Characterizations of PMCA2-interacting complex and its role as a calcium oxalate crystal-binding protein
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Three isoforms of plasma membrane Ca2+-ATPase (PMCA) are expressed in the kidney. While PMCA1 and PMCA4 play major role in regulating Ca2+ reabsorption, the role for PMCA2 remains vaguely defined. To define PMCA2 function, PMCA2-interacting complex was characterized by immunoprecipitation followed by nanoLC-ESI-Qq-TripleTOF MS/MS (IP-MS). After subtracting non-specific binders using isotype-controlled IP-MS, 474 proteins were identified as PMCA2-interacting partners. Among these, eight were known and 20 were potential PMCA2-interacting partners based on bioinformatic prediction, whereas other 446 were novel and had not been previously reported/predicted. Quantitative immuno-co-localization assay confirmed the association of PMCA2 with these partners. Gene ontology analysis revealed binding activity as the major molecular function of PMCA2-interacting complex. Functional validation using calcium oxalate monohydrate (COM) crystal-protein binding, crystal-cell adhesion, and crystal internalization assays together with neutralization by anti-PMCA2 antibody compared to isotype-controlled IgG and blank control, revealed a novel role of PMCA2 as a COM crystal-binding protein that was crucial for crystal retention and uptake. In summary, a large number of novel PMCA2-interacting proteins have been defined and a novel function of PMCA2 as a COM crystal-binding protein sheds light onto its involvement, at least in part, in kidney stone pathogenesis.
KeywordsCrystal adhesion Crystal internalization Immuno-co-localization Interactomics IP-MS Kidney stone Renal calculi Renal tubular cells
We thank Phornpimon Tipthara and Kedsarin Fong-ngern for their technical assistance. This study was supported by Mahidol University research grant, Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative, and the Thailand Research Fund (IRN60W0004 and IRG5980006). AV is supported by Siriraj Graduate Thesis Scholarship, whereas VT is supported by “Research Staff” Grant.
AV and VT designed research; AV performed experiments; AV and VT analyzed data; AVand VT wrote the manuscript; all authors reviewed the manuscript.
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Conflict of interest
The authors declare no conflict of interest.
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