Lec1A Chinese hamster ovary cell mutants appear to arise from a structural alteration in N-acetylglucosaminyltransferase I

W. Chaney, P. Stanley

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40 Scopus citations

Abstract

The biochemical and kinetic properties of UDP-GlcNAc:α-D-mannoside (GlcNAc to Manα1,3) β1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) have been investigated in the Chinese hamster ovary glycosylation mutant Lec1A. Previous studies showed that, whereas Lec1A cells synthesize complex carbohydrates at levels consistent with partial GlcNAc-TI action, no GlcNAc-TI activity was detected in Lec1A cell-free extracts (Stanley, P., and Chaney, W. (1985) Mol. Cell. Biol. 5, 1204-1211). It is now reported that, under altered reaction conditions, GlcNAc-TI activity can be measured in Lec1A cell extracts. The GlcNAc-TI enzyme in Lec1A.2C has a pH optimum of 7.5 (compared with 6.25 for the parental enzyme) and apparent K(m) values for Man5GlcNAc2Asn and UDP-GlcNAc that are, respectively, 21- and 44-fold higher than the apparent K(m) values of GlcNAc-TI from parental Chinese hamster ovary cells. Two independent Lec1A mutants possess GlcNAc-TI activities with similarly altered biochemical and kinetic properties. In fact, under optimal assay conditions for each cell line, the level of GlcNAc-TI in Lec1A extracts is equal to that of parental Chinese hamster ovary cell extracts. Interestingly, the two glycosylation sites of the G glycoprotein of vesicular stomatitis virus are processed quite differently in Lec1A cells. The glycopeptide nearest the carboxyl-terminal appears to be a preferred substrate for the Lec1A GlcNAc-TI activity. The combined data suggest that the Lec1A mutation affects the gene that codes for GlcNAc-TI, giving rise to a structurally altered glycosyltransferase with different biochemical properties.

Original languageEnglish (US)
Pages (from-to)10551-10557
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number23
StatePublished - 1986

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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