Carbonic Anhydrase II Binds to the Carboxyl Terminus of Human Band 3, the Erythrocyte Cl−/HCO3−Exchanger

Abstract

In this study, we provide evidence that the 33-residue carboxyl-terminal (Ct) region of the human erythrocyte chloride/bicarbonate exchanger, band 3, binds carbonic anhydrase II (CAII). Immunofluorescence showed that tomato lectin-mediated clustering of band 3 in ghost membranes caused a similar clustering of CAII, indicating an in situ association. CAII cosolubilized and coimmunoprecipitated with band 3, suggesting that the two proteins form a complex. Band 3 (K 1/2 ‫؍‬ 70 nM) or the membrane domain of band 3 (K 1/2 ‫؍‬ 100 nM) bound saturably to immobilized CAII in a solid phase binding assay. The interaction with CAII was specifically blocked by an antibody to the Ct of band 3. Affinity blotting showed that a glutathione S-transferase (GST)-fusion protein (GST-Ct) containing the last 33 residues of human band 3 bound to CAII. The solid phase binding assay showed that binding of GST-Ct to immobilized CAII was saturable (K 1/2 ‫؍‬ 20 nM). The binding rate was slow (t 1/2 ‫؍‬ 12 h) at physiological ionic strength and pH but was enhanced at low ionic strength or acidic pH. Intact band 3 (K i ‫؍‬ 15 nM), the membrane domain of band 3 (K i ‫؍‬ 100 nM), or antibodies to the Ct of band 3 were able to block GST-Ct binding to CAII, confirming the specificity of the interaction. Affinity chromatography showed that CAII bound to immobilized GST-Ct with a 1:1 stoichiometry. This work indicates that CAII, the bicarbonate supplier, is directly coupled to band 3, the chloride/bicarbonate exchanger in red blood cells.

A Transport Metabolon

“…Extracellular lectin caused agglutination of AE1 and a similar redistribution of CAII on the cytosolic surface of the erythrocyte membrane (30), suggesting a physical interaction of AE1 with CAII. CAII can be co-immunoprecipitated with solubilized AE1, and finally, a sensitive microtiter assay showed that CAII but not CAI interacts with the carboxyl terminus of AE1 (30). Truncation and point mutation of the AE1 carboxyl terminus led to the identification of the binding site of CAII in human AE1 as LDADD (amino acids 886 -890) (32).…”

“…Binding assays also showed that CAII interacts with the carboxyl-terminal region of AE2 (32), but interaction between AE3 and CAII has not yet been examined. The interaction between AE1 and CAII is pH-dependent (30), which suggested binding of the acidic LDADD motif of AE1 with a basic region of CAII. Truncation and mutagenesis of the basic amino-terminal region of CAII showed that it forms the AE1 binding site (33).…”

“…Binding of erythrocyte membranes to CAII has been show to increase its enzymatic activity (28). This interaction is weak, however, because the bulk of carbonic anhydrase can be readily removed from isolated erythrocyte membranes (29,30). Reaction of an anion transport inhibitor (DIDS) with AE1 altered the binding of a fluorescent inhibitor to carbonic anhydrase, suggesting a physical link between these two proteins (31).…”

“…Reaction of an anion transport inhibitor (DIDS) with AE1 altered the binding of a fluorescent inhibitor to carbonic anhydrase, suggesting a physical link between these two proteins (31). Extracellular lectin caused agglutination of AE1 and a similar redistribution of CAII on the cytosolic surface of the erythrocyte membrane (30), suggesting a physical interaction of AE1 with CAII. CAII can be co-immunoprecipitated with solubilized AE1, and finally, a sensitive microtiter assay showed that CAII but not CAI interacts with the carboxyl terminus of AE1 (30).…”

Genetic ablation of carbonic anhydrase IX disrupts gastric barrier function via claudin‐18 downregulation and acid backflux

“…The gastric surface cells are not only the site of the strongest CAIX expression within the gastric mucosa, but also of the basolateral acid extruders NHE1 and NBC1 2, 37, 38. It has been shown that carbonic anhydrases form ‘bicarbonate metabolons’ with acid/base transporters, by binding close to the ion transporter's intra‐ or extracellular binding site for ${\text{HCO}}_3^{-}$ and augmenting the availability of this ion for transport 9, 39. The organization of the gastric microvasculature is such that it brings bicarbonate enriched blood extruded by parietal cells during acid secretion – the so‐called alkaline tide – directly to the surface cells 40.…”