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. ß

Retinoic acid-induced premature osteoblast-to-preosteocyte transitioning has multiple effects on calvarial development

“…Our data are consistent with recent comparative evolutionary analyses revealing that Spp1 only emerged together with bone mineralization (Venkatesh et al, 2014), which is under control of preosteocytes, rather than osteoblasts (Dallas and Bonewald, 2010;Franz-Odendaal et al, 2006). Nevertheless, an anti-mineralizing role of Spp1 in (mineralizing) preosteocytes might at first sight be surprising, in particular as its 'partner', the endopeptidase Phex, is known to degrade it (Barros et al, 2013). However, it could be regarded as an intrinsic negative-feedback response, similar to the commonly observed induction of cyp26 expression in response to RA signals (Laue et al, 2008;Loudig et al, 2005).…”

Family with sequence similarity member 20C is the primary but not the only kinase for the small‐integrin‐binding ligand N‐linked glycoproteins in bone

“…4A), indicating that this pSer was not phosphorylated by FAM20C. Another study reported several pSers located in non-Ser-X-Glu motifs in BSP (pSer 9 in Ser-Ile-Leu and pSer 18 in SerMet-Lys) (35), which appear unlikely to be phosphorylated by FAM20C. Unfortunately, we were not able to evaluate their phosphorylation status in the KO mice because of the limited MS coverage.…”

“…The ASARM peptides in SIBLINGs are believed to inhibit biomineralization through binding to the hydroxyapatite, and an excess of phosphorylated ASARM peptides derived from the overexpressed OPN in Hyp mice is thought to be associated with hypophosphatemic rickets (17)(18)(19). Although the MS analyses in this study did not retrieve the ASARM peptides from OPN, our MS data of BSP derived from Fam20C-KO mice compared with results reported in a previous study of BSP derived from WT mice (3), as well as our MS analyses of DMP1 derived from both WT and Fam20C-KO mice, clearly showed pSer loss in the ASARM motifs in these SIBLINGs (Fig.…”

“…Supramolecular assembly studies have shown that the phosphorylation of DMP1 significantly affects the size and direction of apatite crystal growth (7,16). In addition, a mineral-inhibiting functional domain identified in bone NCPs-the acidic Ser-and aspartate-rich motif (ASARM)-is believed to bind to hydroxyapatite and inhibit mineralization on a dose-dependent basis (17)(18)(19) and to modulate bone mineralization through phosphaturic effects by binding to and inhibiting a protein encoded by phosphateregulating gene with homologies to endopeptidase on the X chromosome (PHEX) (19,20). The hunt for the kinase that phosphorylates SIBLINGs has continued for several decades.…”

A materials science vision of extracellular matrix mineralization

“…For other mineralization inhibitors, such as Matrix Extracellular Phosphoglycoprotein (MEPE) and the peptide Acidic Serine and Aspartate-Rich Motif (ASARM) found in SIBLING proteins, the reader can be referred to excellent recent reviews 27,[37][38][39] . 40 …”