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.

Abstract

UDP-GlcNAc:a-3-D-mannoside P-1,2-Nacetylglucosaminyltransferase 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 ollgonucleotide-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 AgtlO 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 glycosyltrnsferases, 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.

Expression cloning of a cDNA encoding UDP-GlcNAc:Gal beta 1-3-GalNAc-R (GlcNAc to GalNAc) beta 1-6GlcNAc transferase by gene transfer into CHO cells expressing polyoma large tumor antigen.

“…Besides the cDNA isolated in the present study, cDNA has been obtained for only one other GlcNAc transferase (30,31). Although these enzymes utilize the same nucleotide sugar, UDP-GlcNAc, no homology in primary amino acid sequence could be detected between these two enzymes.…”

The 17-residue transmembrane domain of beta-galactoside alpha 2,6-sialyltransferase is sufficient for Golgi retention

“…Comparison of the primary sequences of GT revealed that the entire transmembrane domain is identical in human and bovine and that 19 of the 20 residues in the transmembrane domain are also conserved in mouse (DAgostaro et al ., 1989 ;Masri et al ., 1988 ;Russo et al ., 1990 ;Shaper et al, 1988). Furthermore, the sequence of the transmembrane domain of NTI is totally conserved between human and rabbit (Kumar et al ., 1990 ;Sarkar et al ., 1991). The high degree of conser-…”

Expression of the developmental I antigen by a cloned human cDNA encoding a member of a beta-1,6-N-acetylglucosaminyltransferase gene family.

“…In addition, sequence comparison with glycosyltransferases cloned by others, including ~-l,2-N-acetylglucosaminyltransferase I (Kumar et al 1990;Sarkar et al 1991) and f3-1,4- 5B, C). However, the homology in the amino acid sequences of this portion is not as long as the aforementioned.…”