Human Molecular Genetics, 2001, Vol. 10, No. 15 1539-1546
© 2001 Oxford University Press
Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein
1Department of Biology, 2Department of Pediatrics and 3Department of Human Genetics, McGill University, Montreal, Quebec, Canada, 4The McGill University - Montreal Children’s Hospital Research Institute, Montreal, Quebec, Canada and 5Department of Biochemistry, Université de Montréal, Montreal, Quebec, Canada
PHEX is homologous to the M13 zinc metallopeptidases, a class of type II membrane glycoproteins. Although more than 140 mutations in the PHEX gene have been identified in patients with X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets, the molecular consequences of disease-causing PHEX mutations have not yet been investigated. We examined the effect of PHEX missense mutations on cellular trafficking of the recombinant protein. Four mutant PHEX cDNAs were generated by PCR mutagenesis: C85R, G579R and S711R, identified in XLH patients, and E581V, previously engineered in neutral endopeptidase 24.11, where it abolished catalytic activity but not plasma membrane targeting. Wild-type and mutant PHEX cDNAs were transfected in HEK(293) cells and PHEX protein expression was characterized. In contrast to the wild-type and E581V PHEX proteins, the C85R, G579R and S711R mutants were completely sensitive to endoglycosidase H digestion, indicating that they were not fully glycosylated. Sequestration of the disease-causing mutant proteins in the endoplasmic reticulum (ER) and plasma membrane localization of wild-type and E581V PHEX proteins was demonstrated by immunofluorescence and cell surface biotinylation. Of the three mutant PHEX proteins, the S711R was the least stable and the only one that could be rescued from the ER to the plasma membrane in cells grown at 26°C. The chemical chaperone glycerol failed to correct defective targeting of all three mutant proteins. Our data provide a mechanism for loss of PHEX function in XLH patients expressing the C85R, G579R and S711R mutations.
+ To whom correspondence should be addressed at: McGill University - Montreal Children’s Hospital Research Institute, 4060 Sainte-Catherine Street West, Room 222, Montreal, Quebec, H3Z 2Z3 Canada. Tel: +1 514 934 4400; Fax: +1 514 934 4331; Email: mdht@www.debelle.mcgill.ca
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