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

Abstract

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.

Clinical, hormonal, biochemical and genetic characteristics of 75 patients with hypophosphatemic rickets

“…В структуре пеп-тидаз выделяют короткий N-концевой фрагмент, ги-дрофобный трансмембранный домен и большой C-внеклеточный домен с двумя Zn-связывающими мотивами и консервативными цистеиновыми остат-ками. Последний участок белка играет роль каталити-ческого центра [3,23]. Полагали, что продукт гена PHEX принимает участие в деградации фосфотонина FGF23.…”

“…Y. Sabbagh и соавт. [23] показали, что при таком дефекте белок менее устойчив к гликозилиро-ванию, быстрее подвергается внутриклеточной де-градации и тем самым теряет способность встраи-ваться в плазматическую мембрану.…”

Monogenic Phosphate Balance Disorders

“…The PHEX (Phosphate-regulating endopeptidase homolog, XB; MIM* 300550) gene consists of 22 exons (Sabbagh, Boileau et al 2003) and was positionally cloned in 1995 (HYP Consortium 1995 integral membrane zinc-dependent endopeptidase family. The gene is expressed in a wide variety of tissues including the kidney with a higher expression in mature osteoblasts and odontoblasts.…”

“…Both the whole-body and bone-specific (osteocalcinpromoted inactivation) knockout mouse model of PHEX as well as the spontaneous Hyp mouse model display increased bone production, increased levels of serum FGF23, decreased kidney membrane NPT2 and osteomalacia (Yuan, Takaiwa et al 2008). Cell studies indicate mechanistic defects both during protein processing in the endoplasmic reticulum and cell membrane (Sabbagh, Boileau et al 2001) and as abrogated catalytic activity (Sabbagh, Boileau et al 2003). There are several mutations associated with PHEX-hypophosphatemia (see the PHEX mutation database: http://www.phexdb.mcgill.ca/) and most of the mutations are located in the region encoding the extracellular domain, but there are also examples of pathological mutations in the 5'UTR (Dixon, Christie et al 1998) and 3'UTR (Ichikawa, Traxler et al 2008) of the gene.…”

“…PHEX gene structure and the corresponding encoded regions. Adapted from (Sabbagh, Boileau et al 2003).…”