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A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
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Sina Ibne Noor1, Somayeh Jamali1, Samantha Ames1, Silke Langer1, Joachim W. Deitmer1
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& Holger M. Becker1,2
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1
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Kaiserslautern, Germany; 2Institute of Physiological Chemistry, University of Veterinary Medicine
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Hannover, D-30559 Hannover, Germany
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For correspondence:
[email protected]
Division of General Zoology, Department of Biology, University of Kaiserslautern, D-67653
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Abstract
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Many tumor cells produce vast amounts of lactate and acid, which have to be removed from the cell
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to avoid intracellular lactacidosis and suffocation of metabolism. In the present study, we show that
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proton-driven lactate flux is enhanced by the intracellular carbonic anhydrase CAII, which is
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colocalized with the monocarboxylate transporter MCT1 in MCF-7 breast cancer cells. Co-expression
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of MCTs with various CAII mutants in Xenopus oocytes demonstrated that CAII facilitates MCT
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transport activity via CAII-Glu69 and CAII-Asp72, which could function as surface proton antennae for
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the enzyme. While CAII-Glu69 and CAII-Asp72 seem to mediate proton transfer between enzyme and
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transporter, CAII-His
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involved in proton shuttling between the two proteins, but mediates binding between MCT and CAII.
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Taken together, the results suggest that CAII features a moiety that exclusively mediates proton
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exchange with the MCT to facilitate transport activity.
, the e tral residue of the e z
e s i tra ole ular proto shuttle, is not
1
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Introduction
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Many tumor cells, especially those which reside in a hypoxic environment, rely on glycolysis to meet
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their increased demand for energy and biosynthetic precursors (Hanahan & Weinberg, 2011; Schulze
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& Harris, 2012; Parks et al., 2013). Thereby they produce considerable amounts of acid and lactate,
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which have to be removed from the cell to avoid intracellular lactacidosis and suffocation of their
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metabolism. Lactate efflux from cancer cells is mediated primarily by the monocarboxylate
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transporters MCT1 (SLC16A1) and MCT4 (SLC16A3), both of which transport lactate together with a
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proton across the cell membrane (Poole & Halestrap, 1993; Parks et al., 2013). This MCT-mediated H+
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efflux can exacerbate extracellular acidification and support the formation of a hostile environment,
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which favors tumor growth and allows tumor cells to escape conventional cancer therapies (Kennedy
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& Dewhirst, 2010; Parks et al., 2011, 2013; Gillies et al., 2012). While expression of MCT1 is not
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altered with varying oxygen tension, expression of MCT4 has been shown to be upregulated in
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different cancer cells under hypoxia (Ullah et al., 2006). However, expression of the two isoforms
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strongly varies among different tumor species and cell lines. In the MCF-7 breast cancer cell line,
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used in this study, lactate flux is exclusively mediated by MCT1 under both normoxic and hypoxic
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conditions (Jamali et al., 2015).
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Another family of key proteins in tumor acid/base regulation is carbonic anhydrases, which catalyze
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the reversible hydration of CO2 to HCO3- + H+. While the role of the cancer-specific isoform CAIX in
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tumor development and progression has been extensively studied, physiological data on CAII in
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cancer cells are still relatively scarce. CAII was found in different types of brain tumors, with the most
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malignant species exhibiting the strongest expression, and was linked to poor prognosis in patients
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suffering from those tumor types (Parkkila et al., 1995; Haapasalo et al., 2007). Furthermore, CAII
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was suggested to mediate malignant behavior of pulmonary neuroendocrine tumors (Zhou et al.,
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2015). Experiments on breast cancer cells demonstrated that CAII is upregulated in the highly
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tumorigenic MDA-MB-231 cell line, when the cells are exposed to the chemotherapeutic drug
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doxorubicin (Mallory et al., 2005). On the other hand, a reduction in CAII expression was proposed to 2
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promote tumor cell motility and contribute to tumor growth and metastasis in non-small cell lung
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cancer and gastric carcinoma (Chiang et al., 2002; Li et al., 2012) and was found to be associated with
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tumor differentiation and poor prognosis in patients with pancreatic cancer (Sheng et al., 2013),
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suggesting a differential function of CAII in different tumor types.
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Catalysis in CA occurs in two distinct and separate stages: the interconversion of CO2 and HCO3-
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followed by the transfer of an H+ to the bulk solution to regenerate the zinc-bound hydroxide, the
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latter of which being the rate-limiting step in the overall reaction (Tu et al., 1989; Lindskok, 1997). In
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CAII, the fastest of the α-CAs (Steiner et al., 1975; Boone et al., 2014), H+ transfer between the zinc-
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bound water and the solvent surrounding the enzyme is facilitated by the side chain of His64, which
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shuttles H+ between the bulk solvent and a network of well-ordered hydrogen-bonded water
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ole ules i the e z
e s a ti e-site cavity (Fisher et al., 2007a). Furthermore, H+ transfer efficiency
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of His64 is fine-tuned by several hydrophilic residues (Tyr7, Asn62 and Asn67), which contribute to
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the stabilization of the intervening water molecules within the active-site cavity and influence
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orientation and the pKa value of His64 (Fisher et al., 2007b). This makes the residue His64 essential
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for CAII to reach full enzymatic activity. By using an algorithm for mapping proton wires in proteins,
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Shinobu & Agmon (2009) proposed that the active site H+ wire exits to the protein surface, and leads
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to Glu69 and Asp72, located on an electronegative patch on the rim of the active site cavity. Based
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on that observation, the authors proposed that positively charged, protonated buffer molecules dock
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in that area, from which a proton is delivered to the active site when the enzyme works in the
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dehydration direction.
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Cytosolic CAII has been found to facilitate transport function of various acid/base transporters
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including the Cl-/HCO3- exchangers AE1 and AE2 (Vince & Reithmeier, 1998, 2000; Sterling et al.,
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2001), the Na+/HCO3- cotransporters NBCe1 and NBCn1 (Gross et al., 2002; Loiselle et al., 2004;
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Becker et al., 2007), and the Na+/H+ exchanger NHE1 (Li et al., 2002, 2006). Augmentation of
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acid/base transport by CAII requires both direct binding between transporter and enzyme as well as
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CAII catalytic activity and has been coined tra sport
eta olo . First evidence for a transport 3
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metabolon, formed between CAII and an acid/base transporter, has been presented in 1993 by Kifor
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et al. (1993) for the Cl-/HCO3- exchanger AE1. CAII could be immunoprecipitated with AE1 when
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antiserum against the N-terminal of AE1 was used, while serum directed against the C-terminal of
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the transporter failed to immunoprecipitate CAII, suggesting that CAII physically binds to the C-
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terminal tail of AE1 (Vince and Reithmeier, 1998). These data were confirmed by affinity blotting and
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a solid phase binding assay with CAII and a GST fusion protein of the AE1 C-terminal (Vince and
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Reithmeier, 1998). Single site mutations identified the acidic cluster D887ADD in the C-terminal tail of
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AE1 as binding site for CAII (Vince and Reithmeier, 2000; Vince et al., 2000). Binding of CAII to this
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cluster would tether the enzyme close to the transporter pore of AE1 near the inner cell surface. This
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location has been suggested to ideally position CAII to hydrate incoming CO2 and directly supply the
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AE1 transporter with a localized substrate pool (Vince and Reithmeier, 2000). Indeed, inhibition of
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CAII catalytic activity decreased transport activity of AE1, heterologously expressed in HEK293 cells
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by up to 60% (Sterling et al., 2001).
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First evidence for a direct interaction between the Na+/HCO3- cotransporter NBCe1 and CAII was
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presented by Gross et al. (2002) using isothermal titration calorimetry. In analogy to the CAII-binding
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cluster D887ADD found in AE1 (Vince and Reithmeier, 2000), the authors suggested the cluster
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D986NDD within the C-terminal of NBCe1 as the putative CAII binding site. Functional interaction
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between NBCe1 and CAII was further shown by heterologous protein expression in Xenopus oocytes
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(Becker and Deitmer, 2007). Both injection and co-expression of CAII increased NBCe1-mediated
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membrane current, membrane conductance and Na+ influx during application of CO2/HCO3- in an
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ethoxzolamide-sensitive manner.
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Evidence for an interaction between NHE1 and intracellular CAII was obtained by measuring the
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recovery from a CO2-induced acid load in AP1 cells, transfected with NHE1 (Li et al., 2002).
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Cotransfection of NHE1 with CAII almost doubled the rate of pH recovery as compared to cells
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expressing NHE1 alone, while cotransfection with the catalytically inactive mutant CAII-V143Y even
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decreased the rate of pH recovery, indicating a physical interaction between NHE1 and catalytically 4
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active CAII. Physical interaction between the two proteins was demonstrated by co-
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immunoprecipitation of heterologously expressed NHE1 and CAII (Li et al., 2002). A micro titer plate
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binding assay with a GST fusion protein of the NHE1 C-terminal tail revealed that CAII binds to the
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penultimate group of 13 amino acids of the C-terminal tail (R790IQRCLSDPGPHP), with the amino acids
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S796 and D797 playing an essential role in binding (Li et al., 2001, 2006).
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While a considerable amount of data indicates a physical and functional interaction between various
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acid/base transporters and carbonic anhydrases, several studies also questioned such transport
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metabolons. Lu et al. (2006) could not observe a CAII-mediated increase in membrane conductance
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in NBCe1-expressing Xenopus oocytes, even when fusing CAII to the C-terminal of NBCe1. In line with
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these experiments, Yamada et al. (2011) could find no increase in the membrane current during
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application of CO2/HCO3- when co-expressing wild-type NBCe1A or the mutant NBCe1-Δ
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the putative CAII binding site D986NDD) with CAII. The concept of a physical interaction between
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HCO3- transporters and CAII has also been challenged by a binding study of Piermarini and colleagues
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(2007). The authors were able to reproduce the findings of other groups by showing that GST fusion
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proteins of the C-terminal tails of NBCe1, AE1 and NDCBE (SLC4A8) can bind to immobilized CAII in a
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micro titer plate binding assay. However, when reversing the assay or using pure peptides, no
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increased binding of CAII to the immobilized GST fusion proteins could be detected (Piermarini et al.,
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2007). It was concluded that a bicarbonate transport metabolon may exist, but that CAII might not
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directly bind to the transporters. That CAII activity could improve substrate supply to bicarbonate
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transporters even without the requirement for a metabolon, involving direct physical interaction,
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was also pointed out in a study on AE1 transport activity by Al-Samir et al. (2013). By using Förster
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resonance energy transfer measurements and immunoprecipitation experiments with tagged
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proteins, the authors showed no binding or close co-localization of AE1 and CAII. Functional
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measurements in red blood cells and theoretical modeling suggested that transport activity of AE1
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can be best supported by CAII, when the enzyme is equally distributed within the cell s cytosol (Al-
p la ki g
5
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Samir et al., 2013). For detailed reviews on transport metabolons see McMurtrie et al., 2004; Moraes
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& Reithmeier, 2012; Deitmer & Becker, 2013; Becker et al., 2014.
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We have previously shown that transport activity of MCT1 and MCT4 is enhanced by CAII, when the
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two proteins are heterologously co-expressed in Xenopus oocytes (Becker et al., 2005, 2010, 2011;
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Becker & Deitmer 2008). In contrast to the transport metabolons described before, enhancement of
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MCT / tra sport fu tio is i depe de t of the e z
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catalytic activity with 6-Ethoxy-2-benzothiazolesulfonamide (EZA) and co-expression of MCT1/4 with
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the catalytically inactive mutant CAII-V143Y failed to suppress the CAII-induced facilitation of MCT1/4
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transport activity (Becker et al., 2005; Becker & Deitmer, 2008). First evidence that this non-catalytic
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interaction between MCT1/4 and CAII requires direct binding between the two proteins was shown
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by injection of CAII that was bound to an antibody prior to the injection. In this experiment CAII was
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not able to enhance transport activity of MCT1 in Xenopus oocytes, suggesting a sterical suppression
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of the interaction by the antibody (Becker & Deitmer, 2008). In the same study, truncation of the
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MCT1 C-terminal tail led to loss of interaction between MCT1 and CAII in Xenopus oocytes (Becker &
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Deitmer, 2008). By introduction of single site mutations in MCT1 and MCT4, the glutamic acidic
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clusters E489EE and E431EE within the C-terminal tail of MCT1 and MCT4, respectively, could be
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identified as binding sites for CAII (Stridh et al., 2012; Noor et al., 2015). In both studies binding was
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confirmed both on the functional level by measuring MCT transport activity in Xenopus oocytes and
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by pull-down assays using GST-fusion proteins. Direct interaction between MCT1 and CAII was not
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only shown in vitro and by heterologous protein expression in Xenopus oocytes, but also in mouse
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astrocytes via an in situ proximity ligation assay (Stridh et al., 2012). Since CA catalytic activity is not
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required to facilitate MCT transport function, it was hypothesized that CAII might utilize parts of its
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intramolecular proton pathway to function as a proton antenna for the transporter (Almquist et al.,
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2010; Becker et al., 2011). Protonatable residues with overlapping Coulomb cages could form proton-
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attractive domains and could share a proton at a very fast rate, exceeding the upper limit of
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diffusion-controlled reactions (Ädelroth et al., 2004; Friedman et al., 2005). When these residues are
e s atal ti a ti it . Both, inhibition of CA
6
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located in proteins or lipid head groups at the plasma membrane, they can collect protons from the
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solution and direct them to the entrance of a proton-transfer pathway of a membrane-anchored
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protei , a phe o e o ter ed proto - olle ti g a te
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2006). The need for such a proton antenna is based on the observation that H+ cotransporters, such
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as MCTs, extract H+ from the surrounding area at rates well above the capacity for simple diffusion to
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replenish their immediate vicinity. Therefore, the transporter must exchange H+ with protonatable
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sites at the plasma membrane, which could function as proton collectors for the transporter
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(Martínez et al., 2010). The finding that the mutant CAII-H64A, missing the central residue of the
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e z
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that CAII may function as proton antenna for the MCT, to provide or subtract protons to or from the
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transporter, respectively, via its intramolecular H+ shuttle (Becker et al., 2011). In line with this, we
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could recently show that integration of a proton antenna, in form of 6 histidine residues, into the C-
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terminal tail of MCT4 could facilitate MCT4 transport activity even in the absence of carbonic
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anhydrase (Noor et al., 2017).
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Transport activity of MCTs was not only shown to be facilitated by intracellular CAII, but also by the
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extracellular CA isoforms CAIV and CAIX (Klier et al., 2011, 2014; Jamali et al., 2015). Hypoxia-
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regulated CAIX is considered to be a key protein in tumor acid/base regulation. CAIX, the expression
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of which is usually linked to poor prognosis, is tethered to the membrane via a transmembrane
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domain, with the catalytic center facing the extracellular site. Like other fast carbonic anhydrases,
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CAIX is equipped with an intramolecular proton shuttle for rapid exchange of H+ between the
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catalytic center and the surrounding bulk solution. We could demonstrate that knockdown of CAIX
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with siRNA reduced MCT1-mediated lactate transport in hypoxic MCF-7 cells, while inhibition of CA
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catalytic activity with EZA had no effect on MCT1 transport activity (Jamali et al., 2015). Co-
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expression of MCT1/4 with CAIX in Xenopus oocytes revealed that CAIX-mediated facilitation of MCT
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transport activity requires the enzymes intramolecular proton shuttle His200 (Jamali et al., 2015).
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Furthermore, knockdown of CAIX, but not inhibition of CA catalytic activity, reduced proliferation of
a Ädelroth et al.,
; Brä dé et al.,
e s i tra ole ular proto shuttle, fails to fa ilitate MCT tra sport a ti it , led to the otio
7
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hypoxic MCF-7 cells, indicating that the CAIX-driven increase in lactate efflux is crucial for proper
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function of cancer cells (Jamali et al., 2015).
