Abstract
Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues. The transport activity of MCT1, MCT2, and MCT4 can be facilitated by the extracellular carbonic anhydrase IV (CAIV) via a noncatalytic mechanism. Combining physiological measurements in HEK-293 cells and Xenopus oocytes with pulldown experiments, we analyzed the direct interaction between CAIV and the two MCT chaperones basigin (CD147) and embigin (GP70). Our results show that facilitation of MCT transport activity requires direct binding of CAIV to the transporters chaperones. We found that this binding is mediated by the highly conserved His-88 residue in CAIV, which is also the central residue of the enzyme's intramolecular proton shuttle, and a charged amino acid residue in the Ig1 domain of the chaperone. Although the position of the CAIV-binding site in the chaperone was conserved, the amino acid residue itself varied among different species. In human CD147, binding of CAIV was mediated by the negatively charged Glu-73 and in rat CD147 by the positively charged Lys-73. In rat GP70, we identified the positively charged Arg-130 as the binding site. Further analysis of the CAIV-binding site revealed that the His-88 in CAIV can either act as H donor or H acceptor for the hydrogen bond, depending on the charge of the binding residue in the chaperone. Our results suggest that the CAIV-mediated increase in MCT transport activity requires direct binding between CAIV-His-88 and a charged amino acid in the extracellular domain of the transporter's chaperone.
Highlights
Monocarboxylate transporters (MCTs) mediate the proton-coupled exchange of high-energy metabolites, including lactate and pyruvate, between cells and tissues
We have previously shown that carbonic anhydrase IV (CAIV) facilitates transport activity of MCT1, MCT2, and MCT4 by a mechanism that is independent from the enzyme’s catalytic activity, presumably by functioning as a proton antenna for the transporter (49 –51)
CAIV was only able to facilitate transport activity of MCT2 when the transporter was co-expressed with its chaperone GP70 [49]
Summary
Facilitation of MCT transport activity by CAIV requires the Glu-73 in the Ig1 domain of hCD147. MCT transport activity was determined by measuring the rate of change in intracellular Hϩ concentration (⌬[Hϩ]/⌬t) during application of 3 and 10 mM lactate in HEK-293 cells, transfected with hCD147 alone or together with CAIV (Fig. 1B). Coexpression of hCD147–WT with CAIV resulted in a significant increase in ⌬[Hϩ]/⌬t, indicating CAIV-mediated facilitation of MCT transport activity in HEK-293 cells (Fig. 1C). E31Q/E73Q were coexpressed with CAIV (Fig. 1, B and C) These results indicate that CAIV-mediated facilitation of MCT transport activity requires Glu-73 in the Ig1 domain of hCD147. To test whether Glu-73 in hCD147 functions as the binding site for CAIV, we performed pulldown experiments with CAIV and GST fusion proteins of the Ig1 domain of hCD147–WT, hCD147–E31Q, and hCD147–E73Q, respectively (Fig. 3A).
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