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

Glycobiology. 2003 Jan;13(1):43-50. doi: 10.1093/glycob/cwg003. Epub 2002 Oct 30.

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 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Northern
  • Blotting, Western
  • CHO Cells / metabolism*
  • Chickens / immunology
  • Codon
  • Cricetinae
  • DNA Primers
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Glycosylation
  • Lectins / metabolism
  • Microsomes / enzymology*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation / genetics*
  • N-Acetylglucosaminyltransferases / deficiency
  • N-Acetylglucosaminyltransferases / genetics*
  • N-Acetylglucosaminyltransferases / metabolism*
  • Oligosaccharides / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Deletion
  • Transfection

Substances

  • Codon
  • DNA Primers
  • DNA, Complementary
  • Lectins
  • Oligosaccharides
  • N-Acetylglucosaminyltransferases
  • alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I

Associated data

  • GENBANK/AF510636
  • GENBANK/AF510637
  • GENBANK/AF510638
  • GENBANK/AF510639
  • GENBANK/AF510640