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

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 1991 Jan 1

PMID 1824724

Citations 31

Authors

M Sarkar

E HULL

Y Nishikawa

R J Simpson

R L Moritz

R Dunn

H SCHACHTER

Affiliations

Soon will be listed here.

Abstract

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.

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