TY - JOUR
T1 - Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I
AU - Chen, Wei
AU - Stanley, Pamela
N1 - Funding Information:
We thank Subha Sundaram for excellent technical support and the preparation of the chicken anti-mouse GlcNAc-TI antibody. We also thank Drs. Harry Schachter, Paul Gleeson, and Tohru Komano for antibodies. This work was supported by a grant from the National Institutes of Health (ROI CA36434 to P.S.) and by partial support from Albert Einstein Cancer Center Grant POI 13330. Accession numbers: Lec1.3C, AF510636; Lec1.1N, AF510637; Lec9.1.3.C, AF510638; Lec3.2.8.1, AF510639; Lec1.1C, AF510640.
PY - 2003/1/1
Y1 - 2003/1/1
N2 - 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 ∼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.
AB - 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 ∼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.
KW - GlcNAc-TI
KW - Lectin resistance
KW - Mgat1 gene mutations
KW - Site-directed mutagenesis
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U2 - 10.1093/glycob/cwg003
DO - 10.1093/glycob/cwg003
M3 - Article
C2 - 12634323
AN - SCOPUS:0037234471
SN - 0959-6658
VL - 13
SP - 43
EP - 50
JO - Glycobiology
JF - Glycobiology
IS - 1
ER -