{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T13:26:03Z","timestamp":1791293163718,"version":"4.1.0"},"reference-count":50,"publisher":"Oxford University Press (OUP)","issue":"4","license":[{"start":{"date-parts":[[2010,4,1]],"date-time":"2010-04-01T00:00:00Z","timestamp":1270080000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/academic.oup.com\/journals\/pages\/open_access\/funder_policies\/chorus\/standard_publication_model"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2010,4,1]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>The SIBLING family (small integrin-binding ligand N-linked glycoproteins) of mineral-regulating proteins, which includes matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN), contains an acidic serine- and aspartate-rich motif (ASARM). X-linked hypophosphatemia caused by inactivating mutations of the PHEX gene results in elevated mineralization-inhibiting MEPE-derived ASARM peptides. Although the OPN ASARM motif shares 60% homology with MEPE ASARM, it is still unknown whether OPN ASARM similarly inhibits mineralization. In this study we have examined the role of OPN ASARM and its interaction with PHEX enzyme using an osteoblast cell culture model, mass spectrometry, mineral-binding assays, and computational modeling. MC3T3-E1 osteoblast cultures were treated with differently phosphorylated OPN ASARM peptides [with 5 phosphoserines (OpnAs5) or 3 phosphoserines (OpnAs3)] or with control nonphosphorylated peptide (OpnAs0). Phosphorylated peptides dose-dependently inhibited mineralization, and binding of phosphorylated peptides to mineral was confirmed by a hydroxyapatite-binding assay. OpnAs0 showed no binding to hydroxyapatite and did not inhibit culture mineralization. Computational modeling of peptide-mineral interactions indicated a favorable change in binding energy with increasing phosphorylation consistent with hydroxyapatite-binding experiments and inhibition of culture mineralization. Addition of PHEX rescued inhibition of mineralization by OpnAs3. Mass spectrometry of cleaved peptides after ASARM-PHEX incubations identified OpnAs3 as a PHEX substrate. We conclude that OPN ASARM inhibits mineralization by binding to hydroxyapatite in a phosphorylation-dependent manner and that this inhibitor can be cleaved by PHEX, thus providing a mechanistic explanation for how loss of PHEX activity in X-linked hyposphosphatemia can lead to extracellular matrix accumulation of ASARM resulting in the osteomalacia. \u00a9 2010 American Society for Bone and Mineral Research<\/jats:p>","DOI":"10.1359\/jbmr.090832","type":"journal-article","created":{"date-parts":[[2009,9,23]],"date-time":"2009-09-23T15:15:02Z","timestamp":1253718902000},"page":"695-705","source":"Crossref","is-referenced-by-count":172,"title":["Phosphorylation-dependent inhibition of mineralization by osteopontin ASARM peptides is regulated by PHEX cleavage"],"prefix":"10.1093","volume":"25","author":[{"given":"William N","family":"Addison","sequence":"first","affiliation":[{"name":"Faculty of Dentistry, McGill University, Montreal, Quebec, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David L","family":"Masica","sequence":"additional","affiliation":[{"name":"Program in Molecular Biophysics, The Johns Hopkins University, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeffrey J","family":"Gray","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, The John Hopkins University, Baltimore, MD, USA"},{"name":"Program in Molecular Biophysics, The Johns Hopkins University, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc D","family":"McKee","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada"},{"name":"Faculty of Dentistry, McGill University, Montreal, Quebec, 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O-glycosylation sites","volume":"4","author":"Sorensen","year":"1995","journal-title":"Protein Sci."},{"issue":"3","key":"2024020401091905000_bib39","doi-asserted-by":"crossref","first-page":"602","DOI":"10.1006\/bbrc.1997.7702","article-title":"Phosphorylation of osteopontin by Golgi apparatus casein kinase","volume":"240","author":"Lasa","year":"1997","journal-title":"Biochem Biophys Res Commun."