MDPI Acta Medica Okayama 2218-273X 11 4 2021 Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target 510 EN Maho Yamamoto Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Rina Kondo Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Haruka Hozumi Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Okayama University Seita Doi Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Miwako Denda Cell Free Sciences Co., Ltd. Masaki Magari Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoki Kanayama Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Naoya Hatano Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University Ryo Morishita Cell Free Sciences Co., Ltd. Hiroshi Tokumitsu Applied Cell Biology, Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-Delta C) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation. No potential conflict of interest relevant to this article was reported.