Evidence against a direct interaction between intracellular carbonic anhydrase II and pure C-terminal domains of SLC4 bicarbonate transporters.
Based on solid-phase binding assays with enzyme-linked immunosorbent assay detection, previous investigators suggested that intracellular carbonic anhydrase II (CA II) interacts at high affinity with the C-terminal (Ct) domains of SLC4 bicarbonate-transport proteins, expressed as glutathione S-transferase (GST) fusion proteins, to form functional HCO3- metabolons. Here we re-evaluated this protein-protein interaction using two solid-phase binding assays. We first compared the ability of the Ct domain of three SLC4 transporters, SLC4-A1 (AE1), SLC4-A4 (NBCe1), and SLC4-A8 (NDCBE), to bind immobilized CA II, using enzyme-linked immunosorbent assay detection. We found that when expressed as GST fusion proteins, all three bind to CA II (Kd 300-600 nM) better than does pure GST. However, we detected no binding of pure SLC4-Ct peptides to immobilized CA II. Second, we reversed assay orientation by immobilizing the SLC4-Ct fusion proteins or peptides. We found that more CA II binds to GST than to any of the three GST-SLC4-Ct fusion proteins. Furthermore, we detected no binding of CA II to any of the immobilized pure SLC4-Ct peptides. Finally, we used surface plasmon resonance to detect possible rapid interactions between CA II and the pure peptides. Although we detected acetazolamide binding to immobilized CA II and specific antibodies binding to immobilized SLC4-Ct peptides, we detected no binding of CA II to immobilized SLC4-Ct or vice versa. Thus, although an HCO3 metabolon may exist, CA II cannot bind directly to pure SLC4-Ct peptides and can bind to GST-SLC4-Ct fusion proteins only when the CA II is immobilized and the fusion protein is soluble, and not vice versa.
Piermarini
Peter M
PM
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA. pmp26@cornell.edu
Kim
Eugene Y
EY
Boron
Walter F
WF
D000595:Amino Acid Sequence; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052616:Solid Phase Extraction
D001457:Anion Exchange Protein 1, Erythrocyte; D011993:Recombinant Fusion Proteins; C498898:SLC4A4 protein, human; C498903:SLC4A8 protein, human; D027982:Sodium-Bicarbonate Symporters; D024402:Carbonic Anhydrase II