Structure and function of disease-causing missense mutations in the PHEX gene
Structure and function of disease-causing missense mutations in the PHEX gene
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DOI：
10.1210/jc.2002-021809
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发表时间：
2003-05-01
影响因子：
5.8
通讯作者：
Tenenhouse, HS
中科院分区：
文献类型：
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作者：
Sabbagh, Y;Boileau, G;Tenenhouse, HS
在X连锁低磷血症患者中突变的phex基因编码一种与锌金属肽酶M13家族同源的蛋白质。本研究旨在评估9个PHEX错义突变对细胞转运、内肽酶活性和蛋白质构象的影响。通过PCR诱变产生野生型和突变型PHEX蛋白的分泌型，其中包括XLH型患者中发现的C85R、D237G、Y317F、G579R、G579V、S711R、A720T和F731Y，以及在中性内肽酶24.11中取消催化活性但不能定位质膜的E581V。野生型和D237G、Y317F、E581V和F731Y蛋白被末端糖基化并分泌到培养基中，而C85R、G579R、G579V、S711R和A720T蛋白被困在细胞内。在26℃的温度下培养细胞可以分泌G579V、S711R和A720T蛋白，尽管挽救的G579V的产量不足以进行进一步分析。利用一种新的内部猝灭的荧光肽底物对分泌和挽救的PHEX蛋白的内肽酶活性进行了评估，结果显示E581V和S711R完全失活，D237G和Y317F表现出50-60%的野生型活性，A720T和F731Y保持完全催化活性。限制性蛋白水解法的构象分析表明，F731Y对胰酶更敏感，而D237G比野生型蛋白更能抵抗内切酶Glu-c。因此，在携带这些错义突变的XLH患者中，蛋白质运输、内肽酶活性和蛋白质构象方面的缺陷导致了PHEX功能的丧失。
The PHEX gene that is mutated in patients with X-linked hypophosphatemia (XLH) encodes a protein homologous to the M13 family of zinc metallopeptidases. The present study was undertaken to assess the impact of nine PHEX missense mutations on cellular trafficking, endopeptidase activity, and protein conformation. Secreted forms of wild-type and mutant PHEX proteins were generated by PCR mutagenesis; these included C85R, D237G, Y317F, G579R, G579V, S711R, A720T, and F731Y identified in XLH patients, and E581V, which in neutral endopeptidase 24.11 abolishes catalytic activity but not plasma membrane localization. The wild-type and D237G, Y317F, E581V, and F731Y proteins were terminally glycosylated and secreted into the medium, whereas the C85R, G579R, G579V, S711R, and A720T proteins were trapped inside the transfected cells. Growing the cells at 26 C permitted the secretion of G579V, S711R, and A720T proteins, although the yield of rescued G579V was insufficient for further analysis. Endopeptidase activity of secreted and rescued PHEX proteins, assessed using a novel internally quenched fluorogenic peptide substrate, revealed that E581V and S711R are completely inactive; D237G and Y317F exhibit 50-60% of wild-type activity; and A720T and F731Y retain full catalytic activity. Conformational analysis by limited proteolysis demonstrated that F731Y is more sensitive to trypsin and D237G is more resistant to endoproteinase Glu-c than the wild-type protein. Thus, defects in protein trafficking, endopeptidase activity, and protein conformation account for loss of PHEX function in XLH patients harboring these missense mutations.