Many tumor cells, especially those that reside in a hypoxic environment, rely on glycolysis to meet their increased demand for energy and biosynthetic precursors (Hanahan and Weinberg, 2011; Schulze and Harris, 2012; Parks et al., 2013). As a consequence, they produce considerable amounts of acid and lactate, which have to be removed from the cell so as to prevent intracellular lactacidosis and suffocation of their metabolism. Lactate efflux from cancer cells is mediated primarily by the monocarboxylate transporters MCT1 (SLC16A1) and MCT4 (SLC16A3), both of which transport lactate together with a proton across the cell membrane (Poole and Halestrap, 1993; Parks et al., 2013). This MCT-mediated H⁺ efflux can exacerbate extracellular acidification and thus support the formation of a hostile environment, which favors tumor growth and allows tumor cells to escape conventional cancer therapies (Kennedy and Dewhirst, 2010; Parks et al., 2011, 2013; Gillies et al., 2012). Expression of MCT1 is not altered with varying oxygen tension, whereas expression of MCT4 has been shown to be upregulated in different cancer cells under hypoxia (Ullah et al., 2006). However, expression of the two isoforms strongly varies among different tumor species and cell lines. In the MCF-7 breast cancer cell line, used in this study, lactate flux is exclusively mediated by MCT1 under both normoxic and hypoxic conditions (Jamali et al., 2015).

Another family of key proteins in tumor acid/base regulation is the carbonic anhydrases, which catalyze the reversible hydration of CO₂ to HCO₃^– + H⁺. The role of the cancer-specific isoform CAIX in tumor development and progression has been studied extensively, but physiological data on CAII in cancer cells are still relatively scarce. CAII has been found in different types of brain tumors, with the most malignant species exhibiting the strongest expression. In addition, this isoform has been linked to poor prognosis in patients suffering from those tumor types (Parkkila et al., 1995; Haapasalo et al., 2007). Furthermore, CAII has been suggested to mediate the malignant behavior of pulmonary neuroendocrine tumors (Zhou et al., 2015). Experiments on breast cancer cells demonstrated that CAII is upregulated in the highly tumorigenic MDA-MB-231 cell line when the cells are exposed to the chemotherapeutic drug doxorubicin (Mallory et al., 2005). On the other hand, a reduction in CAII expression was proposed to promote tumor cell motility and to contribute to tumor growth and metastasis in non-small cell lung cancer and gastric carcinoma (Chiang et al., 2002; Li X-J et al., 2012). CAII expression was also found to be associated with tumor differentiation and poor prognosis in patients with pancreatic cancer (Sheng et al., 2013), suggesting a differential function of CAII in different tumor types.

Catalysis by CA involves two distinct and separate stages: the interconversion of CO₂ and HCO₃^– followed by the transfer of an H⁺ to the bulk solution to regenerate the zinc-bound hydroxide, the latter being the rate-limiting step in the overall reaction (Tu et al., 1989; Lindskog, 1997). In CAII, the fastest of the α-CAs (Steiner et al., 1975; Boone et al., 2014), H⁺ transfer between the zinc-bound water and the solvent surrounding the enzyme is facilitated by the side chain of His64, which shuttles H⁺ between the bulk solvent and a network of well-ordered hydrogen-bonded water molecules in the enzyme’s active-site cavity (Fisher et al., 2007a). Furthermore, the H⁺ transfer efficiency of His64 is fine-tuned by several hydrophilic residues (Tyr7, Asn62 and Asn67), which contribute to the stabilization of the intervening water molecules within the active-site cavity and which influence the orientation and the pK_a value of His64 (Fisher et al., 2007b). These activities mean that the His64 residue is essential for CAII to reach full enzymatic activity. On the basis of work using an algorithm for mapping proton wires in proteins, Shinobu and Agmon (2009) proposed that the active site H⁺ wire exits to the protein surface, and leads to Glu69 and Asp72 residues that are located on an electronegative patch on the rim of the active site cavity. On the basis of that observation, these authors proposed that positively charged, protonated buffer molecules dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction.