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In the present study we investigated whether CAII can facilitate proton-driven lactate transport in
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MCF-7 breast cancer cells. Knockdown of CAII, which is closely co-localized to MCT1 in MCF-7 cells,
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resulted in a significant reduction of MCT transport activity in both normoxic and hypoxic cells. We
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then further analyzed the molecular mechanism underlying the CAII-mediated facilitation of MCT
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transport activity. Our results showed that CAII features a moiety (Glu69 + Asp72) that exclusively
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mediates proton exchange with the transporter, while the central residue of its intramolecular
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proton shuttle (His64) mediates the binding of CAII to MCT1 and MCT4.
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Results
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Intracellular CAII facilitates lactate transport in MCF-7 breast cancer cells
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We have recently shown that extracellular CAIX facilitates lactate transport in hypoxic cancer cells by
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acting as a proton antenna for MCT1/4 at the extracellular face of the plasma membrane (Jamali et
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al., 2015). Since proton-driven lactate transport would also benefit from a proton antenna at the
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cytosolic face of the cell membrane, we now investigated whether proton/lactate cotransport in
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cancer cells is facilitated by intracellular CAII. Therefore, we knocked down CAII in normoxic (20% O2)
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and hypoxic (1% O2) MCF-7 breast cancer cells using siRNA and determined MCT-mediated
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proton/lactate cotransport by measuring changes in intracellular pH (pHi) during application and
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removal of 3 and 10 mM lactate in the nominal absence of CO2/HCO3- (Figure 1A, B). Indeed,
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knockdown of CAII resulted in a significant decrease in the rate of change in pHi ΔpHi/Δt
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application and removal of lactate, indicating a decrease in the lactate transport rate with reduced
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CAII (Figure 1C, D). Interestingly, reduction in transport activity was more pronounced in hypoxic cells
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than under normoxic conditions (25%-33% reduced in normoxic cells, 47%-57% reduced in hypoxic
oth duri g
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cells), even though expression of CAII, in contrast to CAIX, is not upregulated under hypoxia (Figure 1
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- figure supplement 1; Jamali et al., 2015). A possible reason for this effect might be that the increase
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in proton/lactate transport, mediated by extracellular CAIX under hypoxia, challenges the need for a
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faster proton supply at the intracellular site. Knockdown efficiency of CAII was checked by qRT-PCR
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and western blot analysis, showing a 60% reduction in normoxic and hypoxic cells (Figure 1 - figure
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supplement 1). To investigate whether the CAII-induced augmentation in MCT1 transport activity
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requires catalytic activity of the enzyme, lactate transport in MCF-7 cells can be determined in the
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presence of the CA inhibitor EZA. We had shown this experiment in a recent study on MCF-7 cells
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with the same settings as used in the present study (Jamali et al., 2015). In that study application of
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30 µM EZA had no effect on lactate transport, as measured with the lactate-sensitive FRET
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nanosensor Laconic (San Martín et al., 2013) and by determining the rate of change in intracellular
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pH, in normoxic and hypoxic MCF-7 cells, respectively, neither in the presence nor in the absence of
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5% CO2/15 mM HCO3- (see Figures 3 h, i, and S2 in Jamali et al., 2015). Since EZA did not alter lactate
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transport in MCF-7 cells under any condition, it can be concluded that the observed reduction in
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lactate transport by knockdown of CAII is independent of the e z
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Knockdown of CAII decreased the basal pHi, as measured at the beginning of the experiment, by
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approximately 0.1 pH units, both in normoxic and in hypoxic MCF-7 cells (Figure 1 - figure
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supplement 2 A). This acidification indicates that CAII plays an important role in the acid/base
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regulation of MCF-7 cells, independent the oxygen tension, either directly by providing HCO3- to the
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buffer system, or indirectly by interacting with the cell s acid/base transporters. Since an intracellular
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acidification leads to a less favorable gradient for H+-coupled lactate influx, the observed reduction in
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ΔpHi/Δt duri g la tate appli atio
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investigate the influence of pHi on la tate tra sport, e plotted ΔpHi/Δt duri g appli atio of
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lactate (which was always carried out as the first pulse) against the initial pHi (before lactate
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application) for every individual cell (Figure 1 - figure supplement 2 B-E). Under none of the four
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o ditio s a positi e orrelatio
e s catalytic activity.
ould also e the result of this ha ge i the H + gradient. To M
et ee ΔpHi/Δt a d pHi could be observed. From these results it 9
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can be concluded that the decrease in pHi, as induced by knockdown of CAII, seems to play only a
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minor role in the observed reduction in lactate transport.
230 231
CAII is co-localized with MCT1 in cancer cells
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We have recently investigated which MCT isoforms mediate lactate transport in MCF-7 cells under
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normoxic and hypoxic conditions (Jamali et al., 2015). In that study application of 300 nM AR-
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C155858 fully inhibited the lactate-induced acidification in normoxic and of hypoxic MCF-7 cells. AR-
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C155858 has been shown to inhibit transport activity of MCT1 (and MCT2 when expressed without
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its ancillary protein embigin in Xenopus oocytes), but has no effect on MCT4 transport activity (Ovens
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et al., 2010a,b). By measuring the rate of change in pHi during application of different lactate
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concentrations we determined Km values of ~5 mM lactate form both normoxic and hypoxic MCF-7
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cells (Jamali et al., 2015). For MCT1, a Km value between 3–8 mM had been determined in various cell
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types (Carpenter & Halestrap, 1994; Bröer et al., 1997, 1998). For MCT2, the Km value was found to
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be 0.74 (Bröer et al., 1999; Heidtmann et al., 2015), while for MCT4 Km values between 17–35 mM
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have been reported (Dimmer et al., 2000). Western blot analysis for MCT1, MCT2 and MCT4 revealed
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sharp bands only for MCT1 in MCF-7 cells both under normoxic and hypoxic conditions (Jamali et al.,
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2015). Taken together, these results indicate that lactate transport in MCF-7 cells is exclusively
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mediated by MCT1, both under normoxic and hypoxic conditions.
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Antibody staining of MCT1 and CAII revealed a homogenous distribution of both proteins within the
247
cells (Figure 2A). To investigate whether CAII is colocalized with MCT1 in MCF-7 cells, we performed
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an in situ proximity ligation assay (PLA). The PLA indicated close proximity (< 40 nm) between MCT1
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and CAII, both in normoxic and hypoxic cells (Figure 2B1, B2). The amount of PLA signals per nucleus
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did not significantly differ between normoxic and hypoxic cells (Figure 2C). Knockdown of CAII
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resulted in a significant reduction in the signal (Figure 2B3, C). When the PLA was carried out without
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primary antibodies as negative control, no PLA signals could be detected (Figure 2B4, C).
10
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Taken together, these data suggest that CAII directly interacts with MCT1 to facilitate transport
254
activity.
255 256
CAII supports proliferation of cancer cells
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To investigate whether the CAII-mediated augmentation in lactate flux facilitates cancer cell
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proliferation, we determined the number of MCF-7 cells kept under different conditions for up to
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three days (Figure 3A-D). Indeed, knockdown of CAII significantly decreased cell proliferation (as
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compared to cells transfected with non-targeting negative control siRNA) both under normoxia and
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hypoxia, while transfection with non-targeting negative control siRNA had no significant effects on
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cell proliferation. Interestingly, total inhibition of lactate transport with 300 nM AR-C155858
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decreased cell proliferation by the same degree as did knockdown of CAII (as compared to untreated
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cells). Inhibition of CAII catalytic activity with 30 µM EZA, however, had no effect on cell proliferation
265
(as compared to untreated cells). The effects of AR-C155858 and EZA are in line with previous
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findings that inhibition of MCT transport activity significantly reduces proliferation of MCF-7 and
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MDA-MB-231 cells, while inhibition of CA catalytic activity has no effect on cell proliferation (Jamali
268
et al., 2015). The striking effect of CAII knockdown on normoxic and hypoxic cells suggests the
269
possibility that the CAII protein might have yet another role in cell proliferation, in addition to its
270
function as a facilitator of lactate efflux and its catalytic function in cellular pH regulation.
271 272
CAII facilitates a proton-collecting apparatus at its surface to drive MCT transport activity
273
We have previously shown that CAII facilitates transport activity of MCT1 and MCT4 when
274
heterologously expressed in Xenopus oocytes. CAII-mediated facilitation of MCT1/4 transport activity
275
was found to be independent of the e z
276
activity with EZA and co-expression of MCT1/4 with the catalytically inactive mutant CAII-V143Y
277
failed to suppress the CAII-induced facilitation of MCT1/4 transport activity (Becker et al., 2005;
e s atal ti a ti it , si e both inhibition of CA catalytic
11
278
Becker & Deitmer, 2008). Since CA catalytic activity is not required to facilitate MCT transport
279
function, it was hypothesized that CAII might utilize parts of its intramolecular proton pathway to
280
function as a proton antenna for the transporter (Almquist et al., 2010; Becker et al., 2011). To
281
investigate which moieties within the CAII protein could mediate this antennal function, we tested
282
the influence of various CAII mutants on MCT1/4 transport activity using the Xenopus oocyte
283
expression system.
284
By using an algorithm for mapping proton wires in proteins, Shinobu and Agmon (2009) suggested
285
that CAII-Glu69 and CAII-Asp72, located on an electronegative patch on the rim of the active site
286
cavity of CAII, could fu tio as a do ki g statio
287
to the catalytic center. The position of the two amino acids is shown in the cartoon in Figure 4 A. To
288
test whether CAII-Glu69 or CAII-Asp72 are involved in the facilitation of MCT transport activity, we
289
mutated these two amino acids, as well as the nearby Asp71, and co-expressed the resulting mutants
290
with either MCT1 or MCT4 in Xenopus oocytes. Transport activity of MCT1/4 was determined by
291
measuring changes in intracellular proton concentration ([H+]i) during application and removal of 3
292
and 10 mM lactate, while CAII catalytic activity was checked by application of 5% CO2 / 10 mM HCO3-
293
(Figure 4B; Figure 4 – figure supplement 1A). CAII-WT increased transport activity of MCT1 by 80-
294
100%, as measured by the rate of change in [H+]i Δ[H+]i/Δt duri g application and removal of lactate
295
(Figure 4C, D). However, no significant increase in transport activity was observed, when MCT1 was
296
co-expressed with CAII-E69Q or CAII-D72N, while mutation of the nearby Asp71 (CAII-D71N) had no
297
effect on MCT1 transport activity (Figure 4B-D). Co-expression of the CAII mutants with MCT4 gave
298
similar results as those obtained with MCT1 (Figure 4 – figure supplement 1A-C). Western blot
299
analysis showed that protein expression levels were not altered by the mutations in CAII (Figure 4 –
300
figure supplement 2).
301
It has been shown previously that addition of exogenous H+ donors/acceptors, like imidazole or
302
carnosine, can rescue proton shuttling in carbonic anhydrase in vitro (An et al., 2002; Tu et al., 1989).
303
To check whether chemical rescue of the H+ shuttle in CAII-E69Q and CAII-D72N also can restore the
for protonated buffer molecules to deliver protons
12
304
functional interaction between these mutants and MCT1, we injected 4-methylimidazole (4-MI) into
305
oocytes co-expressing MCT1 and CAII-E69Q/CAII-D72N. Oocytes expressing MCT1 alone or co-
306
expressing MCT1 + CAII-WT or MCT1 + CAII-D71N, respectively, were used as control. The effective
307
free volume of a oo te is arou d .
308
400 mM solution of 4-MI should result in an intracellular 4-MI concentration of 30 mM.
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Crystallization studies have shown that 4-MI i ds to CAII ear the His
310
a moiety near Glu69 and Asp72 (Figure 5A; Duda et al., 2001, 2003). After injection of 4-MI, MCT1
311
transport activity was enhanced after co-expressing CAII-E69Q or CAII-D72N to a similar extent as
312
with co-expression with CAII-WT and CAII-D71N (Figure 5B-D). Since 4-MI acts as mobile H+ buffer,
313
the compound is expected to increase the cytosolic buffer strength (βi), which in turn alters changes
314
in [H+]i. Therefore, we calculated net H+ fluxes (JH) from the rate of change in intracellular pH during
315
application of 3 and 10 mM lactate and βi (Figure 5E; Figure 5 – figure supplement 1A). Injection of 4-
316
MI induced a significant increase in lactate-induced JH in oocytes co-expressing MCT1 with CAII-E69Q
317
and D72N, respectively, while leaving JH in oocytes co-expressing MCT1 with CAII-WT or CAII-D71N,
318
as well as oocytes expressing MCT1 alone, unaltered (Figure 5E). Again the same results could be
319
observed with MCT4 (Figure 5 – figure supplements 1 and 2). These data indicate that the exogenous
320
H+ donor/acceptor 4-MI can rescue the facilitation of MCT1/4 transport activity by CAII-E69Q and
321
CAII-D72N.
μl )euthen et al., 2002). Therefore, injection of 27.6 nl of a
π-stacking to Trp5 and to
322 323
CAII-Glu69 and Asp72 are not involved in binding between CAII and MCTs
324
We have previously shown that CAII-mediated enhancement of MCT1/4 transport activity requires
325
direct binding of the enzyme to an acidic cluster within the transporters C-terminal tails (Stridh et
326
at., 2012; Noor et al., 2015). To check whether the failure of CAII-E69Q and CAII-D72N to enhance
327
MCT transport activity is due to loss of binding between transporter and enzyme, we performed a
328
pull-down assay with GST fusion proteins of the C-terminal tail of MCT1 and MCT4, respectively (GST-
329
MCT1-CT, GST-MCT4-CT), and lysates from oocytes expressing CAII-WT, CAII-E69Q, CAII-D71N, and 13
330
CAII-D72N, respectively (Figure 6). All three CA mutants could be pulled down with GST-MCT1-CT and
331
GST-MCT4-CT, with no evident changes in signal intensity between the mutants and CAII-WT. As
332
negative control, CAII-WT was pulled down with GST alone, resulting in no or only very weak signals.
333
These results indicate that the failure of CAII-E69Q and CAII-D72N to enhance MCT transport activity
334
is not due to a loss of direct binding between transporter and enzyme.
335 336
CAII-Glu69 and Asp72 do not support CAII catalytic activity
337
By using an algorithm for mapping proton wires in proteins, Shinobu and Agmon (2009) had shown
338
that Glu69 and Asp72 form an extension of the CAII proton wire. From this model, the authors
339
proposed that positively charged, protonated buffer molecules could dock in that area, from which a
340
proton is delivered to the active site to facilitate catalytic activity. To investigate whether Glu69 and
341
Asp72 play a role in CAII catalytic function, we determined CAII catalytic activity in oocyte lysates by
342
gas analysis mass spectrometry (Figure 7A, B). CA atal ti a ti it is deter i ed
343
e ri h e t LE , which is influenced both by the rate of hydration and dehydration of CO2 (see
344
Materials & Methods). Therefore a change in LE indicates changes of CA catalytic activity both in the
345
hydration and dehydration reaction. Mutation of CAII-Glu69, Asp71, or Asp72 did not alter CAII
346
catalytic activity, as compared to CAII-WT, while mutation of CAII-His64 (the central residue of the
347
CAII intramolecular H+ shuttle) resulted in a 78% reduction in CA activity (Figure 7A, B). Even when
348
both Glu69 and Asp72 were mutated together (CAII-E69Q/D72N), no alterations in CAII catalytic
349
activity could be observed (Figure 7A, B). The results from the mass spectrometry were confirmed my
350
easuri g Δ[H+]i/Δt i
the log
CAII-expressing oocytes during application and removal of CO2/HCO3-.
