PHEX Research Articles

The regulation and function of phosphate in the human body

Inorganic phosphate (Pi) is required for cellular function and skeletal mineralization. Serum Pi level is maintained within a narrow range through a complex interplay between intestinal absorption, exchange with intracellular and bone storage pools, and renal tubular reabsorption. Pi is abundant in the diet, and intestinal absorption of Pi is efficient and minimally regulated. The kidney is a major regulator of Pi homeostasis and can increase or decrease its Pi reabsorptive capacity to accommodate Pi need. The crucial regulated step in Pi homeostasis is the transport of Pi across the renal proximal tubule. Type II sodium-dependent phosphate (Na/Pi) cotransporter (NPT2) is the major molecule in the renal proximal tubule and is regulated by hormones and nonhormonal factors. Recent studies of inherited and acquired hypophosphatemia which exhibit similar biochemical and clinical features, have led to the identification of novel genes, phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and fibroblast growth factor-23 (FGF-23), that play a role in the regulation of Pi homeostasis. The PHEX gene encodes an endopeptidase, predominantly expressed in bone and teeth but not in kidney. FGF-23 may be a substrate of this endopeptidase and inhibit renal Pi reabsorption. In a survey in the United States and in Japan, the amount of phosphorus from food is gradually increasing. It is thought that excess amounts of phosphorus intake for long periods are a strong factor in bone impairment and ageing. The restriction of phosphorus intake seems to be important under low calcium intake to keep QOL on high level.

Differential regulation of PHEX expression in bone and parathyroid gland by chronic renal insufficiency and 1,25-dihydroxyvitamin D3.

Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, a renal phosphate-wasting disorder associated with defective skeletal mineralization. PHEX is predominantly expressed in bones and teeth and in the parathyroid gland of patients with chronic renal failure and tertiary hyperparathyroidism. The purpose of the present study was to examine the effects of renal insufficiency and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the regulation of PHEX expression in rat tibia and parathyroid gland. In rats fed a high-phosphate (Pi) diet, nephrectomy elicited a significant increase in the serum parathyroid hormone (PTH) concentration that was associated with a significant increase in the abundance of PHEX mRNA and protein in the tibia and a significant increase in PHEX mRNA in the parathyroid gland. In contrast, 1,25(OH)2D3 administration to intact rats fed a control diet elicited a significant decrease in the serum PTH concentration that was accompanied by a significant decrease in PHEX mRNA and protein abundance in the tibia and a significant decrease in PHEX mRNA in the parathyroid gland. In addition, the increases in serum PTH levels and PHEX mRNA in the tibia and parathyroid gland in nephrectomized rats fed a high-Pi diet were blunted by 1,25(OH)2D3. Serum PTH concentration was positively and significantly correlated with tibial PHEX mRNA and protein abundance. In summary, we demonstrate that PHEX expression in the tibia and parathyroid gland is increased by chronic renal insufficiency and decreased by 1,25(OH)2D3 administration and suggest that PTH status may play an important role in mediating these changes in PHEX expression.

Effect of gene dose and parental origin on bone histomorphometry in X-linked Hyp mice

X-linked hypophosphatemia (XLH) is characterized by rickets and osteomalacia and arises from mutations in the Phex and PHEX genes in mice ( Hyp) and humans, respectively. The present study was undertaken to examine the effect of gene dose on the skeletal phenotype using a histomorphometric approach. Metrical traits (vertebral length, growth plate thickness, cancellous osteoid volume per bone volume, and cancellous, endocortical, and periosteal osteoid thickness) were compared in caudal vertebrae of mutant female ( Hyp/+, Hyp/ Hyp) and male ( Hyp/Y) mice and their normal female (+/+) and male (+/Y) littermates. Mutant animals had trait values that differed significantly from those of normal animals. However, with the exception of vertebral length and cancellous osteoid thickness, values were not significantly different between the three mutant genotypes. We also examined the effect of gamete-of-origin on histomorphometric parameters in obligate Hyp/+ females derived from male or female transmitting parents. The metrical trait values in both groups of Hyp/+ mice were similar, with the exception of vertebral length and cancellous osteoid volume per bone volume. In summary, we demonstrate that the amount of osteoid per bone volume is similar in the three mutant genotypes and conclude that the extent and magnitude of the mineralization defect is fully dominant and likely not affected by gene dose. The differences in vertebral length in the mutants suggest that rickets and osteomalacia are not the only causes of decreased vertebral growth in Hyp mice and that Phex protein may influence bone growth and mineralization by distinct pathways.

Differential effects of Npt2a gene ablation and X-linked Hyp mutation on renal expression of Npt2c.

