期刊
CELLULAR AND MOLECULAR LIFE SCIENCES
卷 75, 期 8, 页码 1461-1482出版社
SPRINGER BASEL AG
DOI: 10.1007/s00018-017-2699-2
关键词
Crystal adhesion; Crystal internalization; Immuno-co-localization; Interactomics; IP-MS; Kidney stone; Renal calculi; Renal tubular cells
资金
- Mahidol University research grant
- Office of the Higher Education Commission
- Mahidol University under the National Research Universities Initiative
- Thailand Research Fund [IRN60W0004, IRG5980006]
- Siriraj Graduate Thesis Scholarship
- Research Staff Grant
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.
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