351
E pressio of CAII resulted i a sig ifi a t i rease i Δ[H+]i/Δt, as compared to native oocytes (Figure
352
7C, D). Mutation of CAII-Glu69, Asp71, Asp72, or double mutation of Glu69/Asp72 resulted in no
353
significant change in CA activity, neither during application nor removal of CO2/HCO3-. Only mutation
354
of CAII-His64 induced a significant decrease in catalytic activity. These data indicate that neither
355
Glu69 nor Asp72 are required for full catalytic activity in CAII. Interestingly, mutation of His64 to Ala 14
356
reduced CAII catalytic activity to 25%, when measured by gas analysis mass spectrometry (Figure 7
357
B), but only to 50%, when measured by Δ[H+]i/Δt i i ta t oo tes Figure C, D . These fi di gs i fer
358
that the cytosol of oocytes already contains certain, still unidentified, buffer compounds that can
359
support catalytic activity of the mutant.
360 361
CAII-His64 mediates binding of CAII to MCT1/4, but is not involved in H+ shuttling between the
362
proteins
363
Our previous studies had shown that CAII-mediated facilitation of MCT transport activity requires
364
CAII-His64, the central residue of the enzymes intramolecular H+ shuttle (Becker et al., 2011).
365
Furthermore, our structural models suggest that His64 mediates binding between CAII and an acidic
366
cluster within the C-terminal tails of MCT1 and MCT4 (Figure 8A; Figure 8 – figure supplement 1A;
367
Stridh et al., 2012; Noor et al., 2015). To investigate whether CAII-His64 mediates binding and/or
368
proton shuttling between MCT1/4 and CAII, we co-expressed MCT1/4 with a CAII in which the
369
histidine at position 64 was either mutated to alanine (CAII-H64A) or to lysine (CAII-H64K). Mutation
370
of His to Ala should disrupt both intramolecular H+ shuttling in CAII and binding of the enzyme to the
371
glutamate residues in MCT1/4. Mutation of His to Lys does also disable H + shuttling, due to the
372
increase in pKa, but should still allow binding of CAII to the glutamate residues in the C-terminal tail
373
of MCT1/4. Indeed, tra sport a ti it
374
application and removal of lactate, was not enhanced by co-expression with CAII-H64A, while CAII-
375
H64K enhanced MCT transport activity to the same extent as did CAII-WT (Figure 8B-D; Figure 8 –
376
figure supplement 1B-D).
377
Binding of the CAII mutants to MCT1/4 was evaluated by a pull-down assay with GST fusion proteins
378
of the C-terminal tail of MCT1 and MCT4, respectively (GST-MCT1-CT, GST-MCT4-CT) and lysates
379
from oocytes expressing CAII-WT, CAII-H64A, and CAII-H64K, respectively (Figure 8E, F; Figure 8 –
380
figure supplement 1E, F). CAII-H64A was not able to bind to the C-terminal tail of MCT1 and MCT4,
of MCT
a d MCT , as
easured
Δ[H+]i/Δt duri g
15
381
respectively, showing only 12 and 14% signal intensity of the band for CAII-WT, and therefore not
382
being significantly different from the negative control, in which CAII-WT was pulled down with GST
383
alone. In contrast, CAII-H64K did still bind to the C-terminal tail of MCT1 and MCT4, with no
384
significant alterations in signal intensity as compared to the band for CAII-WT.
385
Catalytic activity of CAII-H64A and CAII-H64K was determined in oocyte lysates by gas analysis mass
386
spectrometry (Figure 8 – figure supplement 2A, B). Both mutations of His64 to Ala and Lys resulted in
387
a significant decrease in CA catalytic activity with a reduction to 53% (CAII-H64K) and 23% (CAII-
388
H64A), respectively, as compared to CAII-WT. However, mutation of His64 did not change the
389
expression level of CAII in oocytes (Figure 8 – figure supplement 2C, D). The finding that catalytic
390
activity of CAII-H64K is strongly reduced, compared to CAII-WT, but still significantly higher than the
391
activity of CAII-H64A indicates that the lysine at position 64 might still be able to function as
392
intramolecular H+ shuttle, even though with significantly less efficacy than histidine.
393
Taken together, these results indicate that His64, the central residue of the CAII intramolecular H+
394
shuttle, mediates i di g of CAII to the tra sporter s C-terminal tail rather than H+-transfer between
395
the two proteins. However, since the H64K mutation still seems to retain some shuttling activity, a
396
role of His64 in H+-transfer cannot be fully ruled out.
397 398
The histidines in the N-terminus of CAII are not involved in binding of CAII to MCT1
399
It was previously proposed by Vince et al. (2000) that CAII binds to the C-terminal tail of the Cl-/HCO3-
400
anion exchanger AE1 via a cluster of five histidine and one lysine residues in the enzyme s N-terminal
401
domain.
402
H3P/H4Q/K9A/H10K/H15K/H17S;
403
(which does not bind to AE1) failed to bind to the transporter. Indeed the same CAII-mutant was also
404
not able to facilitate MCT1 transport activity (Becker et al., 2008). To investigate the role of His3,
405
His4, Lys9, His10, and His15 in the facilitation of MCT transport activity, we mutated every single one
This
assumption
was
based
oi ed
on
the
finding
that
a
CAII-mutant
(CAII-
CAII-HEX ), mimicking the N-terminal domain of CAI
16
406
of these amino acids, either alone or in combination, and co-expressed the resulting mutants with
407
MCT1 in Xenopus oocytes. However, none of the mutations resulted in a loss of functional interaction
408
between MCT1 and CAII (Figure 9), indicating that none of the histidines or lysine in the N-terminal of
409
CAII is directly involved in the facilitation of MCT transport activity. A possible reason for the inability
410
of CAII-HEX to facilitate MCT transport activity might be that the introduction of charged and bulky
411
amino acids into the N-terminal domain may prohibit access of the MCT C-terminal tail to the binding
412
site CAII-His64.
413 414 415
Discussion
416
Our previous studies have shown that CAII facilitates transport activity of MCT1 and MCT4
417
independent of the enzyme s catalytic activity, since both inhibition of CA activity with EZA and
418
molecular disruption of the catalytic center (CAII-V143Y) did not reduce the CAII-induced increase in
419
MCT transport activity (Becker et al., 2005; 2008). Since CA catalytic activity is not required to
420
facilitate MCT transport function, we hypothesized that CAII might utilize parts of its intramolecular
421
proton pathway to function as a proton antenna for the transporter (Almquist et al., 2010; Becker et
422
al., 2011). The proton pathway of CAII consists of a water wire, coordinated by Asn62 and Asn67,
423
which extends from the active site to the histidine residue at position 64 (Fisher et al., 2007a,b).
424
From there the proton is passed on between the imidazole ring in His64 and exogenous buffer
425
molecules surrounding the enzyme (Fisher et al., 2007a,b). In addition, Shinobu and Agmon (2009)
426
presented a model in which the active site H+ wire exits to the protein surface, and leads to Glu69
427
and Asp72, located on an electronegative patch on the rim of the active-site cavity. Based on this
428
model, the authors proposed that positively charged, protonated buffer molecules could dock in that
429
area, from which a proton is delivered to the active site, when the enzyme works in the dehydration
430
direction. However, this assumption is purely based on mathematical modelling and has, to our 17
431
knowledge, not yet been evaluated experimentally. In the present study, we could show that both
432
Glu69 and Asp72 are essential for CAII-mediated facilitation of MCT1/4 transport activity both in the
433
influx and efflux direction. Mutation of Glu69 and Asp72 did, however, not alter CAII catalytic
434
activity, as demonstrated in vitro by gas analysis mass spectrometry and in vivo by determining the
435
rate of change in intracellular H+ concentration in oocytes during application and removal of CO2.
436
These results suggest that this proton-collecting moiety is not involved in the facilitation of CAII
437
catalytic activity, but rather mediates a second, independent function, which is the rapid supply and
438
removal of protons from the pore of the adjacent transporter, rendering CAII a bona fide proton
439
antenna. Whether this kind of interaction does only exist between CAII and MCTs, or whether CAII
440
could also function as proton antenna for other proton-coupled membrane transporters, like proton-
441
coupled amino acid transporters or Na+/H+ exchangers, remains to be shown.
442
Our previous studies had shown that CAII-mediated facilitation of MCT transport activity requires
443
CAII-His64, which mediates the exchange of protons between catalytic center and surrounding bulk
444
solution during the CAII catalytic cycle. However, our structural models suggested that His64 rather
445
mediates binding of CAII to Glu489/Glu491 in MCT1 (Stridh et al., 2012) and Glu431/Glu433 in MCT4
446
(Noor et al., 2015). To investigate whether His64 mediates binding or proton shuttling between MCT
447
and CAII, or both, we exchanged the histidine at position 64 either to alanine or to lysine. Mutation
448
of His to Ala should disable both intramolecular H+ shuttling in CAII and binding of the enzyme to the
449
glutamate residues in MCT1/4. Mutation of His to Lys does also disable H+ shuttling, since lysine has a
450
considerably higher pKa than the imidazole ring in histidine, which does not allow fast
451
protonation/deprotonation reactions at physiological pH. However, lysine should still be able to form
452
hydrogen bonds with the glutamate residues in the C-terminal tail of MCT1 and MCT4, and should
453
therefore still enable CAII to bind to the transporter. While MCT transport activity was not enhanced
454
by CAII-H64A, CAII-H64K facilitated MCT transport activity to the same extent as CAII-WT. These
455
results let us conclude that His64 may not be involved in proton transfer between MCT and CAII, but
456
instead mediates binding of the enzyme to the transporters C-terminal tail. However, since CAII18
457
H64K still displayed a higher catalytic activity than CAII-H64A, it cannot be ruled out that CAII-H64K
458
still retains some proton shuttling activity. Indeed, it has been shown that lysine residues that are
459
buried in the protein interior could display considerably lower pKa values than in water (Isom et al.,
460
2011). Therefore, it might be possible that CAII-H64K can still retain enough shuttling activity to
461
facilitate MCT-mediated lactate transport.
462
Taken together, these results suggest that CAII facilitates MCT transport activity by a completely
463
different mechanism than the catalytic activity: Catalytic activity of CAII is facilitated by proton
464
shuttling via His64, while Glu69 and Asp72 are ineffective in supporting CA activity. When interacting
465
with MCT1/4, His64 does function as binding site, while proton transfer between transporter and
466
enzyme seems to be mediated by Glu69 and Asp72 (Figure 10).
467
The physiological need for CAII serving as such a proton antenna for MCTs might derive from the low
468
apparent diffusion rate of protons in a strongly buffered solution like the cytosol. By applying a
469
mathematical model of Brownian diffusion, Martinez et al. (2010) determined the maximum supply
470
capacity of substrates to various transporters and enzymes. For the MCT1 the authors calculated that
471
the diffusion rate of lactate, which is present in the cell within the millimolar range, exceeds the
472
turnover rate of the transporter by several magnitudes, supporting the notion that for this molecule
473
the cytosol is a well-mixed compartment. For protons, however, the authors calculated that the
474
maximum supply rate is approximately 15 times lower than the apparent turnover number of MCT1,
475
as calculated by Ovens et al. (2010a). In other words, the model demonstrates that MCT1 extracts H+
476
from the cytosol at rates well above the capacity for simple diffusion to replenish its immediate
477
vicinity. This paradoxical result implies that the transporter does not extract H+ substrates directly
478
from the bulk cytosol, but fro
479
aqueous phase and the transport site (Martinez et al., 2010). The findings of the present study
480
suggest that CAII could serve as such a harvester which captures protons from protonatable residues
481
at the plasma membrane and funnels them to the transporter pore (Figure 10). By this, CAII would
a i ter ediate har esti g
o part e t located between the
19
482
counteract the local depletion of protons in the direct vicinity of the transporter to support transport
483
activity. Since the direction of proton movement is depending on the ion gradient, this principle can
484
be applied both in the influx and in the efflux direction. In case of proton/lactate efflux, CAII would
485
harvest H+ from protonatable residues near the plasma membrane and shuttle them to the
486
transporter, in case of proton/lactate influx, CAII would capture the H+ from the transporter pore
487
and distribute them to protonatable residues near the plasma membrane, following the
488
electrochemical H+ gradient. Indeed, we could previously show that protons, which are focally
489
applied to MCT1-expressing oocytes, moved faster along inner face of the plasma membrane when
490
CAII was injected, suggesting that CAII could allocate H+ along the plasma membrane and thereby
491
increase the area in which H+ can be released from the membrane into the bulk solution (Becker &
492
Deitmer, 2008). By this, CAII would counteract the accumulation of protons at the transporter pore,
493
which would result in reduction of transport activity in the influx direction.
494
Even though the mathematical model from Martinez et al. (2010) suggests a 15 fold lower supply
495
rate of H+, our measurements in Xenopus oocytes have shown that CAII enhances transport activity
496
of MCT a d MCT
497
activity is either facilitated by other proton-collecting molecules, or - more likely - that MCTs are
498
already able to extract protons from adjacent protonatable sites, even though not as efficient as
499
when cooperating with CAII.
500
The relevance of this proton antenna for proper cell function becomes evident from our physiological
501
experiments on MCF-7 breast cancer cells. First evidence for a functional interaction between MCT
502
and CAII comes from the finding that knockdown of CAII reduces lactate transport in MCF-7 cells.
503
That this decrease in transport activity is due to a loss of CAII catalytic activity could be ruled out,
504
since we could show in a recent study that inhibition of CA activity by application of EZA did not alter
505
lactate flux in MCF-7 cells under the same conditions (Jamali et al., 2015). Therefore, it can be
506
assumed that CAII facilitates lactate flux in cancer cells by a non-catalytic, direct interaction. Indeed,
o l
arou d t o fold. This i plies that in the absence of CAII MCT transport
20
507
CAII is in close co-localization with MCT1 in MCF-7 cells, as shown by in situ proximity ligation assay.
508
Thus it appears likely that CAII can form a protein complex with MCT1 in the cells to facilitate lactate
509
flux by functioning as a proton antenna for the transporter. In line with these findings, we previously
510
showed that CAII also facilitates lactate transport in mouse cerebellar and white matter astrocytes,
511
i depe de t fro
512
and CAII are in close proximity in astrocytes as shown by in situ PLA. Interestingly, colocalization
513
could only be detected when an antibody directed against the intracellular loop between TM 6 and 7
514
of MCT1 was used. When the PLA was carried out using an antibody against the C-terminal of MCT1,
515
carrying the CAII binding site, no proximity between MCT1 and CAII could be detected (Stridh et al.,
516
2012). Astrocytes are highly glycolytic cells, which are widely believed to export lactate as an energy
517
fuel for adja e t euro s, a phe o e o
518
Magistretti, 1994; Magistretti, 2006; Barros and Deitmer, 2010). These findings infer that CA-
519
mediated facilitation of MCT1/4 transport activity might be a more general, or at least common,
520
phenomenon in glycolytic cells which have to extrude high amounts of lactate.
