Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein

PHEX is homologous to the M13 zinc metallopeptidases, a class of type II me mbrane glycoproteins. Although more than 140 mutations in the PHEX gene hav e been identified in patients with X-linked hypophosphatemia (XLH), the mos t prevalent form of inherited rickets, the molecular consequences of diseas e-causing PHEX mutations have not yet been investigated. We examined the ef fect of PHEX missense mutations on cellular trafficking of the recombinant protein. Four mutant PHEX cDNAs were generated by PCR mutagenesis: C85R, G5 79R 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 transfe cted in HEK(293) cells and PHEX protein expression was characterized. In co ntrast to the wild-type and E581V PHEX proteins, the C85R, G579R and S711R mutants were completely sensitive to endoglycosidase H digestion, indicatin g that they were not fully glycosylated. Sequestration of the disease-causi ng mutant proteins in the endoplasmic reticulum (ER) and plasma membrane lo calization of wild-type and E581V PHEX proteins was demonstrated by immunof luorescence and cell surface biotinylation. Of the three mutant PHEX protei ns, the S711R was the least stable and the only one that could be rescued f rom the ER to the plasma membrane in cells grown at 26 degreesC. The chemic al chaperone glycerol failed to correct defective targeting of all three mu tant proteins. Our data provide a mechanism for loss of PHEX function in XL H patients expressing the C85R, G579R and S711R mutations.