Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I

Wei Chen, Pamela Stanley

Research output: Contribution to journalArticle

84 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)43-50
Number of pages8
JournalGlycobiology
Volume13
Issue number1
DOIs
StatePublished - Jan 1 2003

Fingerprint

CHO Cells
Genes
Mutation
Nonsense Codon
Cohort Effect
Mutagenesis
X ray crystallography
X Ray Crystallography
Membrane Glycoproteins
Site-Directed Mutagenesis
DNA Sequence Analysis
Lectins
Polysaccharides
alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I
Exons
Nucleotides
Complementary DNA
Amino Acids
Polymerase Chain Reaction
DNA

Keywords

  • GlcNAc-TI
  • Lectin resistance
  • Mgat1 gene mutations
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Five Lec1 CHO cell mutants have distinct Mgat1 gene mutations that encode truncated N-acetylglucosaminyltransferase I. / Chen, Wei; Stanley, Pamela.

In: Glycobiology, Vol. 13, No. 1, 01.01.2003, p. 43-50.

Research output: Contribution to journalArticle

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