521
In the present study, knockdown of CAII resulted in a more pronounced reduction in MCT1 transport
522
activity in hypoxic than in normoxic cancer cells, even so expression of CAII was not increased under
523
hypoxia. This effect might be attributed to extracellular CAIX, which functions as a proton antenna
524
for MCT1 in MCF-7 cells under hypoxic conditions (Jamali et al., 2015). CAIX operates only on the
525
extracellular side of the membrane, which might lead to the formation of a proton micro-domain on
526
the cytosolic side of the membrane in the absence of intracellular CAII, since supply or removal of H+
527
is enhanced on the extracellular side, leading to an increase in MCT transport activity. This would
528
result in increased accumulation or depletion of H+ at the cytosolic side, depending on transport
529
direction, which would impair MCT transport rate. With a proton antenna on both sides of the
530
membrane, formation of H+ micro-domains would be suppressed both at the cis- and at the trans-
531
side. In such a scenario, extracellular CAIX and intracellular CAII would cooperate
532
pri iple , providing protons to the transporter on one side and removing them on the other side of
the e z
e s atal ti a ti it
“tridh et al.,
. Like in the cancer cells, MCT1
oi ed Astrocyte-to-Neuron Lactate Shuttle Pelleri a d
a push a d pull
21
533
the cell membrane (Figure 10). By this mechanism, intracellular CAII and extracellular CAIX could
534
cooperate to enhance lactate transport in cancer cells under hypoxic conditions. Since CAIX is already
535
expressed in normoxic MCF-7 cells at a low level (Jamali et al., 2015), this push and pull principle
536
could also occur in normoxic cells, even though to a lower extend. The lower, yet robust, expression
537
of extracellular CAIX under normoxia could also explain the reduced effect of CAII knockdown on
538
lactate transport in normoxic MCF-7 cells. In line with this notion, we previously found in Xenopus
539
oocytes that intracellular CAII can also work in concert with another extracellular carbonic
540
anhydrase, CAIV, to ensure rapid shuttling of protons and lactate across the cell membrane (Klier et
541
al., 2014).
542
In CAII, H+ shuttling between enzyme and transporter is mediated by Glu69 and Asp72. CAIX seems to
543
lack an analogue moiety within its catalytic domain. However, it features a 59 amino acids long
544
proteoglycan-like (PG) domain that is unique to CAIX among the CA family (Pastorek & Pastorekova,
545
2015). The PG domain of human CAIX contains 18 glutamate and 8 aspartate residues, which have
546
been suggested to function as an intramolecular proton buffer, which could support CAIX catalytic
547
activity when operating in an acidic environment (Innocenty et al., 2009). We could recently show
548
that both truncation of the CAIX PG domain and application of a PG-binding antibody (M75)
549
decreased CAIX-induced facilitation in Xenopus oocytes and breast cancer cells (Ames et al., 2018).
550
Those findings let us to the conclusion that the CAIX PG domain could function as extracellular
551
proton antenna for monocarboxylate transporters within the acidic environment of a solid tumor.
552
The importance of CAII for cell viability was demonstrated by a cell proliferation assay. Knockdown of
553
CAII reduced proliferation of MCF-7 cells to the same extent as did full inhibition of lactate transport
554
with AR-C155858, while inhibition of CA catalytic activity with EZA had no effect on proliferation.
555
These results are correspond to previous findings that knockdown of CAIX or interference with the
556
CAIX PG domain by an antibody, but not inhibition of CA catalytic activity, decreases cell proliferation
557
in hypoxic MCF-7 and MDA-MB-231 cancer cells (Jamali et al., 2015; Ames et al., 2018). Apparently,
22
558
removal of the proton antenna on one side of the membrane is sufficient to reduce MCT-mediated
559
proton/lactate efflux from the cell. This in turn could lead to intracellular lactacidosis and impairment
560
of metabolism, which will ultimately result in a decrease in cell proliferation.
561
Knockdown of CAII decreased, but did not abolish lactate transport in MCF-7 cells. However,
562
knockdown of CAII decreased cell proliferation to the same extend as did inhibition of MCT1
563
transport activity with AR-C155858. This striking effect of CAII knockdown on normoxic and hypoxic
564
cells suggests that the CAII protein might have another role in cell proliferation, which is beyond its
565
function as a facilitator of lactate efflux. Since cell proliferation is not decreased by inhibition of CA
566
activity with EZA, the supporting effect of CAII seems to be independent of the enzyme s catalytic
567
activity. Zhou et al. (2015) showed that knockdown of CAII in NCI-H727 and A549 cancer cell lines
568
resulted in significant reduction in clonogenicity in vitro, and marked suppression of tumor growth in
569
vivo. By using an apoptosis gene array, the authors found a novel association of CAII-mediated
570
apoptosis with specific mitochondrial apoptosis–associated proteins. These findings suggest that the
571
CAII protein features multiple functions within the cell, including cellular acid/base regulation,
572
facilitation of lactate transport, and regulation of apoptosis.
573
In summary, our results show that CAII supports lactate flux in cancer cells, presumably by
574
functioning as a proton antenna for MCTs. Therefore, CAII features a molecular moiety that
575
exclusively mediates proton exchange with the transporter, while parts of its intramolecular proton
576
shuttle, pivotal for catalytic activity, mediate binding to the MCT. This enhancement of proton
577
movement does not only drive basic lactate flux, but is also a prerequisite for further facilitation of
578
lactate transport by CAIX under hypoxic conditions, as increased glycolytic activity requires even
579
higher lactate transport capacity in cancer cells.
580 581 582 23
583
Materials and Methods
584 585
Key resources table Reagent type (species) or resource
Designation
cell line (homo sapiens)
MCF-7
recombinant DNA reagent
pGHJ-CAII
recombinant DNA reagent
pGHJ-MCT1
recombinant DNA reagent
pGHJ-MCT4
recombinant DNA reagent
pGEX-MCT1-CT
recombinant DNA reagent
pGEX-MCT4-CT
antibody antibody antibody antibody antibody
antibody antibody antibody marker
rabbit anti-carbonic anhydrase II polyclonal antibody goat anti-MCT1 polyclonal antibody mouse anti-β-Actin monoclonal antibody mouse anti-GST Tag monoclonal antibody goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody Alexa Fluor 546 donkey anti-rabbit IgG Alexa Fluor 488 donkey anti-goat IgG Alexa Fluor 488 Phalloidin
Source or reference German Collection of Microorganisms and Cell Cultures (DSMZ) Becker et al., 2015 PMID: 16174776 Bröer et al., 1998 PMID: 9639576 Dimmer et al., 2000 PMID: 10926847 Stridh et al., 2012 PMID: 22451434 Noor et al., 2015 PMID: 25561737
Identifiers
Additional information
ACC-115
Millipore
AB1828
1:500
Santa Cruz Biotechnology
sc-14917
1:300
Santa Cruz Biotechnology
sc-47778
1:2500
Millipore
05-782
1:400
Santa Cruz Biotechnology
sc2004
1:2000
Santa Cruz Biotechnology
sc-2031
1:2000
Invitrogen
A10040
1:1000
Invitrogen
A-11055
1:1000
Life Technologies
A12379
1:500
586 587
Cultivation of MCF-7 cells
588
The human breast adenocarcinoma cell line MCF-7 was purchased from the German Collection of
589
Microorganisms and Cell Cultures DSMZ, Braunschweig, Germany (DSMZ-No. ACC-115). Cells were
590
cultured i
591
#353001, Fisher Scientific) in RPMI-1640 medium (Sigma-Aldrich, Schnelldorf, Germany),
592
supplemented with 10% fetal calf seru , % Mi i al Eagle s Mediu , .
593
glucose, 16 mM NaHCO3, and 1% penicillin/streptomycin, pH 7.2, at 37°C in 5% CO2, 95% air
594
(normoxia) or 5% CO2, 1% O2, 94% N2 (hypoxia) in humidified cell culture incubators. Cells were
tissue ulture dishes Cor i g™ Fal o ™ Eas -Grip Tissue Culture Dish
M hu a i suli ,
M
24
595
subcultivated for a maximum of 15 passages. The cell line was tested negative for contamination
596
with mycoplasma.
597 598
siRNA-mediated knockdown of CAII
599
CAII was knocked down in MCF-7 cells, using siRNA (Ambion Silencer® Select anti CA2 siRNA, s2249,
600
Life Technologies). Non-targeting negative control siRNA (Ambion Silencer® Select Negative Control
601
No. 1 siRNA) was used as control. Cells were transfected with 50 pmol of siRNA, using Lipofectamine
602
RNAiMAX transfection reagent (Life Technologies) in OptiMEM medium (Thermo Fisher). Transfected
603
cells were incubated for 3 days under normoxic or hypoxic conditions. Knockdown efficiency was
604
calculated by measurement of CAII RNA levels with quantitative real-time PCR and CAII protein levels
605
with western blot analysis.
606 607
Quantitative real-time PCR
608
Total RNA was extracted from MCF-7 cells, using the RNeasy MinElute Cleanup Kit (Qiagen GmbH,
609
Hilden, Germany). cDNA was synthesized with SuperScript III Reverse Transcriptase (Life
610
Technologies) and random hexamer primers (200 ng; #SO142, Fisher Scientific GmbH, Schwerte,
611
Germany). Real time PCR was carried out with SYBR Green (Po erUp™ SYBR Green Master Mix,
612
applied biosystems) on an AriaMx real-time PCR system (Agiland Technologies). All samples were run
613
in duplicate. RPL27 was used as a reference gene. The characteristics of the primers are shown in
614
Table 1. Calculation of the gene expression level was carried out using the comparative threshold
615
method 2-ΔΔCT.
616 617
Western blot analysis of CAII
618
To determine expression levels of CAII, MCF-7 cells were harvested by trypsinization and lysed in lysis
619
buffer (50 mM NaCl, 25 mM Tris, 0.5% (v/v) Triton X-100) with protease inhibitor (‘o he O plete™, 25
620
Mini, EDTA-free Protease Inhibitor Cocktail Tablets) for 1h at 4°C. The lysate was cleared from cell
621
debris by centrifugation. Protein concentration was determined with Bradford Reagent (Protein
622
Assay Dye Reagent Concentrate, Bio-Rad). 20 µg of total protein were separated on a 12%
623
polyacrylamide gel and blotted on a polyvinylidene membrane (Roti®-PVDF, Carl-Roth). Unspecific
624
binding sites were blocked with 5% milk powder, dissolved in PBS for 2 h. CAII was detected using
625
primary anti-CAII antibody (dilution 1:500; rabbit anti-carbonic anhydrase II polyclonal antibody,
626
AB1828, Millipore) and a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody
627
(dilution 1:2000; sc2004, Santa Cruz Biotechnology). After documentation the membrane was
628
washed 2x 10 min with stripping buffer (1.5% (w/v) glycine, 0.1% (w/v) SDS, 1% (v/v) Tween20, pH
629
. . As loadi g o trol β-Actin was labelled with Mouse-Anti-β-Actin (1:2500; sc-47778; Santa Cruz
630
Biotechnology). Quantification of the band intensity was carried out with the software ImageJ. The
631
sig al for CAII as or alized to the sig al for β-Actin in the same lane.
632 633
pH imaging in MCF-7 cells
634
Changes in intracellular pH in MCF-7 cells were measured with the pH sensitive dye
635
Seminaphthorhodafluor 5- and 6- ar o li a id F I itroge ™ “NA‘F F-AM, Life Technologies)
636
using a confocal laser scanning microscope (LSM 700 with AxioExaminer.D1 upright microscope,
637
equipped with a 40x water immersion objective (Zeiss C-Apochromat 40x/1.20 W, Carl Zeiss
638
Microscopy GmbH, Frankfurt, Germany). SNARF-5F is a ratiometric pH indicator which exhibits a
639
significant pH-dependent emission shift from yellow-orange to deep red fluorescence when
640
switching from acidic and basic pH (Han & Burgess, 2010). With a pKa of 7.2 SNARF-5 is
641
recommended for measuring cytosolic pHi (Han & Burgess, 2010). The fluorescence emission
642
spectrum of SNARF-5 has an isosbestic point at ~590 nm. The isosbestic point is the wavelength at
643
which fluorescence emission of SNARF-5 does not change under varying pH values, which provides
644
the possibility of ratiometric imaging. For ratiometric imaging SNARF-5 was excited at 555 nm with a
645
scanning frequency of 0.4 Hz. The emitted light was separated with a variable dichroic mirror at 590 26
646
nm in a 590 nm fraction and the signals of the 590 nm fraction.
648
MCF-7 cells were loaded with 10 µM of the acetoxymethyl ester of SNARF 5 for 10-15 minutes at RT
649
in a 35 x 10 mm tissue culture dish which was also used as bath chamber. Loading efficiency was
650
checked by taking snapshots from time to time in the setup. After loading, cells were constantly
651
perfused with medium at room temperature at a rate of 2 ml/min using a tubing system. The
652
medium had the following composition: 143 mM NaCl, 5 mM KCl, 2 mM CaCl 2, 1 mM MgSO4, 1 mM
653
Na2HPO4, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.2. In lactate-
654
containing media NaCl was substituted by Na-L-lactate in equimolar amounts. To reproducibly
655
measure the rate of lactate-induced acidification, cells were depleted of lactate for at least 15
656
minutes prior to lactate application. For application of lactate, the application tube was switched
657
between different beakers containing the desired solutions.
658
Data analysis was carried out with ImageJ (National Institutes of Health, USA). For analysis a region of
659
interest (ROI) was drawn around each cell (Figure 1 – Figure Supplement 3 A3). The intensity of each
660
ROI was recorded for each frame and the values were stored digitally in a spread sheet.
661
In order to convert the fluorescent ratio into pH values, the system was calibrated by the use of
662
nigericin (10 µM; Life Technologies) as recently described (Forero-Quintero et al., 2017). Nigericin is a
663
microbial toxin derived from Streptomyces hygroscopicus, which functions as a K+/H+ exchanging
664
ionophore (Kovbasnjuk et al., 1991). At high K+ concentrations (130 mM K+ used in this study),
665
nigericine allows to equilibrate intracellular and extracellular pH. For calibration SNARF-5 loaded cells
666
were subsequently superfused with saline, adjusted to pH 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, in
667
the presence of nigericin (Figure 1 – Figure Supplement 3 B). Steady state values were calculated by
668
exponential regression fitting for every single cell (Figure 1 – Figure Supplement 3 B). The fluorescent
669
ratio was blotted against the extracellular pH and the data were fitted using a Boltzmann function: y=
A − A
1+�
�−� ��
+A 27
670
with A1 = initial value, A2 = final value, x0 = center, dx = time constant. The resulting values are
671
given in the inset in Figure 1 – Figure Supplement 3 C.
672
Based on these data, pH values were calculated from the fluorescent ratio (R) for every data point
673
using the formula
674
pH = x + dx ∗ ln
675
A −A R−A
−1 .
The resulting pH values were plotted against the time, as exemplarily shown in Figure 1 A. From the
676
resulting curve the maximum rate of change in pHi ΔpHi/Δt duri g appli atio of la tate i flu a d
677
withdrawal (efflux) of lactate was determined for every single cell by linear regression fitting using
678
OriginPro 8.6.
679
For all imaging experiments the number of repetitions is given as n = number of cells / number of
680
batches.
681 682
Antibody staining of MCF-7 cells
683
MCF-7 cells, growing on glass cover slips, were rinsed twice in phosphate buffered saline (PBS) and
684
fixated in 4% paraformaldehyde in PBS for 20 min. Cells were permeabilized with 0.5% Triton X-100
685
and unspecific binding sites were blocked with 3% bovine serum albumin (BSA) and 1% normal goat
686
serum (NGS) for 2h at room temperature. Cells were incubated with primary antibodies (rabbit anti-
687
CAII polyclonal antibody, AB1828, Millipore, dilution 1:500 and goat anti-MCT1 polyclonal antibody,
688
sc-14917, Santa Cruz, dilution 1:300) overnight at 7°C. Cells were washed with PBS and incubated
689
with the secondary antibody (1:1000; Alexa Fluor 546 donkey anti-rabbit IgG and Alexa Fluor 488
690
donkey anti-goat IgG; Thermo Fisher). Cells were mounted on a microscope slide using mounting
691
ediu
ith DAPI ProLo g™ Gold A tifade Mou ta t ith DAPI, Ther o Fisher a d analyzed with
692
a confocal laser scanning microscope (Leica TCS SP5, Leica Microsystems, Wetzlar, Germany) with a
693
63x objective (HCX PL APO lambda blue 63x 1.4 OIL, Leica Microsystems).