Cytosolic CAII has been found to facilitate the transport function of various acid/base transporters including the Cl^–/HCO₃^– exchangers AE1 and AE2 (Vince and Reithmeier, 1998, 2000; Sterling et al., 2001), the Na⁺/HCO₃^– cotransporters NBCe1 and NBCn1 (Gross et al., 2002; Loiselle, 2004; Becker and Deitmer, 2007), and the Na⁺/H⁺ exchanger NHE1 (Li et al., 2002, 2006). Augmentation of acid/base transport by CAII requires both direct binding between transporter and enzyme and CAII catalytic activity, and the complex involved has been coined ‘transport metabolon’. The first evidence for a transport metabolon, formed between CAII and an acid/base transporter, was presented in 1993 by Kifor et al. (1993) for the Cl^–/HCO₃^– exchanger AE1. CAII could be immunoprecipitated with AE1 when antiserum against the N-terminal of AE1 was used, whereas serum directed against the C-terminal of the transporter failed to immunoprecipitate CAII, suggesting that CAII physically binds to the C-terminal tail of AE1 (Vince and Reithmeier, 1998). These findings were confirmed by affinity blotting and by a solid-phase binding assay with CAII and a glutathione S-transferase (GST) fusion protein of the AE1 C-terminal (Vince and Reithmeier, 1998). Single-site mutations identified the acidic cluster D⁸⁸⁷ADD in the C-terminal tail of AE1 as the binding site for CAII (Vince and Reithmeier, 2000; Vince et al., 2000). Binding of CAII to this cluster would tether the enzyme close to the transporter pore of AE1 near the inner cell surface. This location has been suggested to position CAII ideally to hydrate incoming CO₂ and to supply the AE1 transporter directly with a localized substrate pool (Vince and Reithmeier, 2000). Indeed, inhibition of CAII catalytic activity decreased the transport activity of AE1, which is heterologously expressed in HEK293 cells, by up to 60% (Sterling et al., 2001).

First evidence for a direct interaction between the Na⁺/HCO₃^- cotransporter NBCe1 and CAII was presented by Gross et al. (2002), based on isothermal titration calorimetry. In analogy to the CAII-binding cluster D⁸⁸⁷ADD found in AE1 (Vince and Reithmeier, 2000), the cluster D⁹⁸⁶NDD within the C-terminal of NBCe1 was suggested as the putative CAII binding site. Functional interaction between NBCe1 and CAII was further shown by heterologous protein expression in Xenopus oocytes (Becker and Deitmer, 2007). Both injection and co-expression of CAII increased NBCe1-mediated membrane current, membrane conductance and Na⁺ influx when CO₂ and HCO₃^– is applied in an ethoxzolamide-sensitive manner.

Evidence for an interaction between NHE1 and intracellular CAII was obtained by measuring the recovery from a CO₂-induced acid load in AP1 cells transfected with NHE1 (Li et al., 2002). Cotransfection of NHE1 with CAII almost doubled the rate of pH recovery as compared to that in cells expressing NHE1 alone, whereas cotransfection with the catalytically inactive mutant CAII-V143Y even decreased the rate of pH recovery, indicating a physical interaction between NHE1 and catalytically active CAII. Physical interaction between the two proteins was demonstrated by co-immunoprecipitation of heterologously expressed NHE1 and CAII (Li et al., 2002). A micro titer plate binding assay with a GST fusion protein of the NHE1 C-terminal tail revealed that CAII binds to the penultimate group of 13 amino acids of the C-terminal tail (R⁷⁹⁰IQRCLSDPGPHP), with the amino acids S⁷⁹⁶ and D⁷⁹⁷ playing an essential role in binding (Li et al., 2002, 2006).

