Characterization of PHEX endopeptidase catalytic activity:: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors
Characterization of PHEX endopeptidase catalytic activity:: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors
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DOI：
10.1042/bj3550707
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发表时间：
2001-05-01
影响因子：
4.1
通讯作者：
Crine, P
中科院分区：
文献类型：
--
作者：
Boileau, G;Tenenhouse, HS;Crine, P
PHEX基因（与bar染色体上（X）上的内肽酶具有同源性的磷酸盐调节基因）的突变是X连锁低磷酸盐血症的原因，在人类疾病Hyp小鼠模型中的研究表明，该基因产物与肾磷酸盐（P-i）重吸收和骨矿化的调节有关。尽管PHEX作用的机制尚不清楚，但与内肽酶M13家族成员的结构同源性表明PHEX蛋白在参与控制肾β-i转运和基质矿化的肽因子的活化或降解中具有功能。为了确定PHEX是否具有内肽酶活性，我们产生了重组可溶性分泌形式的人PHEX（secPHEX），并用几种肽底物（包括各种骨相关肽）测试了纯化蛋白的活性。我们发现甲状旁腺激素相关肽（107-139）是secPHEX的底物，并且该酶在肽内的三个位置切割，所有位置都位于天冬氨酸残基的N-末端。此外，我们表明，骨钙素，PPi和P-i，所有这些都是丰富的骨，是secPHEX活性的抑制剂。骨钙素对secPHEX活性的抑制作用在Ca 2+存在下被消除。我们认为，PHEX的活性和矿化可能在体内由PPi/P-i和Ca 2+控制，在后一种情况下，调节需要骨钙素的参与。
Mutations in the PHEX gene (phosphate regulating gene with homologies to endopeptidases on the (X) over bar chromosome) are responsible for X-linked hypophosphataemia, and-studies in the Hyp mouse model of the human disease implicate the gene product in the regulation of renal phosphate (P-i) reabsorption and bone mineralization. Although the mechanism for PHEX action is unknown, structural homologies with members of the M13 family of endopeptidases suggest a function for PHEX protein in the activation or degradation of peptide factors involved in the control of renal P-i transport and matrix mineralization. To determine whether PHEX has endopeptidase activity, we generated a recombinant soluble, secreted form of human PHEX (secPHEX) and tested the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hormone-related peptide(107-139) is a substrate for secPHEX and that the enzyme cleaves at three positions within the peptide, all located at the N-terminus of aspartate residues. Furthermore, we show that osteocalcin, PPi and P-i, all of which are abundant in bone, are inhibitors of secPHEX activity. Inhibition of secPHEX activity by osteocalcin was abolished in the presence of Ca2+. We suggest that PHEX activity and mineralization may be controlled in vivo by PPi/P-i and Ca2+ and, in the latter case, the regulation requires the participation of osteocalcin.