Title : Role of glycosylation in the renal electrogenic Na+-HCO3- cotransporter (
NBCe1 )
Abstract :
- The electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1 is important for the regulation of intracellular pH (pH(i)) and for epithelial HCO(3)(-) transport in many tissues, including kidney, pancreas, and brain
- In the present study, we investigate glycosylation sites in NBCe1
- Treatment of rat kidney membrane extracts with peptide N-glycosidase F ( PNGase F) shifted the apparent molecular weight (MW) of NBCe1 from 130 to 116, the MW predicted from the deduced amino acid sequence
- Treatment with endoglycosidase F(2) or H or O-glycosidase did not affect the MW of NBCe1
- Lectin-binding studies, together with the enzyme data, suggest that the N-linked carbohydrates are of tri- or tetra-antennary type
- To localize glycosylation sites , we individually mutated the seven consensus N-glycosylation sites by replacing asparagine (N) with glutamine (Q) and assessing mutant transporters in Xenopus laevis oocytes
- Immunoblotting of oocyte membrane extracts treated with PNGase F indicates that NBCe1 is normally glycosylated at N597 and N617 (both on the third extracellular loop)
- However, N592 (on the same loop) is glycosylated when the other two sites are mutated
- The triple mutant (N592Q/N597Q/N617Q) is completely unglycosylated but, based on microelectrode measurements of membrane potential and pH(i) in oocytes, preserves the Na(+) and HCO(3)(-) dependence and electrogenicity of wild-type NBCe1