While a considerable amount of data indicates a physical and functional interaction between various acid/base transporters and carbonic anhydrases, several studies have also questioned such transport metabolons. Lu et al. (2006) did not observe a CAII-mediated increase in membrane conductance in NBCe1-expressing Xenopus oocytes, even when fusing CAII to the C-terminal of NBCe1. In line with these findings, Yamada et al. (2011) found no increase in the membrane current during application of CO₂ and HCO₃^– when co-expressing wild-type NBCe1A or the mutant NBCe1-Δ65bp (lacking the putative CAII binding site D⁹⁸⁶NDD) with CAII. The concept of a physical interaction between HCO₃^– transporters and CAII has also been challenged by a binding study carried out by Piermarini et al. (2007). These authors were able to reproduce the findings of other groups by showing that sequences in the C-terminal tails of NBCe1, AE1 and NDCBE (SLC4A8) that are fused to GST can bind to immobilized CAII in a micro titer plate binding assay. However, when reversing the assay or using pure peptides, no increased binding of CAII to the immobilized GST fusion proteins could be detected (Piermarini et al., 2007). It was concluded that a bicarbonate transport metabolon may exist, but that CAII might not bind directly to the transporters. That CAII activity could improve substrate supply to bicarbonate transporters even without the requirement for a metabolon, or the involvement of direct physical interaction, was also pointed out in a study on AE1 transport activity by Al-Samir et al. (2013). By using Förster resonance energy transfer measurements and immunoprecipitation experiments with tagged proteins, the authors showed no binding or close co-localization of AE1 and CAII. Functional measurements in red blood cells and theoretical modeling suggested that the transport activity of AE1 can be best supported by CAII, when the enzyme is equally distributed within the cell’s cytosol (Al-Samir et al., 2013). For detailed reviews on transport metabolons see McMurtrie et al. (2004), Moraes and Reithmeier (2012), Deitmer and Becker (2013), Becker et al. (2014).

We have previously shown that transport activity of MCT1 and MCT4 is enhanced by CAII, when the two proteins are heterologously co-expressed in Xenopus oocytes (Becker et al., 2005, 2010, 2011; Becker and Deitmer, 2008). In contrast to the transport metabolons described before, enhancement of MCT1/4 transport function is independent of the enzyme’s catalytic activity. Both, inhibition of CA catalytic activity with 6-ethoxy-2-benzothiazolesulfonamide (EZA) and co-expression of MCT1/4 with the catalytically inactive mutant CAII-V143Y failed to suppress the CAII-induced facilitation of MCT1/4 transport activity (Becker et al., 2005; Becker and Deitmer, 2008). The first evidence that this non-catalytic interaction between MCT1/4 and CAII requires direct binding between the two proteins was provided by injection of CAII that was bound to an antibody prior to the injection. In this experiment, CAII was not able to enhance the transport activity of MCT1 in Xenopus oocytes, suggesting a sterical suppression of the interaction by the antibody (Becker and Deitmer, 2008). In the same study, truncation of the MCT1 C-terminal tail led to loss of the interaction between MCT1 and CAII in Xenopus oocytes (Becker and Deitmer, 2008). By introduction of single site mutations in MCT1 and MCT4, the glutamic acidic clusters E⁴⁸⁹EE and E⁴³¹EE within the C-terminal tail of MCT1 and MCT4, respectively, could be identified as binding sites for CAII (Stridh et al., 2012; Noor et al., 2015). In both studies, binding was confirmed both on the functional level by measuring MCT transport activity in Xenopus oocytes and by pull-down assays using GST-fusion proteins. Direct interaction between MCT1 and CAII was shown not only in vitro and by heterologous protein expression in Xenopus oocytes, but also in an in situ proximity ligation assay in mouse astrocytes (Stridh et al., 2012). As CA catalytic activity is not required to facilitate MCT transport function, it was hypothesized that CAII might utilize parts of its intramolecular proton pathway to function as a proton antenna for the transporter (Almquist et al., 2010; Becker et al., 2011). Protonatable residues with overlapping Coulomb cages could form proton-attractive domains and could share a proton at a very fast rate, exceeding the upper limit of diffusion-controlled reactions (Ädelroth and Brzezinski, 2004; Friedman et al., 2005). When these residues are located in proteins or lipid head groups at the plasma membrane, they can collect protons from the solution and direct them to the entrance of a proton-transfer pathway of a membrane-anchored protein, a phenomenon termed a ‘proton-collecting antenna’ (Ädelroth and Brzezinski, 2004; Brändén et al., 2006). The need for such a proton antenna is based on the observation that H⁺ co-transporters, such as MCTs, extract H⁺ from the surrounding area at rates well above the capacity of simple diffusion to replenish their immediate vicinity. Therefore, the transporter must exchange H⁺ with protonatable sites at the plasma membrane, which could function as proton collectors for the transporter (Martínez et al., 2010). The finding that the mutant CAII-H64A, which is missing the central residue of the enzyme’s intramolecular proton shuttle, fails to facilitate MCT transport activity, led to the notion that CAII may function as a proton antenna for the MCT. In doing so, it would provide protons to or or subtract protons from the transporter via its intramolecular H⁺ shuttle (Becker et al., 2011). In line with this, we showed recently that integration of a proton antenna, in the form of six histidine residues, into the C-terminal tail of MCT4 could facilitate MCT4 transport activity even in the absence of carbonic anhydrase (Noor et al., 2017).

