Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain.
NCBE (SLC4A10) is a member of the SLC4 family of bicarbonate transporters, several of which play important roles in intracellular-pH regulation and transepithelial HCO(3)(-) transport. Here we characterize a new antibody that was generated in rabbit against a fusion protein consisting of maltose-binding protein and the first 135 amino acids (aa) of the N-terminus of human NCBE. Western blotting--both of purified peptides representing the initial approximately 120 aa of the transporters and of full-length transporters expressed in Xenopus oocytes--demonstrated that the antibody is specific for NCBE versus the two most closely related proteins, NDCBE (SLC4A8) and NBCn1 (SLC4A7). Western blotting of tissue in four regions of adult mouse brain indicates that NCBE is expressed most abundantly in cerebral cortex (CX), cerebellum (CB) and hippocampus (HC), and less so in subcortex (SCX). NCBE protein was present in CX, CB, and HC microdissected to avoid choroid plexus. Immunocytochemistry shows that NCBE is present at the basolateral membrane of embryonic day 18 (E18) fetal and adult choroid plexus. NCBE protein is present by Western blot and immunocytochemistry in cultured and freshly dissociated HC neurons but not astrocytes. By Western blot, nearly all NCBE in mouse and rat brain is highly N-glycosylated (approximately 150 kDa). PNGase F reduces the molecular weight (MW) of natural NCBE in mouse brain or human NCBE expressed in oocytes to approximately the predicted MW of the unglycosylated protein. In oocytes, mutating any one of the three consensus N-glycosylation sites reduces glycosylation of the other two, and the triple mutant exhibits negligible functional expression.
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. liming.chen@yale.edu
L-M
LM
Chen
M L
ML
Kelly
J D
JD
Rojas
M D
MD
Parker
H S
HS
Gill
B A
BA
Davis
W F
WF
Boron
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D015153:Blotting, Western; D001923:Brain Chemistry / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D027963:Chloride-Bicarbonate Antiporters / Q000737:chemistry / Q000378:metabolism*; D005822:Genetic Vectors; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000737:chemistry; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000378:metabolism*; D013045:Species Specificity; D014018:Tissue Distribution; D014982:Xenopus laevis
D000906:Antibodies; D027963:Chloride-Bicarbonate Antiporters; D011993:Recombinant Fusion Proteins; C526208:Slc4a10 protein, mouse; D027982:Sodium-Bicarbonate Symporters; D049452:Green Fluorescent Proteins; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase