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 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.