The transport activity of MCTs was shown to be facilitated not only by intracellular CAII but also by the extracellular CA isoforms CAIV and CAIX (Klier et al., 2011, 2014; Jamali et al., 2015). Hypoxia-regulated CAIX is considered to be a key protein in tumor acid/base regulation. CAIX, the expression of which is usually linked to poor prognosis, is tethered to the membrane by a transmembrane domain, with its catalytic center facing the extracellular site. Like other fast carbonic anhydrases, CAIX is equipped with an intramolecular proton shuttle for rapid exchange of H⁺ between the catalytic center and the surrounding bulk solution. We were able to demonstrate that knockdown of CAIX with short interfering RNA (siRNA) reduced MCT1-mediated lactate transport in hypoxic MCF-7 cells, whereas inhibition of CA catalytic activity with EZA had no effect on MCT1 transport activity (Jamali et al., 2015). Co-expression of MCT1/4 with CAIX in Xenopus oocytes revealed that CAIX-mediated facilitation of MCT transport activity requires the enzyme's intramolecular proton shuttle, His200 (Jamali et al., 2015). Furthermore, knockdown of CAIX, but not inhibition of CA catalytic activity, reduced the proliferation of hypoxic MCF-7 cells, indicating that the CAIX-driven increase in lactate efflux is crucial for the proper functioning of cancer cells (Jamali et al., 2015).

In the present study, we investigated whether CAII can facilitate proton-driven lactate transport in MCF-7 breast cancer cells. Knockdown of CAII, which is closely co-localized with MCT1 in MCF-7 cells, resulted in a significant reduction of MCT transport activity in both normoxic and hypoxic cells. We then further analyzed the molecular mechanism underlying the CAII-mediated facilitation of MCT transport activity. Our results showed that CAII features a moiety (Glu69 + Asp72) that exclusively mediates proton exchange with the transporter, while the central residue of its intramolecular proton shuttle (His64) mediates the binding of CAII to MCT1 and MCT4.

Our previous studies have shown that CAII facilitates the transport activities of MCT1 and MCT4 independent of the enzymes' catalytic activity, as both inhibition of CA activity with EZA and molecular disruption of the catalytic center (CAII-V143Y) did not reduce the CAII-induced increase in MCT transport activity (Becker et al., 2005; Becker and Deitmer, 2008). As CA catalytic activity is not required to facilitate MCT transport function, we hypothesized that CAII might utilize parts of its intramolecular proton pathway to function as a proton antenna for the transporter (Almquist et al., 2010; Becker et al., 2011). The proton pathway of CAII consists of a water wire, coordinated by Asn62 and Asn67, which extends from the active site to the histidine residue at position 64 (Fisher et al., 2007a, 2007b). From there, the proton is passed on between the imidazole ring in His64 and exogenous buffer molecules surrounding the enzyme (Fisher et al., 2007a, 2007b). In addition, Shinobu and Agmon (2009) presented a model in which the active site H⁺ wire exits to the protein surface and leads to Glu69 and Asp72, which are located in an electronegative patch on the rim of the active-site cavity. On the basis of this model, the authors proposed that positively charged, protonated buffer molecules could dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction. However, this assumption is based purely on mathematical modelling and has, to our knowledge, not yet been evaluated experimentally. In the present study, we were able to show that both Glu69 and Asp72 are essential for CAII-mediated facilitation of MCT1/4 transport activity, in both the influx and the efflux direction. Mutation of Glu69 and Asp72 did not, however, alter CAII catalytic activity, as demonstrated in vitro by gas analysis mass spectrometry and in vivo by determining the rate of change in intracellular H⁺ concentration in oocytes during the application and removal of CO₂. These results suggest that this proton-collecting moiety is not involved in the facilitation of CAII catalytic activity, but rather mediates a second, independent function, which is the rapid supply and removal of protons from the pore of the adjacent transporter, rendering CAII a bona fide proton antenna. Whether this kind of interaction only exists between CAII and MCTs, or whether CAII could also function as proton antenna for other proton-coupled membrane transporters, such as proton-coupled amino acid transporters or Na⁺/H⁺ exchangers, remains to be shown.

