Two chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose

James Ripka, Anthony Adamany, Pamela Stanley

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Abstract

A biochemical basis for the pea and lentil lectin resistance of two Chinese hamster ovary (CHO) cell mutants, Lec13 and Lec13A, was investigated. Studies of the G glycopeptides of vesicular stomatitis virus grown in the mutants indicated that Lec13 cells essentially lack the ability to add fucose to complex carbohydrates while Lec13A cells synthesize significant proportions of fucosylated, complex moieties. However, both mutants were known to be reverted to lectin sensitivity by growth in l-fucose, making them similar to the mouse lymphoma mutant, PLR1.3, which is defective in the conversion of GDP-manose to GPD-fucose [M. L. Reitman, I. S. Trowbridge, and S. Kornfeld (1980) J. Biol. Chem. 255, 9900-9906]. Optimal conditions for the production of GDP-fucose from GDP-mannose by CHO cytosol were found to occur at pH 8 in the presence of 7.5 μm GDP-mannose, 15 mm Mg2+, 0.2 mm NAD+, 0.2 mm NADPH, 10 mm niacinamide, 5 mm ATP, and 50 mm Tris-HCl. Under these conditions, Lec13 cytosol produced no detectable GDP-fucose nor GDP-sugar intermediates while Lec13A cytosol produced significant quantities of both. Mixing experiments with Lec13 cytosol identified the first enzyme of the conversion pathway (GDP-mannose 4,6-dehydratase, EC 4.2.1.47) as the site of the block. In addition to being markedly reduced, the Lec13A 4,6-dehydratase activity was relatively insensitive to changes in pH in comparison to the activity in parental cytosol, suggesting that Lec13A cells might possess a structurally altered GDP-mannose 4,6-dehydratase enzyme.

Original languageEnglish (US)
Pages (from-to)533-545
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume249
Issue number2
DOIs
StatePublished - 1986

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Guanosine Diphosphate Fucose
Guanosine Diphosphate Mannose
Glycosylation
Hydro-Lyases
Cricetulus
Cytosol
Fucose
Ovary
Guanosine Diphosphate Sugars
GDPmannose 4,6-dehydratase
Niacinamide
Glycopeptides
Enzymes
NADP
Viruses
Vesicular Stomatitis
Lectins
NAD
Adenosine Triphosphate
Cells

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Two chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose. / Ripka, James; Adamany, Anthony; Stanley, Pamela.

In: Archives of Biochemistry and Biophysics, Vol. 249, No. 2, 1986, p. 533-545.

Research output: Contribution to journalArticle

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