Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-Man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Low expression of lipid-linked oligosaccharide due to a functionally altered Dol-P-Man synthase reduces protein glycosylation in cAMP-dependent protein kinase deficient Chinese hamster ovary cells

Chinese hamster ovary cells express a wide variety of glycoproteins with Mr ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate...

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Bibliographic Details
Published in:Glycoconjugate journal Vol. 21; no. 8-9; pp. 479 - 486
Main Authors: Banerjee, Dipak K, Aponte, Eira, Dasilva, Jaime J
Format: Journal Article
Language:English
Published: United States Springer Nature B.V 01-08-2004
Subjects:
Animals
CHO Cells
Cricetinae
Cricetulus
Cyclic AMP-Dependent Protein Kinase Type II
Cyclic AMP-Dependent Protein Kinases - genetics
Cyclic AMP-Dependent Protein Kinases - metabolism
Deoxyglucose - metabolism
Gene Expression Regulation - physiology
Glycosylation
Kinases
Lipopolysaccharides - biosynthesis
Mannosyltransferases - metabolism
Mutation
Proteins
Rodents
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Summary:Chinese hamster ovary cells express a wide variety of glycoproteins with Mr ranging from 15,000 to 200,000 dalton and higher. Glycosylation of these proteins was much less in cAMP-dependent protein kinase (PKA)-deficient mutants which expressed either (i) a defective C-subunit with altered substrate specificity and having no detectable type II kinase (mutant 10215); or (ii) an altered RI subunit and having no detectable type II kinase (mutant 10248); or (iii) exhibited the lowest level of total kinase with no detectable type I kinase but having a small amount of type II kinase (mutant 10260). Addition of 8Br-cAMP enhanced protein glycosylation index in wild type cells 10001 by 120% but only 7 to 23% in the mutant cells. The rate of lipid-linked oligosaccharide (LLO) biosynthesis was linear for 1 h in all cell types, but the total amount of LLO expressed was much less in PKA-deficient mutants. Pulse-chase experiments indicated that the t1/2 for LLO turnover was also twice as high in PKA-deficient cells as in the wild type. Size exclusion chromatography of the mild-acid released oligosaccharide confirmed that both wild type and the mutant cells synthesized Glc3Man9GlcNAc2-PP-Dol as the most predominating species with no accumulation of Man5GlcNAc2-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a Km for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the kcat for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the kcat for DPMS in CHO cell mutants; however, in vitro protein phosphorylation with a cAMP-dependent protein kinase restored their kinetic activity to the level of the wild type.
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ISSN:0282-0080
1573-4986
DOI:10.1007/s10719-004-5538-2