Our previous studies had shown that CAII-mediated facilitation of MCT transport activity requires CAII-His64, which mediates the exchange of protons between the catalytic center and the surrounding bulk solution during the CAII catalytic cycle. Our structural models suggested, however, that His64 mediates binding of CAII to Glu489/Glu491 in MCT1 (Stridh et al., 2012) and to Glu431/Glu433 in MCT4 (Noor et al., 2015). To investigate whether His64 mediates binding or proton shuttling between MCT and CAII, or both, we exchanged the histidine at position 64 to either alanine or lysine. Mutation of His to Ala should disable both intramolecular H⁺ shuttling in CAII and binding of the enzyme to the glutamate residues in MCT1/4. Mutation of His to Lys also disables H⁺ shuttling, as lysine has a considerably higher pK_a than the imidazole ring in histidine, which does not allow fast protonation/deprotonation reactions at physiological pH. However, lysine should still be able to form hydrogen bonds with the glutamate residues in the C-terminal tail of MCT1 and MCT4, and should therefore still enable CAII to bind to the transporter. Although MCT transport activity was not enhanced by CAII-H64A, CAII-H64K facilitated MCT transport activity to the same extent as CAII-WT. These results allowed us to conclude that His64 may not be involved in proton transfer between MCT and CAII, but instead mediates binding of the enzyme to the transporter's C-terminal tail. However, as CAII-H64K still displayed a higher catalytic activity than CAII-H64A, we cannot rule out the possibility that CAII-H64K retains some proton shuttling activity. Indeed, it has been shown that lysine residues that are buried in the protein interior could display considerably lower pK_a values than in those that are exposed to water (Isom et al., 2011). Therefore, it might be possible that CAII-H64K can retain enough shuttling activity to facilitate MCT-mediated lactate transport.

Taken together, these results suggest that CAII facilitates MCT transport activity by a mechanism that is completely different from the catalytic activity. Catalytic activity of CAII is facilitated by proton shuttling via His64, whereas Glu69 and Asp72 are ineffective in supporting CA activity. When interacting with MCT1/4, His64 does function as binding site, whereas proton transfer between transporter and enzyme seems to be mediated by Glu69 and Asp72 (Figure 10).

Figure 10

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The physiological need for CAII's role as a proton antenna for MCTs might derive from the low apparent diffusion rate of protons in a strongly buffered solutionsuch as the cytosol. By applying a mathematical model of Brownian diffusion, Martínez et al. (2010) determined the maximum supply capacity of substrates to various transporters and enzymes. For MCT1, they calculated that the diffusion rate of lactate, which is present in the cell at concentrations within the millimolar range, exceeds the turnover rate of the transporter by several magnitudes. This supports the notion that, for this molecule, the cytosol is a well-mixed compartment. For protons, however, Martínez et al. (2010) calculated that the maximum supply rate is approximately 15 times lower than the apparent turnover rate of MCT1, as calculated by Ovens et al. (2010a). In other words, the model demonstrates that MCT1 extracts H⁺ from the cytosol at rates well above the capacity for simple diffusion to replenish its immediate vicinity. This paradoxical result implies that the transporter does not extract H⁺ substrates directly from the bulk cytosol, but from an intermediate ‘harvesting’ compartment located between the aqueous phase and the transport site (Martínez et al., 2010). The findings of the present study suggest that CAII could serve as such a harvester, which captures protons from protonatable residues at the plasma membrane and funnels them to the transporter pore (Figure 10). If this is the case, then CAII would counteract the local depletion of protons in the direct vicinity of the transporter and thus would support transport activity. As the direction of proton movement depends on the ion gradient, this principle can be applied both in the influx and in the efflux direction. In the case of proton/lactate efflux, CAII would harvest H⁺ from protonatable residues near the plasma membrane and shuttle them to the transporter. In the case of proton/lactate influx, CAII would capture the protons from the transporter pore and distribute them to protonatable residues near the plasma membrane, following the electrochemical H⁺ gradient. Indeed, we have previously been able to show that protons that are focally applied to MCT1-expressing oocytes moved faster along the inner face of the plasma membrane when CAII was injected, suggesting that CAII could allocate H⁺ along the plasma membrane and thereby increase the area in which H⁺ can be released from the membrane into the bulk solution (Becker and Deitmer, 2008). In this case, CAII would counteract the accumulation of protons at the transporter pore, which would result in a reduction in transport activity in the influx direction.

