Role of glycosylation in the renal electrogenic Na+-HCO3- cotransporter (NBCe1).
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.
Choi
Inyeong
I
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. ichoi@physio.emory.edu
Hu
Lihui
L
Rojas
José D
JD
Schmitt
Bernhard M
BM
Boron
Walter F
WF
D000581:Amidohydrolases / Q000737:chemistry; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism*; D037102:Lectins; D008839:Microelectrodes; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics / Q000502:physiology; D009844:Oligosaccharides / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D013045:Species Specificity; D014982:Xenopus laevis
D037102:Lectins; D009844:Oligosaccharides; C422004:Slc4a4 protein, rat; D027982:Sodium-Bicarbonate Symporters; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase