Glycosylation defect in Lec1 Chinese hamster ovary mutant is due to a point mutation in N-acetylglucosaminyltransferase I gene.
The Lec1 Chinese hamster ovary (CHO) mutant is a leuco-phytohemagglutinin resistant cell line unable to synthesize complex and hybrid N-glycans due to the lack of N-acetylglucosaminyltransferase I (GnTI) activity. Here we have identified the lec1 mutation. Using specific antibodies to GnTI we demonstrate that Lec1 cells synthesize an inactive GnTI protein identical in size to the wild-type CHO enzyme. We have cloned and sequenced the gene coding GnTI from parental CHO and Lec1 mutant cells. Comparison of GnTI sequences detected three mutations within the luminal domain of Lec1 GnTI, each resulting in an amino acid substitution. The effect of each mutation on enzyme activity was analyzed by site-directed mutagenesis of wild-type rabbit GnTI and transient expression in COS cells. One of the three mutations (Cys123 --> Arg123) resulted in complete loss of activity, whereas the other two mutations had no apparent effect on enzyme activity. This conclusion was confirmed by expression of GnTI mutants in the GnTI null background of Saccharomyces cerevisiae. Both Lec1 GnTI and the GnTI mutant (Cys123 --> Arg123) are correctly localized to the Golgi apparatus, indicating that the inactive GnTI molecules are sufficiently well folded for efficient transport from the endoplasmic reticulum. These results demonstrate that the lec1 mutation is a point mutation and that Cys123 is a critical residue for GnTI activity.
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria 3181, Australia. pag@cobra.path.monash.edu.au
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Puthalakath
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Burke
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PA
Gleeson
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001483:Base Sequence; D016466:CHO Cells; D002999:Clone Cells; D006224:Cricetinae; D003545:Cysteine; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006031:Glycosylation; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017351:N-Acetylglucosaminyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D017510:Protein Folding; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D001120:Arginine; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D003545:Cysteine