},{"issue":"1\u20132","key":"2024020401091905000_bib40","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1016\/0014-5793(96)00136-6","article-title":"Golgi apparatus mammary gland casein kinase: monitoring by a specific peptide substrate and definition of specificity determinants","volume":"382","author":"Lasa-Benito","year":"1996","journal-title":"FEBS Lett."},{"issue":"3","key":"2024020401091905000_bib41","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1096\/fj.02-0473rev","article-title":"One-thousand-and-one substrates of protein kinase 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Biol. Chem."},{"issue":"26","key":"2024020401091905000_bib43","doi-asserted-by":"crossref","first-page":"20197","DOI":"10.1074\/jbc.M909174199","article-title":"Phosphorylation of osteopontin is required for inhibition of vascular smooth muscle cell calcification","volume":"275","author":"Jono","year":"2000","journal-title":"J Biol Chem."},{"key":"2024020401091905000_bib44","article-title":"Role of Phosphate Groups in Inhibition of Calcium Oxalate Crystal Growth by Osteopontin","author":"Hunter","year":"2008","journal-title":"Cells Tissues Organs."},{"issue":"3","key":"2024020401091905000_bib45","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/0169-6009(91)90071-7","article-title":"Differential regulation of the 55 and 44 kDa forms of secreted phosphoprotein 1 (SPP-1, osteopontin) in normal and transformed rat bone cells by osteotropic hormones, growth factors and a tumor promoter","volume":"13","author":"Kasugai","year":"1991","journal-title":"Bone Miner."},{"issue":"2","key":"2024020401091905000_bib46","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1006\/bbrc.2000.4146","article-title":"Flexible Structures of SIBLING Proteins, Bone Sialoprotein, and Osteopontin","volume":"280","author":"Fisher","year":"2001","journal-title":"Biochemical and Biophysical Research Communications."},{"issue":"44","key":"2024020401091905000_bib47","doi-asserted-by":"crossref","first-page":"16083","DOI":"10.1073\/pnas.0607193103","article-title":"Folding of the C-terminal bacterial binding domain in statherin upon adsorption onto hydroxyapatite crystals","volume":"103","author":"Goobes","year":"2006","journal-title":"Proc Natl Acad Sci USA."},{"issue":"1","key":"2024020401091905000_bib48","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/S0006-291X(02)02125-3","article-title":"Inhibition of MEPE cleavage by PHEX","volume":"297","author":"Guo","year":"2002","journal-title":"Biochemical and Biophysical Research Communications."},{"issue":"1","key":"2024020401091905000_bib49","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/j.bone.2004.09.015","article-title":"Surface plasmon resonance (SPR) confirms that MEPE binds to PHEX via the MEPE-ASARM motif: a model for impaired mineralization in X-linked rickets (HYP)","volume":"36","author":"Rowe","year":"2005","journal-title":"Bone."},{"issue":"2","key":"2024020401091905000_bib50","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1006\/jmbi.1999.3492","article-title":"Structure of human neutral endopeptidase (Neprilysin) complexed with phosphoramidon","volume":"296","author":"Oefner","year":"2000","journal-title":"J Mol Biol."}],"container-title":["Journal of Bone and Mineral Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1359%2Fjbmr.090832","content-type":"unspecified","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/academic.oup.com\/jbmr\/article-pdf\/25\/4\/695\/56579923\/jbmr90832.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/jbmr\/article-pdf\/25\/4\/695\/56579923\/jbmr90832.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,3,13]],"date-time":"2024-03-13T14:53:22Z","timestamp":1710341602000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/jbmr\/article\/25\/4\/695-705\/7600358"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2010,4,1]]},"references-count":50,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2010,4,9]]},"published-print":{"date-parts":[[2010,4,1]]}},"URL":"https:\/\/doi.org\/10.1359\/jbmr.090832","relation":{},"ISSN":["0884-0431","1523-4681"],"issn-type":[{"value":"0884-0431","type":"print"},{"value":"1523-4681","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2010,4,1]]},"published":{"date-parts":[[2010,4,1]]}}}