Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brazil.
J Bone Miner Res. 2013 Mar;28(3):688-99. doi: 10.1002/jbmr.1766.
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.
X 连锁低磷血症（XLH/HYP）-伴有肾磷酸盐丢失、低磷血症、佝偻病和牙脓肿-是由锌金属肽酶 PHEX 基因（与 X 染色体上的内肽酶同源的磷酸盐调节基因）的突变引起的。PHEX 在矿化组织细胞中高度表达。PHEX 的失活突变导致远端肾脏效应（暗示分泌、循环的磷酸盐排泄因子的积累）和在骨骼和牙齿中积累矿化抑制、富含酸性丝氨酸和天冬氨酸的基序（ASARM）肽，这些肽是从 SIBLING 家族（小，整合素结合配体 N-连接糖蛋白）的矿化结合基质蛋白中蛋白水解衍生而来的。尽管后一种观察表明 PHEX 具有局部、直接的基质效应，但尚未鉴定其生理相关的底物蛋白。在这里，我们研究了两个含有 ASARM 基序的 SIBLING 蛋白-骨桥蛋白（OPN）和骨涎蛋白（BSP）-作为 PHEX 的潜在底物。通过切割试验、凝胶电泳和质谱分析，我们报告 OPN 是 PHEX 的全长蛋白底物。OPN 的降解基本完全，包括 ASARM 基序的水解，仅产生非常小的残留片段。没有 PHEX 活性的 Hyp（人类 XLH 的鼠同源物）鼠骨提取物的 Western 印迹清楚地显示出一种约 35 kDa 的 OPN 片段的积累，该片段不存在于野生型鼠骨中。Hyp 骨中的 OPN 的免疫组织化学和免疫金标记（电子显微镜）同样显示出 OPN 和/或其片段的积累与正常野生型骨相比。将 Hyp 鼠骨提取物与 PHEX 孵育可导致这些片段的完全降解。总之，这些结果确定全长 OPN 及其片段是 PHEX 的新型、生理相关的底物，表明矿化抑制的 OPN 片段的积累可能导致 XLH/HYP 特征性佝偻病骨骼中的矿化缺陷。
