Mouse UDP-GlcNAc: dolichyl-phosphate N-acetylglucosaminephosphotransferase. Molecular cloning of the cDNA, generation of anti-peptide antibodies and chromosomal localization

Abstract

A cDNA encoding UDP-GlcNAc-dolichyl-phosphate N-acetylglucosaminephosphotransferase (GPT; EC 2.7.8.15), an enzyme that catalyses the first step in the synthesis of dolichol-linked oligosaccharides, was isolated from mRNA prepared from mouse mammary glands. The cDNA contains an open reading frame that codes for a protein of 410 amino acids with a predicted molecular mass of 46.472 kDa. Mouse GPT has two copies of a putative dolichol-recognition sequence that has so far been identified in all eukaryotic enzymes which interact with dolichol, and four consensus sites for asparagine-linked glycosylation. It shows a high degree of conservation with yeast and hamster GPTs at the amino acid level. The mouse GPT cDNA recognized a single mRNA species of about 2 kb in mouse mammary glands when used as a probe in Northern blot analysis. An antiserum raised against a 15-residue peptide, derived from the predicted amino acid sequence of the cloned mouse cDNA, specifically precipitated the activity of GPT from solubilized mouse mammary gland microsomes, and detected a protein of about 48 kDa on Western blot. This size is in good agreement with that predicted from the cDNA sequence, and also with that (46 and 50 kDa) of purified bovine GPT. With the use of a panel of mouse/hamster somatic-cell hybrids and a specific probe derived from the 3'-non-coding region of the mouse cDNA, the GPT gene was mapped to mouse chromosome 17.