Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia
Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia
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DOI：
10.1002/jbmr.1766
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发表时间：
2013-03-01
影响因子：
6.2
通讯作者：
McKee, Marc D.
中科院分区：
文献类型：
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作者：
Barros, Nilana M. T.;Hoac, Betty;McKee, Marc D.
由锌金属肽酶PHEX基因(磷酸盐调节基因，与X染色体上的内肽酶同源)突变引起的X连锁低磷血症(XLH/HYP)合并肾磷酸盐消耗、低磷血症、骨软化和牙齿脓肿。矿化组织细胞高度表达PHEX。PHEX基因的失活突变会导致远端肾脏效应(意味着一种分泌的循环磷酸因子的积累)，并在骨骼和牙齿中积累含有矿化抑制、酸性丝氨酸和富含天冬氨酸的基序(ASARM)的多肽，这些多肽是从兄弟家族的矿物结合基质蛋白(小的整合素结合配体N-连接糖蛋白)蛋白水解性衍生出来的。尽管后一种观察结果提示了PHEX的局部、直接的基质效应，但其生理相关底物蛋白(S)尚未被鉴定。在这里，我们研究了两种同胞蛋白，它们包含ASARM基序骨桥蛋白(OPN)和骨涎蛋白(BSP)作为PHEX的潜在底物。利用切割分析、凝胶电泳法和质谱法，我们报道了OPN是PHEX的全长蛋白质底物。OPN的降解基本完成，包括ASARM基序的水解，导致只有很小的残留片段。对没有PHEX活性的Hyp(人XLH的小鼠同系物)小鼠骨提取液的Western blotting显示，野生型小鼠骨中没有积累大约35 kDa的OPN片段。免疫组织化学和免疫金标记法(电子显微镜)显示，与正常野生型骨相比，Hyp骨中OPN和/或其片段也有积聚。将Hyp小鼠骨提取液与苯丙氨酸孵育后，这些片段完全降解。综上所述，这些结果确定全长OPN及其片段是PHEX的新的、生理上相关的底物，提示矿化抑制OPN片段的积累可能与XLH/HYP特征的骨软化骨的矿化缺陷有关。(C)2013年美国骨与矿物研究学会。
X-linked hypophosphatemia (XLH/HYP)with renal phosphate wasting, hypophosphatemia, osteomalacia, and tooth abscessesis 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 motifosteopontin (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 approximate to 35kDa 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. (c) 2013 American Society for Bone and Mineral Research.