Even though the mathematical model from Martínez et al. (2010) suggests a 15-fold lower supply rate of H⁺, our measurements in Xenopus oocytes have shown that CAII enhances the transport activity of MCT1 and MCT4 by ‘only’ around two fold. This implies that, in the absence of CAII, MCT transport activity is either facilitated by other proton-collecting molecules or — more likely — that MCTs are already able to extract protons from adjacent protonatable sites, even though not as efficiently as when cooperating with CAII.

The relevance of this proton antenna for proper cell function becomes evident from our physiological experiments on MCF-7 breast cancer cells. First, evidence for a functional interaction between MCT and CAII comes from the finding that knockdown of CAII reduces lactate transport in MCF-7 cells. We can rule out the possibility that this decrease in transport activity is due to a loss of CAII catalytic activity as, in a recent study, we were able to show that inhibition of CA activity by application of EZA did not alter lactate flux in MCF-7 cells (Jamali et al., 2015). Therefore, it can be assumed that CAII facilitates lactate flux in cancer cells by a non-catalytic, direct interaction. Indeed, CAII is in close co-localization with MCT1 in MCF-7 cells, as shown by an in situ proximity ligation assay. Thus, it appears likely that CAII can form a protein complex with MCT1 in these cells to facilitate lactate flux by functioning as a proton antenna for the transporter. In line with these findings, we previously showed that CAII also facilitates lactate transport in mouse cerebellar and white matter astrocytes, independent of the enzyme’s catalytic activity (Stridh et al., 2012). As in the cancer cells, MCT1 and CAII are in close proximity in astrocytes, as shown by in situ PLA. Interestingly, colocalization could only be detected when an antibody directed against the intracellular loop between TM 6 and 7 of MCT1 was used. When the PLA was carried out using an antibody against the C-terminal of MCT1, which carries the CAII binding site, no proximity between MCT1 and CAII could be detected (Stridh et al., 2012). Astrocytes are highly glycolytic cells, which are widely believed to export lactate as an energy fuel for adjacent neurons, a phenomenon coined an ‘Astrocyte-to-Neuron Lactate Shuttle’ (Pellerin and Magistretti, 1994; Magistretti, 2006; Barros and Deitmer, 2010). These findings infer that CA-mediated facilitation of MCT1/4 transport activity might be a more general, or at least common, phenomenon in glycolytic cells that have to extrude large amounts of lactate.

In the present study, knockdown of CAII resulted in a more pronounced reduction in MCT1 transport activity in hypoxic than in normoxic cancer cells, but even so, the expression of CAII was not increased under hypoxia. This effect might be attributed to extracellular CAIX, which functions as a proton antenna for MCT1 in MCF-7 cells under hypoxic conditions (Jamali et al., 2015). CAIX operates only on the extracellular side of the membrane, which might lead to the formation of a proton micro-domain on the cytosolic side of the membrane in the absence of intracellular CAII because the supply or removal of H⁺ is enhanced on the extracellular side, leading to an increase in MCT transport activity. This would result in increased accumulation or depletion of H⁺ at the cytosolic side, depending on transport direction, which would impair MCT transport rate. With a proton antenna on both sides of the membrane, formation of H⁺ microdomains would be suppressed both at the cis- and at the trans-side. In such a scenario, extracellular CAIX and intracellular CAII would cooperate by a ‘push and pull principle’, providing protons to the transporter on one side and removing them on the other side of the cell membrane (Figure 10). Through this mechanism, intracellular CAII and extracellular CAIX could cooperate to enhance lactate transport in cancer cells under hypoxic conditions. As CAIX is already expressed in normoxic MCF-7 cells at a low level (Jamali et al., 2015), this push and pull principle could also occur in normoxic cells, though to a lower extent. The lower, yet robust, expression of extracellular CAIX under normoxia could also explain the reduced effect of CAII knockdown on lactate transport in normoxic MCF-7 cells. In line with this notion, we previously found in Xenopus oocytes that intracellular CAII can also work in concert with another extracellular carbonic anhydrase, CAIV, to ensure rapid shuttling of protons and lactate across the cell membrane (Klier et al., 2014).