694 695 28
696
In situ proximity ligation assay in MCF-7 cells
697
Colocalization of MCT1 and CAII was examined using the Duolink in situ Proximity Ligation Assay kit
698
(Sigma-Aldrich), as described by the manufacturer and by Söderberg et al., (2008). In short, MCF-7
699
cells, growing on glass coverslips were fixated in 4% paraformaldehyde solution (Roti-Histofi
700
Roth, Karlsruhe, Germany) for 30 min. After fixation, u spe ifi binding sites were blocked with 3%
701
bovine serum albumin (BSA; Sigma-Aldrich) and 1% normal goat serum (NGS; Sigma-Aldrich) for 2
702
hours at RT in a humid chamber. Cells were incubated with primary antibodies (rabbit anti-CAII
703
polyclonal antibody, AB1828, Millipore, dilution 1:200 and goat anti-MCT1 polyclonal antibody, sc-
704
14917, Santa Cruz, dilution 1:200) for 2 hours at room temperature. The following steps were
705
perfor ed a ordi g to the
706
incubated with the PLA probes for one hour at 37°C in a humid chamber. After washing, cells were
707
incubated with ligation-ligase solution. The ligation reaction was carried out at 37°C for 30 min in a
708
humid chamber. After washing, cells were incubated with the amplification-polymerase solution for
709
100 min at 37°C in a darkened humid chamber. For better visualization of the cells, F-actin was
710
stained with Alexa Fluor 488 Phalloidin (1:500; A12379, Life technologies). Cells were washed again
711
and mounted on a microscope slide using the mounting media supplied with the kit. Cells incubated
712
without primary antibodies were used as procedure controls. The resulting staining was visualized
713
using a confocal laser-scanning microscope (LSM 700; Carl Zeiss GmbH, Oberkochen, Germany),
714
equipped with a Zeiss EC Plan-Neofluar 40x/1.3 objective. Images were analyzed and the PLA signals
715
quantified using ImageJ.
%;
a ufa turer s proto ol. Briefl , the ells were washed two times and
716 717
Measurement of cell proliferation
718
120 µl of MCF-7 cell suspension (5x105 cells/ml) was added to 24-well plates, containing 70 µl
719
OptiMEM medium (Thermo Fisher), supplemented either with siRNA (Ambion Silencer® Select anti
720
CA2 siRNA, s2249 or Ambion Silencer® Select Negative Control No. 1 siRNA) and Lipofectamine
721
RNAiMAX transfection reagent (Life Technologies), or with the CA inhibitor EZA (Sigma Aldrich) and 29
722
the MCT1 inhibitor AR-C155858 (Santa Cruz Biotechnology), respectively. Cells were incubated for
723
3.5 h under normoxic conditions in a cell culture incubator. After incubation, the wells were filled up
724
to 0.75 µl with RPMI-1640 medium (Sigma-Aldrich), supplemented with 10% fetal calf serum, 2%
725
Mi i al Eagle s Mediu ,
726
penicillin/streptomycin, pH 7.2, and placed at 37°C in 5% CO2, 95% air (normoxia) or 5% CO2, 1% O2,
727
94% N2 (hypoxia) in humidified cell culture incubators. After addition of RPMI-1640 medium the
728
inhibitors were present at a concentration of 30 µM (EZA) and 300 nM (AR-C155858), respectively.
729
Cells were counted after 0 days (immediately after the 3.5 h incubation period), 1 day, 2 days, and 3
730
days. Cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min. After
731
washing, nuclei were stained with 10 µM of the intercalating fluorescent dye Hoechst 33342
732
(ThermoFisher) for 20 minutes. Pictures were taken with a fluorescent microscope (Leica DM IRB),
733
equipped with a 10x objective (Leica 10x PH1). For every condition four wells were used. Three
734
images were taken from each well at random locations, yielding 12 pictures for every condition
735
(n=12/4). The number of nuclei per image was counted using the program ImageJ. Therefore each
736
picture was converted to a monochrome image (with threshold set to 30 – 255). Fused nuclei were
737
separated
738
set to 0-infinity, circularity set to 0.00-1.00). After counting the number of nuclei per picture was
739
converted to number of nuclei per mm2.
ith the o
.
a d
M hu a
i suli ,
atershed a d ou ted
M glu ose,
ith the o
M NaHCO3, and 1%
a d A al ze Parti les size
740 741
Site directed mutation of CAII
742
Site directed mutation of CAII was carried out by PCR using Phusion High-Fidelity DNA Polymerase
743
(Thermo Fisher) and modified primers, which contained the desired mutation. Primers used for
744
creation of the different mutants are shown in Table 2. Human CAII, cloned in the oocyte expression
745
vector pGEM-He-Juel, was used as template. PCR was cleaned up using the GeneJET PCR Purification
746
Kit (Thermo Fisher) and the template was digested with DpnI (Fermentas FastDigest DpnI, Thermo
747
Fisher) before transformation into E.coli DH5α cells. 30
748
Heterologous protein expression in Xenopus oocytes
749
cDNA coding for human CAII-WT or mutants of CAII, rat MCT1, and rat MCT4, respectively, cloned
750
into the oocyte expression vector pGEM-He-Juel was transcribed in vitro with T7 RNA-Polymerase
751
(mMessage mMachine, Ambion Inc., Austin, USA) as described earlier (Becker et al., 2004; Becker,
752
2014). Xenopus laevis females were purchased from the Radboud University, Nijmegen, Netherlands.
753
Segments of ovarian lobules were surgically removed under sterile conditions from frogs
754
anaesthetized with 1 g/l of ethyl 3-aminobenzoate methanesulfonate (MS-222, Sigma-Aldrich), and
755
rendered hypothermic. The procedure was approved by the Landesuntersuchungsamt Rheinland-
756
Pfalz,
757
Verbraucherschutz und Lebensmittelsicherheit, Oldenburg (33.19-42502-05-17A113). As described
758
earlier (Becker et al., 2004; Becker, 2014), oocytes were singularized by collagenase (Collagenase A,
759
Roche, Mannheim, Germany) treatment in Ca2+-free oocyte saline (pH 7.8) at 28°C for 2 h. The
760
singularized oocytes were left overnight in an incubator at 18°C in Ca2+-containing oocyte saline (pH
761
7.8) to recover. Oocytes of the stages V and VI were injected with 5 ng of cRNA coding for MCT1 or
762
MCT4, either together with 12 ng of cRNA coding for CAII or alone. Measurements were carried out 3
763
to 6 days after injection of cRNA. 4-MI was dissolved in DEPC-H2O at a concentration of 400 mM. 27.6
764
nl of the 4-MI solution were injected on the day of the experiments.
765
The oocyte saline had the following composition: 82.5 mM NaCl, 2.5 mM KCl, 1 mM CaCl 2, 1 mM
766
MgCl2, 1 mM Na2HPO4, 5 mM HEPES; titrated with NaOH to the desired pH. In CO2/HCO3-- and
767
lactate-containing saline, NaCl was substituted by NaHCO3 or Na-L-lactate in equimolar amounts.
Koblenz
(23
177-07/A07-2-003
§6)
and
the
Niedersächsisches
Landesamt
für
768 769
Measurement of intracellular H+ concentration in Xenopus oocytes
770
Changes in intracellular H+ concentration in oocytes were determined with ion-sensitive
771
microelectrodes under voltage-clamp conditions, using single-barreled microelectrodes. For
772
production of the pH-sensitive electrode a borosilicate glass capillary with filament, 1.5 mm in
31
773
diameter, was pulled to a micropipette with a tip opening of 0.5 – 1 µm. To achieve a hydrophobic
774
inner surface, the tip of the micropipette is backfilled with a drop of 5% tri-N-butylchlorosilane in
775
99.9% pure carbon-tetrachloride using a thin glass capillary and baked for 4.5 min at 450°C on a hot
776
plate under a fume hood. Silanized micropipettes were backfilled with a drop (approximately 0.5 µl)
777
of H+-selective cocktail (Hydrogen ionophore I - cocktail A, 95291, Sigma-Aldrich), using a thin glass
778
capillary and stored in a wet chamber for at least 30 min, so that the viscous cocktail can reach the
779
front end of the tip. Afterwards the cocktail is covered with 0.1 M Na-citrate solution, pH 6.0, to form
780
a liquid membrane. The electrode was connected to an amplifier (custom build at the University of
781
Kaiserslatern) with a chloride silver wire. For production of the reference and current electrodes
782
borosilicate glass capillaries with filament, 1.5 mm in diameter, were pulled to micropipettes with a
783
tip opening of 1 – 2 µm, backfilled with 3 M KCl and connected to an Axoclamp 2B amplifier (Axon
784
Instruments). For calibration of the pH-sensitive electrode, it was superfused with oocyte saline,
785
adjusted to pH 7.0, followed by superfusion with saline adjusted to pH 6.4. Calibration of the
786
electrode was carried out before every single experiment. After calibration of the pH-sensitive
787
electrode, the bath perfusion was topped and a single oocyte was placed into the bath. The
788
reference and current clamp electrodes were fist impaled into the oocyte. Then the pH-sensitive
789
ele trode
790
direction of flow of the bath perfusion. Since diffusion of protons within the cell is very slow, it is of
791
major importance to position the pH-sensitive microelectrode as close as possible to the inner face of
792
the plasma membrane in order to measure fast changes in intracellular H+-concentration (Bröer et
793
al., 1998). All experiments were carried out at room temperature (22-25°C). During the whole
794
experiment the oocyte was superfused with oocyte saline at a flow rate of 2 ml/min using a tubing
795
system. For application of lactate and CO2/HCO3-, respectively, the application tube was switched
796
between different beakers containing the desired solutions. The measurements were stored digitally
797
using custom made PC software (Neumann, 2018; copy archived at https://github.com/General-
798
Zoology/iClamp) based on the program LabView (National Instruments). A step by step instruction
as i paled a d positio ed to the fro t of the oo te
e
ra e, dire tl i to the
32
799
for the production of ion-sensitive microelectrodes and their use in Xenopus oocytes can be found in
800
Becker (2014).
801
For calculation of pH, the electrode potential (Ve), recorded during the calibration, was plotted
802
against the pH of the two calibration solutions (pH 7.0 and pH 6.4) and a linear fit was created, using
803
the spread sheet program OriginPro 8.6. Afterwards, the pH values for every recorded data point
804
were calculated using the formula
805
pH = intercept + slope * Ve.
806
Since pH is defined as -log ([H+]), the change in [H+] at a given change in pH depends on the baseline
807
pH, which could lead to misinterpretation of the real change in proton concentration. For this reason
808
proton concentration was calculated using the formula
809
[H+] = 10-pH x 109 (nM)
810
for every recorded data point.
811
Transport activity of the MCT was then determined by measuring the rate of change in [H +]i
812
(Δ[H+]i/Δt) during application or removal of substrate by linear fitting.
813
To make sure that the change in [H+]i during application of lactate is the result of MCT transport
814
activity and does not depend on passive diffusion of lactic acid or endogenous lactate transporters,
815
we previously determined Δ[H+]i/Δt duri g appli atio of la tate i Xenopus oocytes, injected with
816
H2O instead of cRNA (Becker et al., 2004). In H2O-injected oocytes no lactate-induced changes in [H+]i
817
could be observed, indicating that the lactate-induced acidification in MCT-expressing oocytes is the
818
result of MCT-mediated proton-coupled lactate transport (Becker et al., 2004).
819 820
Cal ulation of int insi
821
I tri si
822
carried out at the end of each experiment (for example see Figure 4B . βi was calculated from the
823
ha ge i i tra ellular pH ΔpHi) and the change in intracellular HCO3- concentration (Δ[HCO3-]i)
824
uffer apa it
uffe apa ity (βi) and lactate-induced proton flux (JH) βi) of oocytes was determined from the pulse of 5% CO2 / 10 mM HCO3-,
during application of CO2 using the formula 33
825
βi = ΔpHi / Δ[HCO3-]i (mM).
826
The exact calculations are shown on page three in the Figure Source Data 1 from Figure 5.
827
Since all experiments were carried out in the nominal absence of CO2, the CO2/HCO3--dependent
828
uffer apa it βCO2) was omitted from the calculation.
829
Lactate-induced proton flux (JH) was calculated by multiplying the rate of change in intracellular pH
830
duri g la tate appli atio
831
JH = ΔpH/t βi (mM).
832
The al ulatio of βi and JH is shown on page two in the Figure Source Data 1 from Figure 5.
ΔpH/t
ith βi using the formula
833 834
Pull-down of CAII
835
Pull-down of CAII with GST-fusion proteins of the C-termini of MCT1 and MCT4 has been described in
836
detail previously (Noor et al., 2015). In brief C-termini of MCT1 and MCT4 were cloned into the
837
expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells.
838
Protein expression was induced by addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG). 3
839
hours after induction, cells were harvested, resuspended in phosphate-buffered saline (PBS) and
840
lysed in lysis buffer (PBS, supplemented with 2 mM MgCl2, 1% Triton X-100, and protease inhibitor
841
cocktail tablets from Roche). Bacterial lysates were centrifuged for 15 min at 4°C, 12000 x g and the
842
supernatant, containing the GST-fusion protein (bait protein), was collected for further use. CAII-WT
843
and mutants of CAII, respectively, were expressed in Xenopus oocytes. For each experiment 25
844
oocytes were lysed in lysis buffer. Oocyte lysates were centrifuged for 15 min at 4°C, 12000 x g and
845
the supernatant (prey protein) was collected for further use.
846
The pull-down experiment was carried out using the Pierce GST protein interaction pull-down kit
847
(Thermo Fisher). Briefly, for immobilization of GST-fusion protein, bacterial lysate was added to
848
glutathione agarose and incubated for 2 hours at 4°C with end-over-end mixing. After incubation, the
849
excess bait protein was removed by centrifugation and the beads were washed five times with wash
850
buffer (1 PBS : 1 lysis buffer). 400 µl of oocyte lysate, containing CAII, was added to the column and 34
851
incubated for 2 hours at 4 °C with end-over-end mixing. After incubation, the excess prey protein was
852
removed by centrifugation and the beads were washed five times with wash buffer. Protein was
853
eluted fro
854
To determine the relative amount of GST and CAII, an equal volume of the samples was analyzed by
855
western blotting. GST was detected using a primary anti-GST antibody (dilution 1:400; anti-GST tag
856
mouse monoclonal IgG, no. 05-782, Millipore) and a goat anti-mouse IgG horseradish peroxidase-
857
conjugated secondary antibody (dilution 1:2000; sc-2031, Santa Cruz). CAII was detected using
858
primary anti-CAII antibody (dilution 1:400; rabbit anti-carbonic anhydrase II polyclonal antibody,
859
AB1828, Millipore) and a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody
860
(dilution 1:2000; Santa Cruz). Quantification of the band intensity was carried out with the software
861
ImageJ. To overcome variations in the signal intensity between different blots, signal intensity of
862
each band for CAII was normalized to the signal intensity of the band from the pull-down of CAII-WT
863
with the GST-fusion protein of the C-terminal tail. To account for variations in the amount of GST-
864
fusion protein, each normalized signal for CAII was divided by the corresponding, normalized signal
865
for GST.
the eads ith
μl of elutio
uffer
M glutathio e i PB“, pH . .
