Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I.
Lec1 CHO cell mutants lack N-acetylglucosaminyltransferase I (GlcNAc-TI) activity and do not synthesize complex or hybrid N-glycans. The origins of six independent lec1 mutations are shown to reside in the coding region of the Mgat1 gene, proving that GlcNAc-TI is mutated in Lec1 mutants. One mutant has Mgat1 gene transcripts of reduced size, whereas the others possess transcripts of approximately normal size and amount containing a unique insertion or transition mutation that leads to a premature stop codon in the Mgat1 gene coding region. The lec1 mutation in the Lec3.2.8.1 mutant, a line used to generate minimally glycosylated membrane glycoproteins for X-ray crystallography, is a G insertion that leads to a nonsense codon after amino acid 391. The Pro-Lec1.3C line from the ATCC and in laboratory stocks, a line used widely for diverse purposes, possesses a C insertion in the Mgat1 gene coding exon, causing a frame shift and producing a stable, truncated approximately 24-kDa product. Mgat1 gene mutations were confirmed by sequencing genomic DNA PCR products. Mutant cDNAs were reverted by site-directed mutagenesis and shown to confer wild-type lectin binding and GlcNAc-TI activity on Lec1 transfectants. Surprisingly, three Mgat1 gene nucleotide changes previously reported in Pro-Lec1.3C cells (Puthalakath et al. [1996] J. Biol. Chem., 271, 27818-27822) were not detected in this study. These Lec1 mutants provide a novel cohort for investigating the effects on Golgi trafficking and kin recognition of deletion mutants of GlcNAc-TI expressed at endogenous rather than nonphysiological levels.
Chen
Wei
W
Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, New York, NY 10461, USA.
Stanley
Pamela
P
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism*; D002645:Chickens / Q000276:immunology; D003062:Codon; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D037102:Lectins / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D017351:N-Acetylglucosaminyltransferases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection
D003062:Codon; D017931:DNA Primers; D018076:DNA, Complementary; D037102:Lectins; D009844:Oligosaccharides; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I