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 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.
Banerjee
Dipak K
DK
Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, PR, 00936-5067, USA. dbanerjee@rcm.upr.edu
Aponte
Eira
E
Dasilva
Jaime J
JJ
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006031:Glycosylation; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000378:metabolism*; D009154:Mutation
D008070:Lipopolysaccharides; C023023:lipid-linked oligosaccharides; D003847:Deoxyglucose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases