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Two Chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose
Authors:J Ripka  A Adamany  P Stanley
Institution:1. Department of Cell Biology Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461 USA;2. Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, New York 10461 USA;1. Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047;2. Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas 66047;3. Department of Chemistry, University of Kansas, Lawrence, Kansas 66045;1. Department of Medicine, University of Southern California, Los Angeles, CA, USA;2. Molecular Biomarkers Laboratory, The Biodesign Institute, Arizona State University, Tempe, AZ, USA;3. Atherosclerosis Research, Children''s Hospital Oakland Research Institute, Oakland, CA, USA;1. Area for Molecular Function, Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan;2. Medical Innovation Research Unit (MiU), Advanced Institute of Innovative Technology (AIIT), Saitama University, Sakura, Saitama 338-8570, Japan
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-mannose 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 microM 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.
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