In CAII, H⁺ shuttling between enzyme and transporter is mediated by Glu69 and Asp72. CAIX seems to lack an analogue moiety within its catalytic domain. However, it features a 59-amino-acid long proteoglycan-like (PG) domain that is unique to CAIX among the CA family (Pastorek and Pastorekova, 2015). The PG domain of human CAIX contains 18 glutamate and eight aspartate residues that have been suggested to function as an intramolecular proton buffer, which could support CAIX catalytic activity when operating in an acidic environment (Innocenti et al., 2009). We were recently able to show that both the truncation of the CAIX PG domain and the application of a PG-binding antibody (M75) decreased CAIX-induced facilitation in Xenopus oocytes and breast cancer cells (Ames et al., 2018). Those findings led us to the conclusion that the CAIX PG domain could function as an extracellular proton antenna for monocarboxylate transporters within the acidic environment of a solid tumor.

The importance of CAII for cell viability was demonstrated by a cell proliferation assay. Knockdown of CAII reduced the proliferation of MCF-7 cells to the same extent as did full inhibition of lactate transport with AR-C155858, whereas inhibition of CA catalytic activity with EZA had no effect on proliferation. These results correspond to previous findings that knockdown of CAIX or interference with the CAIX PG domain by an antibody, but not inhibition of CA catalytic activity, decreases cell proliferation in hypoxic MCF-7 and MDA-MB-231 cancer cells (Jamali et al., 2015; Ames et al., 2018). Apparently, removal of the proton antenna on one side of the membrane is sufficient to reduce MCT-mediated proton/lactate efflux from the cell. This in turn could lead to intracellular lactacidosis and impairment of metabolism, which will ultimately result in a decrease in cell proliferation.

Knockdown of CAII decreased, but did not abolish, lactate transport in MCF-7 cells. However, knockdown of CAII decreased cell proliferation to the same extend as did inhibition of MCT1 transport activity with AR-C155858. This striking effect of CAII knockdown on normoxic and hypoxic cells suggests that the CAII protein might have another role in cell proliferation, which is beyond its function as a facilitator of lactate efflux. As cell proliferation is not decreased by the inhibition of CA activity with EZA, the supporting effect of CAII seems to be independent of the enzyme’s catalytic activity. Zhou et al. (2015) showed that knockdown of CAII in NCI-H727 and A549 cancer cell lines resulted in significant reduction in clonogenicity in vitro, and in marked suppression of tumor growth in vivo. By using an apoptosis gene array, Zhou et al. (2015) found a novel association of CAII-mediated apoptosis with specific mitochondrial apoptosis–associated proteins. These findings suggest that the CAII protein has multiple functions within the cell, including cellular acid/base regulation, facilitation of lactate transport, and regulation of apoptosis.

In summary, our results show that CAII supports lactate flux in cancer cells, presumably by functioning as a proton antenna for MCTs. Therefore, CAII features a molecular moiety that exclusively mediates proton exchange with the transporter, while parts of its intramolecular proton shuttle, which is pivotal for catalytic activity, mediates binding to the MCT. This enhancement of proton movement not only drives basic lactate flux but is also a prerequisite for further facilitation of lactate transport by CAIX under hypoxic conditions, as increased glycolytic activity requires even higher lactate transport capacity in cancer cells.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-9.

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Author details

1. Sina Ibne Noor

   Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   Conceptualization, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Somayeh Jamali

   Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

3. Samantha Ames

   Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Silke Langer

   Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Joachim W Deitmer

   Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing—review and editing

   Competing interests

   No competing interests declared

6. Holger M Becker

   1. Division of General Zoology, Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany
   2. Institute of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany

   Contribution

   Conceptualization, Supervision, Funding acquisition, Visualization, Investigation, Writing—original draft, Writing—review and editing

   For correspondence

   Holger.Becker@tiho-hannover.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2700-6117

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