Structural analyses of sugars on secreted glycoproteins performed about 30 years ago revealed bi-, tri-, and tetraantennary N-glycans in which GlcNAc residues linked to a conserved trimannosyl core initiated each antenna. These same structures were lectin binding sites on red cell glycoproteins (Kornfeld and Kornfeld 1970), prompting the search for the GlcNAc-transferases that catalyzed the addition of each GlcNAc residue. N-Acetylglucosaminyltransferase-I (GnT-I) was the first N-glycan branching GlcNAc- transferase for which an assay was developed (Gottlieb et al. 1975; Stanley et al. 1975). It is a Type II transmembrane protein of ~447 amino acids (Kumar et al. 1990; Sarkar et al. 1991) that resides in the medial/trans Golgi. GnT-I catalyzes the transfer of GlcNAc from UDP-GlcNAc to the terminal α-1,3-linked Man in Man5GlcNAc2Asn to initiate the synthesis of hybrid and complex N-linked glycans in multicellular organisms (reviewed in Kornfeld and Kornfeld 1985). It is not found in yeast or bacteria. The human gene encoding GnT-I is termed MGAT1 and resides on chromosome 5q35 (Kumar et al. 1992; Tan et al. 1995), and the mouse gene, Mgat1, is on chromosome 11 (Pownall et al. 1992). Two transcripts of ~2.9kb and ~3.3 kb are observed in most mammalian tissues, with the shorter transcript predominating in liver, and the longer transcript in brain (Yang et al. 1994; Yip et al. 1997; Fukada et al. 1998). In mammals, the coding region is in a single exon and the Mgatl gene is ubiquitously expressed. Mutant mice with a targeted Mgatl gene mutation that inactivates GnT-I die at mid-gestation (Ioffe and Stanley 1994; Metzler et al. 1994). However, cultured cells (Gottlieb et al. 1975; Meager et al. 1975; Stanley et al. 1975) and plants (von Schaewen et al. 1993) lacking GnT-I are viable and healthy.
KeywordsChinese Hamster Ovary Cell GlcNAc Residue Lectin Binding Site Chinese Hamster Ovary Cell Mutant Glycosylation Mutant
Unable to display preview. Download preview PDF.
- Chen W, Unligil UM, Rini JM, Stanley P (2001) Independent Lec1A CHO glycosylation mutants arise from point mutations in N-acetylglucosaminyltransferase I that reduce affinity for both substrates. Molecular consequences based on the crystal structure of GlcNAc-TI. Biochem 40:8765–8772CrossRefGoogle Scholar
- Narasimhan S, Stanley P, Schachter H (1977) Control of glycoprotein synthesis. Lectinresistant mutant containing only one of two distinct N-acetylglucosaminyltransferase activities present in wild-type Chinese hamster ovary cells. J Biol Chem 252:3926–3933Google Scholar
- Sarkar M, Hull E, Nishikawa Y, Simpson RJ, Moritz RL, Dunn R, Schachter H (1991) Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine: α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase I. Proc Natl Acad Sci USA 88:234–238PubMedCrossRefGoogle Scholar
- Stanley P, Narasimhan S, Siminovitch L, Schachter H (1975) Chinese hamster ovary cells selected for resistance to the cytotoxicity of phytohemagglutinin are deficient in a UDP-N-acetylglucosamine-glycoprotein N-acetylglucosaminyltransferase activity. Proc Natl Acad Sci USA 72:3323–3327PubMedCrossRefGoogle Scholar
- Tan J, D’Agostaro AF, Bendiak B, Reck F, Sarkar M, Squire JA, Leong P, Schachter H (1995) The human UDP-N-acetylglucosamine: α-6-D-mannoside-β-1,2-N-acetylgluco-saminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein. Eur J Biochem 231:317–328PubMedCrossRefGoogle Scholar
- Wenderoth I, von Schaewen A (2000) Isolation and characterization of plant N-acetyl glu-cosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant in antisense plants. Plant Physiol 123:1097–1108Google Scholar
- Yang J, Bhaumik M, Liu Y, Stanley P (1994) Regulation of n-linked glycosylation. Neuronal cell-specific expression of a 5′-extended transcript from the gene encoding N-acetyl-glucosaminyltransferase I. Glycobiology 4:703–712Google Scholar