title: Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia.
abstract: X-linked hypophosphatemia (XLH/HYP)-with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscesses-is caused by mutations in the zinc-metallopeptidase PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X chromosome). PHEX is highly expressed by mineralized tissue cells. Inactivating mutations in PHEX lead to distal renal effects (implying accumulation of a secreted, circulating phosphaturic factor) and accumulation in bone and teeth of mineralization-inhibiting, acidic serine- and aspartate-rich motif (ASARM)-containing peptides, which are proteolytically derived from the mineral-binding matrix proteins of the SIBLING family (small, integrin-binding ligand N-linked glycoproteins). Although the latter observation suggests a local, direct matrix effect for PHEX, its physiologically relevant substrate protein(s) have not been identified. Here, we investigated two SIBLING proteins containing the ASARM motif-osteopontin (OPN) and bone sialoprotein (BSP)-as potential substrates for PHEX. Using cleavage assays, gel electrophoresis, and mass spectrometry, we report that OPN is a full-length protein substrate for PHEX. Degradation of OPN was essentially complete, including hydrolysis of the ASARM motif, resulting in only very small residual fragments. Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts having no PHEX activity clearly showed accumulation of an ∼35 kDa OPN fragment that was not present in wild-type mouse bone. Immunohistochemistry and immunogold labeling (electron microscopy) for OPN in Hyp bone likewise showed an accumulation of OPN and/or its fragments compared with normal wild-type bone. Incubation of Hyp mouse bone extracts with PHEX resulted in the complete degradation of these fragments. In conclusion, these results identify full-length OPN and its fragments as novel, physiologically relevant substrates for PHEX, suggesting that accumulation of mineralization-inhibiting OPN fragments may contribute to the mineralization defect seen in the osteomalacic bone characteristic of XLH/HYP.
authors.affiliation: Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil. nilana.barros@unifesp.br
authors.forename: Nilana M T
authors.initials: NM
authors.lastname: Barros
authors.forename: Betty
authors.initials: B
authors.lastname: Hoac
authors.forename: Raquel L
authors.initials: RL
authors.lastname: Neves
authors.forename: William N
authors.initials: WN
authors.lastname: Addison
authors.forename: Diego M
authors.initials: DM
authors.lastname: Assis
authors.forename: Monzur
authors.initials: M
authors.lastname: Murshed
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.forename: Marc D
authors.initials: MD
authors.lastname: McKee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism*; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel; D053098:Familial Hypophosphatemic Rickets / Q000378:metabolism*; D040181:Genetic Diseases, X-Linked; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D053495:Osteopontin / Q000737:chemistry / Q000378:metabolism*; D053402:PHEX Phosphate Regulating Neutral Endopeptidase / Q000378:metabolism*; D059748:Proteolysis
chemical_list: D053495:Osteopontin; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506232:Phex protein, mouse