866 867
Determination of CA catalytic activity via mass spectrometry
868
Catalytic activity of CAII in Xenopus oocytes was determined by monitoring the
869
doubly labelled 13C18O2 through several hydration and dehydration steps of CO2 and HCO3- at 24°C
870
(Silverman, 1982; Sültemeyer et al., 1990). The reaction sequence of 18O loss from 13C18O18O (m/z =
871
49) over the intermediate product 13C18O16O (m/z = 47) and the end product 13C16O16O (m/z = 45) was
872
monitored with a quadrupole mass spectrometer (OmniStar GSD 320; Pfeiffer Vacuum, Asslar,
873
Germany). The relative 18O enrichment was calculated from the measured 45, 47, and 49 abundance
874
as a function of time according to: log enrichment = log [49x100/(49+47+45)]. For the calculation of
875
CA activity, the rate of
876
over the time, using OriginPro 8.6. The rate was compared with the corresponding rate of the non-
18
18
O depletion of
O degradation was obtained from the linear slope of the log enrichment
35
877
catalyzed reaction. Enzyme activity in units (U) was calculated from these two values as defined by
878
Badger and Price (1989). From this definition, one unit corresponds to 100% stimulation of the non-
879
catalyzed 18O depletion of doubly labelled 13C18O2. For each experiment a batch of 20 native oocytes
880
or 20 oocytes expressing CAII-WT or a mutant of CAII, were lysed in 80 µl oocyte saline, pipetted into
881
the cuvette and the catalyzed degradation was determined for 10 min.
882 883
Calculation and statistics
884
Statistical values are presented as means ±standard error of the mean. Number of repetitions (n) is
885
indicated in each figure panel. For western blot analysis number of technical replicates (blots) /
886
number of biological replicates (cell lysates or pull-down eluates) are separated by a dash. For
887
calculation of significance in differences, Student´s t-test was used. In the figures shown, a
888
sig ifi a e le el of p ≤ .
is
arked ith *, p ≤ .
ith ** a d p ≤ .
ith ***.
889 890 891
Funding
892
The work was funded by the Deutsche Forschungsgemeinschaft (BE 4310/6-1; to H.M.B.), the
893
Stiftung Rheinland-Pfalz für Innovation (961-386261/957; to H.M.B.) the Research Initiative BioComp
894
(to H.M.B. and J.W.D), and the Landesschwerpunkt Membrantransport (to H.M.B. and J.W.D).
895 896
Conflict of interest
897
The authors declare no conflict of interest.
36
898
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899
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900
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901
10.1016/j.bbabio.2003.10.018.
902
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904
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909
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910
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917
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918
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Tables
1125 1126
Table 1: Characteristics of primers used for qRT-PCR Gene CA II RPL27
Primer
Se 5’→ 3’
Tm (°C)
forward reverse forward reverse
AAACAAAGGGCAAGAGTGCTGACT TTTCAACACCTGCTCGCTGCTG GGTGGTTGCTGCCGAAATGGG TGTTCTTCACGATGACAGCTTTGCG
57.2 58.3 58.9 58.6
Amplicon size (bp) 173 101
Location 680-703 831-852 30-50 106-130
1127 1128 1129 1130
Table 2: Characteristics of primers used for single-site mutation of CAII
1131
Shown are the sense primers for single-site mutation CAII. Nucleotides that differ from the wild-type
1132
sequence are labelled in blue. The antisense primers had the inverse complement sequence of the
1133
sense primers. Multiple mutations of CAII were created from single-site mutants, using the primers
1134
given in the table. CAII-H64A, also used in this study, was kindly provided by Dr. Robert McKenna,
1135
University of Florida, Gainesville, U.S.A.
Mutation
Se 5’→ 3’
Tm (°C)
CAII-H3A
CCGAGGATGTCCGCTCACTGGGGGTACGGC
76.3
CAII-H4A
CCGAGGATGTCCCATGCCTGGGGGTACGGC
76.3
CAII-H3A/H4A
CCGAGGATGTCCGCTGCCTGGGGGTACGGC
77.7
CAII-K9A
GGGTACGGCGCACACAACGGACCTGAG
72.6
CAII-H10A
GGGTACGGCAAAGCCAACGGACCTGAG
71.0
CAII-H15A
GGACCTGAGGCCTGGCATAAGGACTTCC
71.0
CAII-H64K
CAACAATGGTAAAGCTTTCAACG
57.1
CAII-E69Q
CATGCTTTCAACGTGCAGTTTGAT
59.3
CAII-D71N
GGAGTTTAATGACTCTCAGG
55.2
CAII-D72N
TTTGATAACTCTCAGGACAAAGCA
57.6
1136
47
1137
Figure Legends
1138
Figure 1. CAII facilitates lactate-induced proton flux in MCF-7 breast cancer cells. (A, B) Original
1139
recordings of lactate-induced changes in intracellular pH (pHi) in normoxic (A) and hypoxic (B) MCF-7
1140
breast cancer cells either treated with negative control siRNA (control, black traces) or CAII-siRNA
1141
(CAII knockdown, blue traces). (C, D ‘ate of ha ge i i tra ellular pH ΔpHi/Δt) during application
1142
(C) and withdrawal (D) of lactate in normoxic and hypoxic MCF-7 breast cancer cells either treated
1143
with negative control siRNA or CAII-siRNA (mean + SEM). Knockdown of CAII results in a significant
1144
reduction of lactate-induced pH change both under normoxic and hypoxic conditions. The black
1145
asterisks above the bars for CAII knockdown cells refer to the corresponding bars of the control cells.
1146
The blue and gray significance indicators above the bars for hypoxic cells refer to the corresponding
1147
bars of normoxic cells. *p≤ .
1148
The following figure supplement is available for figure 1:
1149
Figure 1 - figure supplement 1. Determination of CAII knockdown efficiency in MCF-7 cells.
1150
Figure 1 - figure supplement 2. Influence of pHi on lactate transport.
1151
Figure 1 - figure supplement 3. Calibration of SNARF-5 in MCF-7 cells.
1152
Figure 1 - source data 1. Original dataset for Figure 1.
, **p≤ .
, ***p≤0.001, n.s. no significance; “tude t s t-test.
1153 1154
Figure 2. MCT1 and CAII are co-localized in MCF-7 breast cancer cells. (A) Antibody staining of CAII
1155
(A1) and MCT1 (A2) in MCF-7 cells. (A3) Overlay of the fluorescence signals for MCT1 (red), CAII
1156
(green) and the nuclei marker DAPI (blue). Specificity of primary antibodies was tested by incubating
1157
MCF-7 cells only with secondary antibodies (A4). (B) in situ proximity ligation assay (PLA) of MCT1 and
1158
CAII in MCF-7 breast cancer cells, incubated under normoxia (B1) and hypoxia (B2), respectively, and
1159
normoxic MCF-7 cells in which CAII was knocked down using siRNA (B3). The red dots indicate co-
1160
localization of MCT1 and CAII with a maximum distance of < 40 nm. (B4) Negative control of an in situ
1161
PLA without primary antibodies. For better visualization of the cells, F-actin was stained with
1162
fluorescence labelled phalloidin (green). (C) Quantification of the PLA signals, shown as signals per 48
1163
nucleus (mean + SEM). The significance indicators above the bars refer to the values of the PLA for
1164
normoxic cells. ***p≤ .
1165
The following figure supplement is available for figure 2:
1166
Figure 2 - source data 1. Original dataset for Figure 2.
, n.s. no significance; “tude t s t-test.
1167 1168
Figure 3. CAII supports proliferation of MCF-7 breast cancer cells. (A, B) Staining of nuclei with
1169
Hoechst 33342 (blue) in MCF-7 cells after 3 days in culture under normoxic (A) or hypoxic (B)
1170
conditions. Cells remained either untreated (control; A1, B1), mock-transfected with non-targeting
1171
negative control siRNA (siNeg; A2, B2), transfected with siRNA against CAII (siCAII; A3, B3), incubated
1172
with 30 µM of the CA inhibitor EZA (A4, B4), or incubated with 300 nM of the MCT1 inhibitor AR-
1173
C155858 (A5, B5). (C, D) Total number of nuclei/mm2 in normoxic (C) and hypoxic (D) MCF-7 cell
1174
cultures, kept for 0–3 days under the conditions as described in (A) and (B). For every data point four
1175
dishes of cells were used and three pictures were taken from each dish at random locations, yielding
1176
12 pictures/data point (n = 12/4). The blue asterisks indicate significance in differences between
1177
siCAII and siNeg, the orange asterisks between control and AR-C155858, and the yellow significance
1178
indicators between control and EZA. *p≤ .
1179
test.
1180
The following figure supplement is available for figure 3:
1181
Figure 3 - source data 1. Original dataset for Figure 3.
, **p≤ .
, ***p≤ .
, .s. o sig ifi a e; “tude t s t-
1182 1183
Figure 4. CAII-Glu69 and Asp72 are crucial for the facilitation of MCT1 transport activity. (A)
1184
Structural model of human CAII (PDB-ID: 2CBA; Håkansson et al., 1992). Glu69 and Asp72, which have
1185
been suggested to form a proton-collecting antenna are labelled in red and green, respectively. The
1186
adjacent Asp71 is labelled in gray. His64, the central residue of the intramolecular proton shuttle, is
1187
labelled in yellow. (B) Original recordings of the change in intracellular H+ concentration in oocytes
1188
expressing MCT1 (black trace), MCT1 + CAII-WT (blue trace), MCT1 + CAII-E69Q (red trace), MCT1 + 49
1189
CAII-D71N (gray trace), and MCT1 + CAII-D72N (green trace), respectively, during application of 3 and
1190
10 mM of lactate and application of 5% CO2 / 10 mM HCO3-. (C, D) Rate of change in intracellular H+
1191
o e tratio
Δ[H+]/Δt as i du ed
appli atio
C a d re o al D of
a d
M la tate,
1192
respectively, in oocytes expressing MCT1 (dark gray), MCT1 + CAII-WT (blue), MCT1 + CAII-E69Q
1193
(red), MCT1 + CAII-D71N (light gray), and MCT1 + CAII-D72N (green), respectively (mean + SEM). The
1194
black significance indicators refer to MCT1, the blue significant indicators refer to MCT1 + CAII-WT.
1195
*p≤ .
1196
The following figure supplement is available for figure 4:
1197
Figure 4 - figure supplement 1. CAII-Glu69 and Asp72 are crucial for the facilitation of MCT4
1198
transport activity.
1199
Figure 4 - figure supplement 2. Expression levels of CAII are not altered by single-site mutation.
1200
Figure 4 - source data 1. Original dataset for Figure 4.
, **p≤0.01, ***p≤ .
, n.s. no significance; “tude t s t-test.
1201 1202
Figure 5. Chemical rescue of the interaction between MCT1 and CAII-E69Q/D72N by 4-
1203
methylimidazole. (A) Structural model of human CAII, complexed with 4-methylimidazole (4-MI)
1204
(PDB-ID: 1MOO; Duda et al., 2003). 4-MI binds near His64 or in a moiety between Glu69 and Asp 72.
1205
Inset: Close-up of the binding moiety. 4-MI can bind in two alternative confirmations (orange and
1206
green) between Leu57, Asn67, Glu69, Asp72, and Ile91. (B) Original recordings of the change in
1207
intracellular H+ concentration in oocytes expressing MCT1 (black trace), MCT1 + CAII-WT (blue trace),
1208
MCT1 + CAII-E69Q (red trace), MCT1 + CAII-D71N (gray trace), and MCT1 + CAII-D72N (green trace),
1209
respectively, during application of 3 and 10 mM lactate. All oocytes were injected with 4-MI (30 mM)
1210
the day the measurements were carried out. (C, D) Rate of change in intracellular H+ concentration
1211
Δ[H+]/Δt as i du ed
appli atio
C a d re o al D of
a d
M la tate, respe ti el , i
1212
oocytes expressing MCT1 (dark gray), MCT1 + CAII-WT (blue), MCT1 + CAII-E69Q (red), MCT1 + CAII-
1213
D71N (light gray), and MCT1 + CAII-D72N (green), respectively (mean + SEM). The black significance
1214
indicators refer to MCT1, the blue significance indicators refer to MCT1+CAII. All oocytes were 50
1215
injected with 4-MI (30 mM) the day the measurements were carried out. (E) Lactate-induced proton
1216
flux (JH), as calculated from the rate of change in pHi a d the ells i tri si
1217
supplement 1), in oocytes expressing MCT1 (dark gray), MCT1 + CAII-WT (blue), MCT1 + CAII-E69Q
1218
(red), MCT1 + CAII-D71N (light gray), and MCT1 + CAII-D72N (green), respectively, either injected
1219
with 4-MI or not (mean + SEM). The significance indicators above the bars from 4-MI-injected
1220
oocytes refer to the cells expressing the same proteins without 4-MI. *p≤ .
1221
***p≤ .
1222
The following figure supplement is available for figure 5:
1223
Figure 5 - figure supplement 1. Intrinsic buffer capacity of oocytes.
1224
Figure 5 - figure supplement 2. Chemical rescue of the interaction between MCT4 and CAII-
1225
E69Q/D72N by 4-methylimidazole.
1226
Figure 5 - source data 1. Original dataset for Figure 5.
uffer apa it
βi; figure
, **p≤ .
,
, n.s. no significance; “tude t s t-test.
1227 1228
Figure 6. CAII-Glu69 and Asp72 do not mediate binding between MCT1/4 and CAII. (A, C)
1229
Representative western blots for CAII (upper panel) and GST (lower panel). CAII-WT, CAII-E69Q, CAII-
1230
D71N and CAII-D72N were pulled down with a GST fusion protein of the C-terminal of (A) MCT1 (GST-
1231
MCT1-CT) and (C) MCT4 (GST-MCT4-CT), respectively. As negative control CAII-WT was pulled down
1232
with GST alone. (B, D) Relative intensity of the fluorescent signal for CAII (mean + SEM). The signal
1233
intensity of CAII, pulled down with GST-MCT1/4-CT was set to 100%. The significance indicators refer
1234
to the original values of CAII-WT. ***p≤0.001, n.s. no significance; “tude t s t-test.
1235
The following figure supplement is available for figure 6:
1236
Figure 6 - source data 1. Original dataset for Figure 6.
1237 1238
Figure 7. CAII-Glu69 and Asp72 do not support CAII catalytic activity. (A) Original recording of the
1239
log enrichment (LE) of either a lysate of 20 native oocytes (black trace) or a lysate of 20 oocytes
1240
expressing CAII-WT (blue), CAII-H64A (yellow), CAII-E69Q (red), CAII-D71N (gray), CAII-D72N (green), 51
1241
or the double mutant CAII-E69Q/D72N (turquois). The beginning of the traces shows the rate of
1242
degradation of the 18O-labelled substrate in the non-catalyzed reaction; the black arrow indicates the
1243
addition of oocyte lysate. (B) Enzymatic activity of CA in units/ml (mean + SEM). The black asterisks
1244
refer to the values from native oocytes, the blue significance indicators refer to the values of oocytes
1245
expressing CAII-WT. (C, D) Rate of change in intracellular H+ concentratio
1246
application (C) and removal (D) of 5% CO2 / 10 mM HCO3- in native oocytes and oocytes expressing
1247
CAII-WT, CAII-H64A, CAII-E69Q, CAII-D71N, CAII-D72N or the double mutant CAII-E69Q/D72N (mean
1248
+ SEM). **p≤ .
1249
The following figure supplement is available for figure 7:
1250
Figure 7 - source data 1. Original dataset for Figure 7.
, ***p≤ .
Δ[H+]/Δt as i du ed
, .s. o sig ifi a e; “tude t s t-test.
