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Biochem Biophys Res Commun
2020 Sep 03;5294:1061-1065. doi: 10.1016/j.bbrc.2020.06.068.
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Identification of the essential extracellular aspartic acids conserved in human monocarboxylate transporters 1, 2, and 4.
Yamaguchi A
,
Narumi K
,
Furugen A
,
Iseki K
,
Kobayashi M
.
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Human monocarboxylate transporters (hMCTs) 1-4 transport monocarboxylates, such as l-lactate and pyruvate, as well as H+ across the plasma membrane. hMCT1, 2, and 4 play important roles in energy balance, pH homeostasis. However, the molecular mechanism of these transporters, especially their pH dependency, remains unknown. The aim of this study was to identify the residues involved in the pH dependence of hMCT1, 2, and 4. Firstly, we focused on the effects of extracellular acids of hMCT1. l-Lactate uptake assay and site-directed mutagenesis revealed that the aspartic acid of hMCT1 (hMCT1 D414) was an important residue conserved in MCT1, 2, and 4 (hMCT2 D398 and hMCT4 D379). Because the functional characteristic of hMCT2-mediated l-lactate transport has not been reported, we built a hMCT2-expressing system using Xenopus laevis oocytes. The transport activity of hMCT2 was enhanced by co-expression with embigin, an ancillary protein, and kinetic analysis of hMCT2-mediated l-lactate uptake revealed that the apparent Km value (0.32 ± 0.02 mM) was lower than that mediated by hMCT1 and 4. Finally, we investigated the conserved aspartic acids of hMCT2 and 4, and revealed that these residues were essential for l-lactate transport. These findings suggested that the extracellular aspartic acids conserved in hMCT1, 2, and 4 played important roles in transport activity and pH dependency, and can function as a first step of substrate and H+ recognition and transport from the extracellular to the intracellular region. These findings contributed to enhance our understanding of the transport process of hMCT1, 2, and 4.
Fig. 1. pH dependence of hMCT1 glutamic acid and aspartic acid mutants. (A, B) The effect of extracellular pH on hMCT1 mutants was assessed after 10 min of incubation with standard buffer at pH 5.5, 6.5, and 7.5 in 1 μM l-lactate. (C) Data were obtained from panel B. All data are mean values ± standard error (S.E.) from three independent experiments, each performed with three to five technical replicates.
Fig. 2. Sequence alignment of MCT1, 2, and 4. Amino acid sequences of MCTs were aligned using the EMBL-EBI web server [21]. Glutamic acids are highlighted in orange, and aspartic acids are highlighted in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Characterization of hMCT2-expressing oocyte. (A) Time course of l-lactate uptake via hMCT2 WT, hMCT2 WT + EMB, EMB, and Water were measured after incubation for the indicated time periods with standard buffer containing 1 μM l-lactate at pH 5.5. (B) The concentration dependence of l-lactate uptake via hMCT2 + EMB was assayed at the indicated concentrations for 10 min. Kinetic analysis was performed by fitting the Michaelis-Menten equation. Eadie-Hofstee plot of the date is exhibited in inset, where the l-lactate uptake rate (V) was plotted against V/[l-lactate] (V/S). (C) The pH dependence of hMCT2 + EMB was measured after 10 min of incubation with standard buffer at pH 5.5, 6.5, and 7.5 added in 1 μM l-lactate. All data are mean values ± S.E. from three independent experiments, each performed with three to five technical replicates.
Fig. 4. pH dependence of hMCT2 and 4 aspartic acid mutants. (A, B) The effect of extracellular pH on hMCT2 and 4 mutants was assessed after 10 min of incubation with standard buffer at pH 5.5, 6.5, and 7.5 added in 1 μM l-lactate. All data are mean values ± S.E. from three independent experiments, each performed with three to five technical replicates.
Fig. S1. 2-Dimensional topology model of hMCT1. 2-Dimensional topology model of hMCT1 with extracellular glutamic acids and aspartic acids highlighted in orange- and red-color, respectively, was visualized using the PROTTER web server. The transmembrane domains were predicted using the CCTOP web server.
Fig. S2. Membrane localization of hMCT1, 2, and 4, and embigin in <span data-format="italic">Xenopus laevis </span>oocytes. The expression of the indicated cRNA in oocytes was examined by fluorescence microscopy using an anti-hMCT1 antibody (A), anti-hMCT2 antibody (B, D), anti-embigin antibody (C), anti-hMCT4 antibody (E).