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

被引:13
作者
Banerjee, DK [1 ]
Aponte, E [1 ]
DaSilva, JJ [1 ]
机构
[1] Univ Puerto Rico, Sch Med, Dept Biochem, San Juan, PR 00936 USA
关键词
Dol-P-Man synthase; Winked glycoprotein; Lipid-linked oligosaccharide; cAMP-dependent protein phosphorylation; Chinese hamster ovary cell mutant; phosphorylation regulation of Dol-P-Man synthase; regulation of asparagine-linked protein glycosylation;
D O I
10.1007/s10719-004-5538-2
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Chinese hamster ovary cells express a wide variety of glycoproteins with M-r 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 t(1/2) for LLO turnover was also twice as high in PKA-cleficient 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 Glc(3)Man(9)GlcNAc(2)-PP-Dol as the most predominating species with no accumulation of Man(5)GlcNAc(2)-PP-Dol in the mutants. Kinetic studies exhibited a reduced mannosylphosphodolichol synthase (DPMS) activity in mutant cells with a K-m for GDP-mannose 160 to 400% higher than that of the wild type. In addition, the k(cat) for DPMS was also reduced 2 to 4-fold in these mutant cells. Exogenously added Dol-P failed to rescue the k(cat) 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.
引用
收藏
页码:479 / 486
页数:8
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