1251 1252
Figure 8. CAII-His64 mediates binding, but no proton transfer between MCT1 and CAII. (A)
1253
Structural model of the binding between the C-terminal tail of MCT1 (raspberry) and CAII (blue).
1254
Binding is mediated by Glu489 (red) and Glu491 (orange) in the MCT1 C-terminal tail and His64
1255
(yellow) in CAII. (B) Original recordings of the change in intracellular H+ concentration in oocytes
1256
expressing MCT1 (black trace), MCT1 + CAII-WT (blue trace), MCT1 + CAII-H64A (yellow trace), and
1257
MCT1 + CAII-H64K (green trace), respectively, during application of 3 and 10 mM of lactate and
1258
application of 5% CO2 / 10 mM HCO3-. (C, D) Rate of change in intracellular H+ concentration
1259
Δ[H+]/Δt as i du ed
appli atio
C a d re o al D of
a d
M la tate, respe ti el , i
1260
oocytes expressing MCT1 (gray), MCT1 + CAII-WT (blue), MCT1 + CAII-H64A (yellow), and MCT1 +
1261
CAII-H64K (green), respectively (mean + SEM). The black significance indicators refer to MCT1, the
1262
blue significance indicators refer to MCT1 + CAII-WT. (E) Representative western blots for CAII (upper
1263
panel) and GST (lower panel). CAII-WT, CAII-H64A, and CAII-H64K were pulled down with a GST
1264
fusion protein of the C-terminal of MCT1 (GST-MCT1-CT). As negative control CAII-WT was pulled
1265
down with GST alone. (F) Relative intensity of the fluorescent signal for CAII (mean + SEM). The signal
52
1266
intensity of CAII, pulled down with GST-MCT1-CT was set to 100%. The significance indicators refer to
1267
the original values of CAII-WT. ***p ≤ .
1268
The following figure supplement is available for figure 8:
1269
Figure 8 - figure supplement 1. CAII-His64 mediates binding, but no proton transfer between MCT4
1270
and CAII.
1271
Figure 8 - figure supplement 2. Catalytic activity and expression levels of CAII His64 mutants.
1272
Figure 8 - source data 1. Original dataset for Figure 8.
, .s. o sig ifi a e, “tude t s t-test.
1273 1274
Figure 9. The histidine residues in the N-terminus of CAII are not involved in the interaction
1275
between CAII and MCT1. (A) Structural model of human CAII (PDB-ID: 1XEV). His3, His4, His10, Lys9,
1276
and His15, which have been suggested to mediate binding of CAII to various acid/base transporters,
1277
are labelled in green. His64, the biding site for MCT1 and MCT4, is labelled in yellow. (B) Rate of
1278
change in intracellular H+ o e tratio
1279
in oocytes expressing MCT1 (dark gray), MCT1 + CAII-WT (blue), or MCT1 + one of the CAII mutants
1280
(green) (mean + SEM). The black significance indicators refer to MCT1, the blue significant indicators
1281
refer to MCT1 + CAII-WT. ***p≤ .
1282
The following figure supplement is available for figure 9:
1283
Figure 9 - source data 1. Original dataset for Figure 9.
Δ[H+]/Δt as i du ed
appli atio of
a d
M la tate,
. .s. o sig ifi a e; “tude t s t-test.
1284 1285
Figure 10. Carbonic anhydrases function as proton antennae for MCTs in glycolytic cancer cells.
1286
Intracellular CAII (blue circle) is anchored to the C-terminal tail of MCT1/4 (raspberry structure) via
1287
CAII-His64 (yellow spot). This binding brings CAII close enough to the transporter pore to shuttle
1288
protons between transporter and surrounding protonatable residues (orange circles). Proton
1289
shuttling is mediated by CAII-Glu69 and CAII-Asp72 (red and green dots). Under hypoxic conditions
1290
CAIX (green circle) binds to MCT1/4 via their chaperon CD147 (ochre structure) to facilitate the
53
1291
exchange of protons between transporter and extracellular protonatable residues (orange circles) via
1292
its proteoglycan-like (PG) domain in a similar fashion as Glu69 and Asp72 in CAII.
1293
The necessity for this proton antenna derives from the slow diffusion of H+ within the highly buffered
1294
cytosol. Lactate, which is produced from glucose (entering the cell by facilitated diffusion via glucose
1295
transporters (light blue structure)) by glycolysis and subsequent conversion of pyruvate, quickly
1296
reaches the MCT by simple diffusion. Protons, however, which are produced during hydrolysis of ATP
1297
(cell work), diffuse very slow within the cell. To allow fast extrusion of protons and lactate from the
1298
cell, the MCT does not extract H+ directly from the bulk cytosol, but from an intermediate harvesting
1299
compartment made up by protonatable residues at the cell membrane and CAII. Like in the
1300
cytoplasm, diffusion of ions in the extracellular space is restricted. Therefore protons have to be
1301
removed from the extracellular site of the transporter by CAIX and shuttled to protonatable residues
1302
at the extracellular face of the cell membrane, from where they can be released to the extracellular
1303
space. By this non-catalytic mechanism intracellular and extracellular carbonic anhydrases could
1304
cooperate non-enzymatically to facilitate proton-driven lactate flux across the cell membrane of
1305
cancer cells.
1306 1307 1308
Figure Supplement Legends
1309
Figure 1 – Figure Supplement 1. Determination of CAII knockdown efficiency in MCF-7 cells. (A ΔCt
1310
values for CAII minus RPL27 of normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII
1311
(blue bars) or non-targeting negative control siRNA (gray bars) (mean + SEM). *p≤ .
1312
test. (B) Relative change in the RNA level of CAII as given by the 2-ΔΔCt values for CAII in normoxic and
1313
hypoxic MCF-7 cells treated with siRNA against CAII compared to cells treated with non-targeting
1314
negative control siRNA (mean + SEM). (C ‘eprese tati e
1315
actin (lower panel) from of normoxic and hypoxic MCF-7 cells, treated with siRNA against CAII or
1316
non-targeting negative control siRNA. (B) Relative intensity of the fluorescent signal for CAII,
ester
; “tude t s t-
lot for CAII upper pa el a d β-
54
1317
normalized to the signal intensity of β-actin in the same probe (mean + SEM). The significance
1318
indicators above the bars for siCAII refer to the values of siNeg. ***p≤ .
; “tude t s t-test.
1319 1320
Figure 1 – Figure Supplement 2. Influence of pHi on lactate transport. (A) Initial intracellular pH
1321
(pHi), as measured at the beginning of the experiment, of normoxic (left row) and hypoxic (right row)
1322
MCF-7 cells, treated with siRNA against CAII (blue) or non-targeting negative control siRNA (gray)
1323
ea ± “EM . ***p≤ .
; “tude t s t-test. (B-E ΔpHi/Δt duri g appli atio of
M la tate i
1324
normoxic (B, C) and hypoxic (D, E) MCF-7 cells, either treated with negative control siRNA (B, D) or
1325
CAII-siRNA (C, E), as shown in Figure 1 B, plotted against the cells initial pHi. Every dot represents one
1326
individual cell. The red lines represent a linear regression fit.
1327 1328
Figure 1 – Figure Supplement 3. Calibration of SNARF-5 in MCF-7 cells. (A) Confocal image of MCF-7
1329
cells, loaded with SNARF-5. (A1) Signal of the emission fraction below 590 nm. (A2) Signal of the
1330
emission fraction above 590 nm. (A3) Signal of the emission fraction below 590 nm divided by the
1331
signal of the emission fraction above 590 nm. (B) Calibration of the fluorescence ratio in the
1332
presence of nigericin and 130 mM K+, at pH 8.0, 7.5, 7.0, 6.5 and 6.0. An exponential equation was
1333
used to calculate the maximum steady state for each pH application, as indicated by the red traces.
1334
Gray traces indicate the confidence bands (for 95% confidence level). (C) Fluorescent ratio plotted
1335
against the extracellular pH (mean ± SEM). A Boltzmann fit (red) was used to calculate the
1336
parameters of conversion (inset). Gray traces indicate the confidence bands (for 95% confidence
1337
level). pH values are calculated as pH = x + dx ∗ ln
1338
A −A R−A
−1 .
1339
Figure 4 – Figure Supplement 1. CAII-Glu69 and Asp72 are crucial for the facilitation of MCT4
1340
transport activity. (A) Original recordings of the change in intracellular H+ concentration in oocytes
1341
expressing MCT4 (black trace), MCT4 + CAII-WT (blue trace), MCT4 + CAII-E69Q (red trace), MCT4 +
1342
CAII-D71N (gray trace), and MCT4 + CAII-D72N (green trace), respectively, during application of 3 and 55
1343 1344
10 mM lactate and application of 5% CO2 / 10 mM HCO3-. (B, C) Rate of change in intracellular H+ o e tratio
Δ[H+]/Δt as i du ed
appli atio
B a d re o al C of
a d
M lactate,
1345
respectively, in oocytes expressing MCT4 (dark gray), MCT4 + CAII-WT (blue), MCT4 + CAII-E69Q
1346
(red), MCT4 + CAII-D71N (light gray), and MCT4 + CAII-D72N (green), respectively (mean + SEM). The
1347
black significance indicators refer to MCT4, the blue significant indicators refer to MCT4 + CAII-WT.
1348
*p≤ .
, p≤ .
, ***p≤0.001. n.s. o sig ifi a e; “tude t s t-test.
1349 1350
Figure 4 – Figure Supplement 2. Expression levels of CAII are not altered by single-site mutation.
1351
(A) Representative western blot for CAII upper pa el a d β-tubulin (lower panel) from native
1352
oocytes and oocytes expressing CAII-WT, CAII-E69Q, CAII-D71N, and CAII-D72N, respectively. (B)
1353
Relative intensity of the fluorescent signal for CAII (mean + SEM). The signal intensity of CAII-WT was
1354
set to 100%. The significance indicators refer to the original values of CAII-WT. ***p≤ .
1355
sig ifi a e; “tude t s t-test.
, .s. o
1356 1357
Figure 5 – Figure Supplement 1. Intrinsic buffer capacity of oocytes. (A, B) Intrinsic buffer capacity
1358
βi), as measured by application of 5% CO2 / 10 mM HCO3-, in oocytes expressing MCT1 (A) or MCT4
1359
(B) either alone or together with CAII-WT, CAII-E69Q, CAII-D71N, and CAII-D72N, respectively, in the
1360
absence and presence of 4-MI in the cytosol (mean + SEM). *p≤ .
1361
test.
, .s. o sig ifi a e; “tude t s t-
1362 1363
Figure 5 – Figure Supplement 2. Chemical rescue of the interaction between MCT4 and CAII-
1364
E69Q/D72N by 4-methylimidazole. (A, B) Rate of change in intracellular H+ o e tratio
1365
as induced by application (A) and removal (B) of 3 and 10 mM lactate, respectively, in oocytes
1366
expressing MCT4 (dark gray), MCT1 + CAII-WT (blue), MCT4 + CAII-E69Q (red), MCT4 + CAII-D71N
1367
(light gray), and MCT4 + CAII-D72N (green), respectively, in the presence of 4-MI in the cytosol (mean
1368
+ SEM). The black significance indicators refer to MCT4, the blue significant indicators refer to MCT4
Δ[H+]/Δt
56
1369 1370
+ CAII-WT. (C) Lactate-induced proton flux (JH), as calculated from the rate of change in pHi and the ells i tri si
uffer apa it
βi; Figure 4 – Figure Supplement 1). The significance indicators above
1371
the bars from 4-MI-injected oocytes refer to the cells expressing the same proteins without 4-MI.
1372
*p≤ .
, **p≤ .
, ***p≤ .
. .s. o sig ifi a e; “tude t s t-test.
1373 1374
Figure 8 – Figure Supplement 1. CAII-His64 mediates binding, but no proton transfer between
1375
MCT4 and CAII. (A) Structural model of the binding between the C-terminal tail of MCT4 (raspberry)
1376
and CAII (blue). Binding is mediated by Glu431 (orange) and Glu433 (red) in the MCT4 C-terminal tail
1377
and His64 (yellow) in CAII. (B) Original recordings of the change in intracellular H+ concentration in
1378
oocytes expressing MCT4 (black trace), MCT4 + CAII-WT (blue trace), MCT4 + CAII-H64A (yellow
1379
trace), and MCT4 + CAII-H64K (green trace), respectively, during application of 3 and 10 mM of
1380
lactate and application of 5% CO2 / 10 mM HCO3-. (C, D) Rate of change in intracellular H+
1381
o e tratio
Δ[H+]/Δt as i du ed
appli atio
C a d re o al D of
a d
M la tate,
1382
respectively, in oocytes expressing MCT4 (gray), MCT4 + CAII-WT (blue), MCT4 + CAII-H64A (yellow),
1383
and MCT4 + CAII-H64K (green), respectively. The black significance indicators refer to MCT4, the blue
1384
significant indicators refer to MCT4 + CAII-WT (mean + SEM). (E) Representative western blots for
1385
CAII (upper panel) and GST (lower panel). CAII-WT, CAII-H64A, and CAII-H64K were pulled down with
1386
a GST fusion protein of the C-terminal of MCT4 (GST-MCT4-CT). As negative control CAII-WT was
1387
pulled down with GST alone. (F) Relative intensity of the fluorescent signal for CAII (mean + SEM).
1388
The signal intensity of CAII, pulled down with GST-MCT4-CT was set to 100%. The significance
1389
indicators refer to the original values of CAII-WT. ***p≤ .
, .s. o sig ifi a e; “tude t s t-test.
1390 1391
Figure 8 – Figure Supplement 2. Catalytic activity and expression levels of CAII His64 mutants. (A)
1392
Original recording of the log enrichment (LE) of either a lysate of 20 native oocytes (black trace) or a
1393
lysate of 20 oocytes expressing CAII-WT (blue), CAII-H64A (yellow), or CAII-H64K (green). The
1394
beginning of the traces shows the rate of degradation of the
18
O-labelled substrate in the non57
1395
catalyzed reaction; the black arrow indicates the addition of oocyte lysate. (B) Enzymatic activity of
1396
CA in units/ml (mean + SEM). The black asterisks refer to the values from native oocytes, the blue
1397
significance indicators refer to the values of oocytes expressing CAII-WT, the yellow significance
1398
indicators refer to the values of oocytes expressing CAII-H64A. (C) Representative western blot for
1399
CAII upper pa el a d β-tubulin (lower panel) from native oocytes and oocytes expressing CAII-WT,
1400
CAII-H64A, and CAII-H64K, respectively. (D) Relative intensity of the fluorescent signal for CAII (mean
1401
+ SEM). The signal intensity of CAII-WT was set to 100%. The significance indicators refer to the
1402
original values of CAII-WT. ***p≤ .
, .s. o sig ifi a e; “tude t s t-test.
58
Figure 1
B
A Normoxia pHi 0.2
Hypoxia pHi 0.2
control
control
3 min
3 min
CAII knock-down
Hypoxia n=41/3
**
n=54/5 n=46/3
***
control
**
n=39/3 n.s.
