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Structure and Function of Disease-Causing Missense Mutations in the PHEX Gene

Departments of Biology (Y.S., H.S.T.), Pediatrics (H.S.T.), and Human Genetics (H.S.T.), McGill University, and The McGill University–Montreal Children’s Hospital Research Institute (Y.S., H.S.T.), Montreal, Quebec, Canada H3Z 2Z3; Department of Biochemistry (G.B.), Université de Montréal, Montréal, Quebec H3C 3J7, Canada; and Department of Biophysics (M.C., A.K.C.), Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, Brazil

Address all correspondence and requests for reprints to: Dr. Harriet S. Tenenhouse, Montreal Children’s Hospital Research Institute, 4060 Ste-Catherine Street West, Room 222, Montreal, Quebec, Canada H3Z 2Z3. E-mail: mdht{at}debelle.mcgill.ca.

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.

This work was supported by grants from the Canadian Institutes of Health Research (MT-14107 to H.S.T. and MT-13052 to G.B.). Yves Sabbagh is the recipient of Studentship Awards from the Canadian Institutes of Health Research and FRSQ-FCAR-Santé.

Abbreviations: Abz, Aminobenzoic acid; Dnp, dinitrophenyl; ECE, endothelin-converting enzyme; endo H, endoglycosidase H; ER, endoplasmic reticulum; NEP, neutral endopeptidase 24.11; secPHEX, secreted form of the wild-type PHEX protein; XLH, X-linked hypophosphatemia.

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