e., a short amino-terminal domain, a transmembrane domain, a "neck" region, and a large carboxyl-terminal catalytic domain. RNA was transcribed off the 2.5-kb cDNA, and in vitro translation with rabbit reticulocyte lysate yielded a 52-kDa protein with GnT I activity.
Sarkar
M
M
Research Institute, Hospital for Sick Children, Toronto, ON, Canada.
Hull
E
E
Nishikawa
Y
Y
Simpson
R J
RJ
Moritz
R L
RL
Dunn
R
R
Schachter
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015723:Gene Library; D005796:Genes; D005964:Glucosyltransferases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D014158:Transcription, Genetic
D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D012333:RNA, Messenger; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I