*** n.s.
*** CAII knock-down
D 0.22
*** control
CAII knock-down
0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0
Efflux
10 mM Lac
*
Normoxia
n=54/5
***
control
Hypoxia
n=41/3 n=39/3
n=46/3
10 mM Lac
Normoxia
3 mM Lac
3 mM Lac
Influx
10 mM Lac
0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0
3 mM Lac
DpHi/Dt (min-1)
C 0.22
10 mM Lac
DpHi/Dt (min-1)
3 mM Lac
CAII knock-down
***
CAII knock-down
** ***
*** * ***
control CAII knock-down
Figure 1 - Figure Supplement 1
A 6
*
*
B 1.0 n=6/3 0.8
(43 kDa)
D 1.2 1.0
n=6/3
0.8 0.6
0.2 0
***
***
0.4
Normoxia
siCAII
b-Actin
siNeg vs siCAII
siNeg
(30 kDa)
Hypoxia
siCAII
siNeg
Normoxia Hypoxia siNeg siCAII siNeg siCAII
CAII
0.0
Hypoxia
siCAII
C
Normoxia
0.4 0.2
Rel. amount of CAII
0
siCAII
1
siNeg
2
0.6 Normoxia
-DDCt
3
2
DCt
4
siNeg
5
n=6/3
Hypoxia
Figure 1 - Figure Supplement 2
B 0.24
7.8
*** n=54/5
*** 7.6
0.08
7.4
7.5
control CAII knock-down
0.12 0.08
7.6
7.7
7.8
7.9
8.0
0
8.1
7.4
7.5
7.9
8.0
8.1
7.9
8.0
8.1
E 0.24 Normoxia
Hypoxia
0.20
CAII knock-down 0.16
n=46/3
0.12 0.08 0.04 7.4
7.8
initial pHi
C 0.24
0
7.7
7.6
initial pHi
0.20
7.4
n=41/3
0.16
0.04
n=46/3
Normoxia
7.2
0.12
0
Hypoxia
7.5
7.3
0.16
0.04
n=39/3
DpHi/Dt (min-1)
initial pHi
7.7
Hypoxia Control
0.20
n=54/5
DpHi/Dt (min-1)
n=41/3
DpHi/Dt (min-1)
0.20 7.9
D 0.24
Normoxia Control
DpHi/Dt (min-1)
A 8.0
CAII knock-down 0.16
n=39/3
0.12 0.08 0.04
7.5
7.6
7.7
7.8
initial pHi
7.9
8.0
8.1
0
7.4
7.5
7.6
7.7
7.8
initial pHi
Figure 1 - Figure Supplement 3
A1
A2
590nm
30 µM
100 80 60 40
8.0
7.5
30 µM
C 100
10 min
20
7.0
590nm
30 µM
Fluorescent ratio (a.u.)
Fluorescent ratio (a.u.)
B
A3
6.5
6.0 pHe 7.5
Model Equation Reduced Chi-Sqr Adj. R-Square
80
Amplitude
Boltzmann y = A2+(A1-A2)/(1+exp((x-x0)/dx)) 3.78483 0.99943 Value Std.Err. 108.50451 5.02114 A1 14.36576 2.7177 A2 6.84642 0.04354 x0 dx 0.06249 0.53247
60 40
n = 53/4
20 6.0
6.5
7.0 pHe
7.5
8.0
Figure 2
A1
A2
A3
A4
Anti-CAII
Anti-MCT1
Anti-CAII Anti-MCT1 DAPI
no prim Ab DAPI
30 µM
30 µM
30 µM
30 µM
B1
B2
B3
B4
normoxia
hypoxia
CAII-siRNA
no primary antibody
15 µM
15 µM
15 µM
n.s.
Normoxia 15
Hypoxia CAII-siRNA
10
*** n=9/3
0
n=12/4
5
(normoxia)
n=12/4
PLA signals / nucleus
C 20
Ab MCT1 + Ab CAII
n=9/3
*** no prim. Ab
15 µM
Figure 3
Normoxia A2 siNeg
control
Normoxia A3 siCAII
300 µM
300 µM
Hypoxia B2 siNeg
B1 control
number of cells / mm2
C 500
300 µM
400
control siNeg
300
siCAII
200
n = 12/4 n.s.
100
** ***
0 0
EZA AR-C
*
*** *** 1 2 time of incubation (days)
300 µM
Hypoxia B4 EZA
*** *** 3
300 µM
Hypoxia B5 AR-C155858
300 µM
n.s.
Normoxia
Normoxia A5 Normoxia AR-C155858
300 µM
Hypoxia B3 siCAII
300 µM
Normoxia A4 EZA
300 µM
D 500 number of cells / mm2
A1
Hypoxia
300 µM
Hypoxia
n.s.
400
control siNeg
300
siCAII
200
n = 12/4
EZA AR-C
n.s. n.s.
100
*** **
0 0
*** *** 1 2 time of incubation (days)
*** *** 3
Figure 4
B MCT1
A
MCT1 + CAII-WT MCT1 + CAII-E69Q MCT1 + CAII-D71N MCT1 + CAII-D72N
hCAII His64
Zn
+
[H ]i 20 nM 3 min
Glu69 Asp72
Asp71
3 mM Lac
30 0
150
n=10
*** n.s.
n=12
*** 3 mM Lac
60
***
***
120 90
D 180
*** n.s.
n=13
*** n.s.
n.s.
**
*** n.s.
MCT1 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
Efflux
60 30 0
***
***
120 90
n=8 n.s.
n=15
n=13
n=10 n=12
*** * ***
10 mM Lac
150
n=8 n.s.
5% CO2/10 mM HCO3
3 mM
n=15
D[H+]i/Dt (nM/min)
Influx
10 mM Lac
D[H+]i/Dt (nM/min)
C 180
10 mM Lac
*** *
*** *
n.s.
**
*** *
MCT1 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
-
Figure 4 - Figure Supplement 1
A
MCT4 MCT4 + CAII-WT MCT4 + CAII-E69Q MCT4 + CAII-D71N MCT4 + CAII-D72N
+
[H ]i 20 nM 4 min
n.s.
***
**
120
*** n.s.
90
30 0
3 mM Lac
60
***
*** n.s.
*** n.s.
n.s.
** *** n.s.
MCT4 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
150
-
Efflux n=8
n.s.
***
**
120
*** n.s.
90 60 30 0
**
10 mM Lac
n=8
D[H+]i/Dt (nM/min)
150
5% CO2/10 mM HCO3
C 180
Influx
10 mM Lac
D[H+]i/Dt (nM/min)
B 180
10 mM Lactate
3 mM
3 mM Lactate
** n.s.
*** n.s.
n.s.
*
** n.s.
MCT4 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
Figure 4 - Figure Supplement 2
A Native
CAIIWT
CAIIE69Q
CAIID71N
CAIID72N
CAII (30 kDa)
b-Tub (50 kDa) n.s.
rel. amounts of CAII (%)
B 125
n.s.
n.s.
n=2/2 100 50 75 25 0
*** Native
CAIIWT
CAIIE69Q
CAIID71N
CAIID72N
Figure 5
B MCT1 + 4-MI
A
MCT1 + CAII-WT + 4-MI MCT1 + CAII-E69Q + 4-MI MCT1 + CAII-D71N + 4-MI MCT1 + CAII-D72N + 4-MI
hCAII 4-MI Ala64
Asn67
Å
3.2Å
Asp71
16 14 12 10 8 6 4 2 0
7.1Å
Leu57
Asp72
3 mM Lac
D 160 n.s.
n.s.
+ 4-MI
n.s.
3 mM Lac
**
***
**
**
**
n.s.
n.s.
**
n.s.
**
**
D[H+]i/Dt (nM/min)
Influx n=8
10 mM Lac
D[H+]i/Dt (nM/min) JH (mM/min)
E
140 120 100 80 60 40 20 0
4-MI
MCT1 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N Influx n=15
140 120 100 80 60 40 20 0
n=8
n=8
**
n.s.
n=12
n=8
n.s.
n.s.
n=10
**
10 mM Lac
Efflux n=8
n.s.
n.s.
**
+ 4-MI
n.s.
*
**
*
*
n.s.
**
n.s.
*
10 mM Lac
8.6
Å
3.3
+
[H ]i 20 nM 2 min
6.7Å
Å
4.9
3 mM
Å
4-MI
Asp72
C 160
Glu69
8Å Å 3.9
2.
4-MI
3.7
Glu69
6. 6Å
Ile91
Zn
n.s.
*
MCT1 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N n=8
n=8
n=8
n.s.
* n=12
n.s.
**
n.s.
MCT1 +4-MI
+CAII- +4-MI WT
+CAII- +4-MI E69Q
+CAII- +4-MI D71N
+CAII- +4-MI D72N
Figure 5 - Figure Supplement 1
4-MI
control
control
4-MI
control
n.s.
n.s.
n.s.
4-MI
n.s.
*
4-MI
4-MI
control
4-MI
control
4-MI
control
4-MI
control
MCT1 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
n=8
control
n.s.
bi (mM)
*
n.s.
4-MI
*
n.s.
20 18 16 14 12 10 8 6 4 2 0
control
B
n=8-15
4-MI
18 16 14 12 10 8 6 4 2 0
control
bi (mM)
A
MCT4 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
Figure 5 - Figure Supplement 2
JH (mM/min)
n.s.
**
**
n.s.
**
Influx
12
n=8
90 60 30 0
n.s.
** n.s.
**
**
n.s.
** n.s.
**
** n.s.
**
MCT4 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
n.s.
10
n.s.
**
+ 4-MI
120
MCT4 +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
14
**
n.s.
*
***
n.s.
8 6
n.s.
*
n=8
150
10 mM Lac
3 mM
30
n.s.
**
**
**
60
n.s.
n.s.
+ 4-MI
90
0
C
**
Efflux
3 mM
120
n=8
D[H+]i/Dt (nM/min)
150
B 180
Influx
10 mM Lac
D[H+]i/Dt (nM/min)
A 180
n.s.
*
n.s.
n.s.
*
4 2 0
MCT4 +4-MI
+CAII- +4-MI WT
+CAII- +4-MI E69Q
+CAII- +4-MI D71N
+CAII- +4-MI D72N
Figure 6
A
GST +CAIIWT
GST-MCT1-CT +CAII- +CAIIWT E69Q
C +CAIID71N
GST +CAIIWT
+CAIID72N
CAII
CAII
(30 kDa)
(30 kDa)
GST
GST
(30 kDa)
(30 kDa)
125
D 150 n=4/3
n.s. n.s. n.s.
100 75 50 25 0
*** GST +CAIIWT
GST-MCT1-CT +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
rel. amounts of CAII (%)
rel. amounts of CAII (%)
B 1 50
GST-MCT4-CT +CAII- +CAIIWT E69Q
125
n=4/3
n.s.
+CAIID71N
n.s.
+CAIID72N
n.s.
100 75 50 25 0
*** GST +CAIIWT
GST-MCT´4-CT +CAII- +CAII- +CAII- +CAIIWT E69Q D71N D72N
Figure 7
D[H+]i/Dt (nM/min)
C 250 200 150
Influx n=8
***
n.s.
n.s.
n.s.
n.s.
***
***
***
** **
50 Native CAII- CAII- CAII- CAII- CAII- CAIIWT H64A E69Q D71N D72N E69Q D72N
n.s.
n.s.
***
***
n.s.
***
20
D 250 200
n=7
n=6
n=6
n=9
*** ***
10 n=6
100
0
***
30
0
***
***
n=7
CA activity (U/mL)
Native CAII-WT CAII-H64A LE CAII-E69Q 0.1 CAII-D71N 1 min CAII-D72N CAII-E69Q/D72N
n.s.
40
n=11
B
oocyte lysate
D[H+]i/Dt (nM/min)
A
Native CAII- CAII- CAII- CAII- CAII- CAIIWT H64A E69Q D71N D72N E69Q D72N Efflux n=8
***
n.s.
n.s.
n.s.
n.s.
***
***
***
***
150 100
** **
50 0
Native CAII- CAII- CAII- CAII- CAII- CAIIWT H64A E69Q D71N D72N E69Q D72N
Figure 8
A
B MCT1
MCT1-CT
MCT1 + CAII-WT MCT1 + CAII-H64A MCT1 + CAII-H64K
CT
Glu489
Glu491 hCAII His64 +
[H ]i 25 nM 2 min
Zn
3 mM Lac
**
120
** n.s. **
60 30 0
E
MCT1
GST +CAIIWT CAII
(30 kDa)
GST (30 kDa)
10 mM Lac
90
+CAIIWT
** n.s.
+CAIIH64A
GST-MCT1-CT +CAII+CAIIWT H64A
n.s.
***
F
n=8
**
n.s.
**
120
** n.s.
90
**
60 30 0
+CAIIH64K
+CAIIH64K
150
Efflux
MCT1
+CAIIWT
+CAIIH64A
1 50 125
n=4/2
n.s.
100 75 50 25 0
*** GST +CAIIWT
n.s.
**
** n.s.
10 mM Lac
**
5% CO2/10 mM HCO3
3 mM Lac
n=8
D[H+]i/Dt (nM/min)
n.s.
rel. amounts of CAII (%)
150
D 180
Influx
3 mM Lac
D[H+]i/Dt (nM/min)
C 180
10 mM Lac
*** GST-MCT1-CT +CAII- +CAII- +CAIIWT H64A H64K
+CAIIH64K
-
Figure 8 - Figure Supplement 1
A
B MCT4
MCT4-CT
MCT4 + CAII-WT MCT4 + CAII-H64A MCT4 + CAII-H64K
CT
hCAII
Glu431 His64 Glu433
+
[H ]i 25 nM 2 min
Zn
3 mM Lac
120
** n.s. ***
60 30 0
E
MCT4
GST +CAIIWT CAII
(30 kDa)
GST (30 kDa)
10 mM Lac
90
+CAIIWT
** n.s.
+CAIIH64A
GST-MCT4-CT +CAII+CAIIWT H64A
n.s.
***
F
n=8
n.s.
***
**
120
*n.s. **
90
***
60 30 0
+CAIIH64K
+CAIIH64K
150
Efflux
MCT4
10 mM Lac
***
5% CO2/10 mM HCO3
3 mM Lac
***
D[H+]i/Dt (nM/min)
n=8
n.s.
rel. amounts of CAII (%)
150
D 180
Influx
3 mM Lac
D[H+]i/Dt (nM/min)
C 180
10 mM Lac
+CAIIWT
1 50 125
** n.s.
**
+CAIIH64A
+CAIIH64K
n.s.
n=4/2
100 75 50 25 0
*** GST +CAIIWT
n.s.
*** GST-MCT4-CT +CAII- +CAII- +CAIIWT H64A H64K
-
Figure 8 - Figure Supplement 2
A
C
oocyte lysate
Native
CAIIWT
CAIIH64A
CAIIH64K
CAII (30 kDa)
Native CAII-WT CAII-H64A CAII-H64K
b-Tub (50 kDa)
1 min
D
50
CA activity (U/ml)
n=8
40
***
n=7
30 20
n=7
10
*** ***
0
*** *** ***
n=6
Native
CAIIWT
CAIIH64A
CAIIH64K
150
rel. amounts of CAII (%)
B
LE 0.2
125
n=4/3
n.s.
n.s.
CAIIH64A
CAIIH64K
100 75 50 25
***
0 Native
CAIIWT
Figure 9
His3
His15
His64
B 160 140 120 100 80 60 40 20 0
n.s.
n.s.
n.s.
***
***
***
***
n.s.
n.s.
n.s.
***
***
n.s.
***
*** n.s.
*** *** 3 mM Lac
His4
His10 Lys9
10 mM Lactate
hCAII
D[H+]/Dt (nM/min)
A
MCT1+ CAII WT n=18 n=28
CAII H3A n=16
n.s.
***
CAII H4A n=15
n.s.
***
n.s.
***
n.s.
n.s.
***
***
n.s.
***
CAII CAII CAII CAII CAII H10A H15A H3/4A H3/4/10A K9A n=12 n=8 n=9 n=6 n=12
Figure 10
Lac
-
H
Gluc
+
H
+
MCT1/4
GLUT intracellular
H64 E69 D72
Gluc
H glycolysis
H
CAIX
CD 147
extracellular
+ PG
Pyr
-
Lac LDH
-
H
+
H
+
+
CAII
cell work