The present study was undertaken to define the mechanisms governing the regulation of the novel renal brush-border membrane (BBM) Na-phosphate (Pi) cotransporter designated type IIc (Npt2c). To address this issue, the renal expression of Npt2c was compared in two hypophosphatemic mouse models with impaired renal BBM Na-Pi cotransport. In mice homozygous for the disrupted Npt2a gene (Npt2-/-), BBM Npt2c protein abundance, relative to actin, was increased 2.8-fold compared with Npt2+/+ littermates, whereas a corresponding increase in renal Npt2c mRNA abundance, relative to beta-actin, was not evident. In contrast, in X-linked Hyp mice, which harbor a large deletion in the Phex gene, the renal abundance of both Npt2c protein and mRNA was significantly decreased by 80 and 50%, respectively, relative to normal littermates. Pi deprivation elicited a 2.5-fold increase in BBM Npt2c protein abundance in Npt2+/+ mice but failed to elicit a further increase in Npt2c protein in Npt2-/- mice. Pi restriction led to an increase in BBM Npt2c protein abundance in both normal and Hyp mice without correcting its renal expression in the mutants. In summary, we report that BBM Npt2c protein expression is differentially regulated in Npt2-/- mice and Hyp mice and that the Npt2c response to low-Pi challenge differs in both hypophosphatemic mouse strains. We demonstrate that Npt2c protein is maximally upregulated in Npt2-/- mice and suggest that Npt2c likely accounts for residual BBM Na-Pi cotransport in the knockout model. Finally, our data indicate that loss of Phex function abrogates renal Npt2c protein expression.

Human recombinant endopeptidase PHEX has a strict S1' specificity for acidic residues and cleaves peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein.

The PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) encodes a protein (PHEX) with structural homologies to members of the M13 family of zinc metallo-endopeptidases. Mutations in the PHEX gene are responsible for X-linked hypophosphataemia in humans. However, the mechanism by which loss of PHEX function results in the disease phenotype, and the endogenous PHEX substrate(s) remain unknown. In order to study PHEX substrate specificity, combinatorial fluorescent-quenched peptide libraries containing o -aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as the donor-acceptor pair were synthesized and tested as PHEX substrates. PHEX showed a strict requirement for acidic amino acid residues (aspartate or glutamate) in S(1)' subsite, with a strong preference for aspartate. Subsites S(2)', S(1) and S(2) exhibited less defined specificity requirements, but the presence of leucine, proline or glycine in P(2)', or valine, isoleucine or histidine in P(1) precluded hydrolysis of the substrate by the enzyme. The peptide Abz-GFSDYK(Dnp)-OH, which contains the most favourable residues in the P(2) to P(2)' positions, was hydrolysed by PHEX at the N-terminus of aspartate with a k(cat)/ K(m) of 167 mM(-1) x s(-1). In addition, using quenched fluorescence peptides derived from fibroblast growth factor-23 and matrix extracellular phosphoglycoprotein sequences flanked by Abz and N -(2,4-dinitrophenyl)ethylenediamine, we showed that these physiologically relevant proteins are potential PHEX substrates. Finally, our results clearly indicate that PHEX does not have neprilysin-like substrate specificity.

Structure and function of disease-causing missense mutations in the PHEX gene.

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.

Regulation of the murine PHEX gene by vitamin D3

Regulation of the murine PHEX gene by vitamin D3

Partial rescue of the Hyp phenotype by osteoblast-targeted PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) expression.

Inactivating mutations and/or deletions of PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans and in the murine homolog Hyp. The predominant osteoblastic expression of Phex has implicated a primary metabolic osteoblast defect in the pathophysiology of this disorder. By targeting PHEX expression to osteoblasts in the Hyp genetic background, we aimed to correct the corresponding biochemical and morphological abnormalities and obtain information on their pathogenetic mechanism. When transgene Phex expression, driven by a mouse pro-alpha1(I) collagen gene promoter, was crossed into the Hyp background, it improved the defective mineralization of bone and teeth but failed to correct the hypophosphatemia and altered vitamin D metabolism associated with the disorder. Ex vivo bone marrow cultures confirmed the amelioration in the Hyp-associated matrix mineralization defect after Phex expression. These findings suggest that while the Hyp bone and teeth abnormalities partially correct after PHEX gene transfer, additional factors and/or sites of PHEX expression are likely critical for the elaboration of the appropriate molecular signals that alter renal phosphate handling and vitamin D metabolism in this disorder.

Glucocorticoid regulation of the murine PHEX gene.

The phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) is a member of the neutral endopeptidase family, which is expressed predominantly on the plasma membranes of mature osteoblasts and osteocytes. Although it is known that the loss of PHEX function results in X-linked hypophosphatemic rickets, characterized by abnormal bone matrix mineralization and renal phosphate wasting, little is known about how PHEX is regulated. We therefore sought to determine whether the murine PHEX gene is regulated by glucocorticoids (GCs), which are known to influence phosphate homeostasis and bone metabolism. Northern blot analysis revealed increased PHEX mRNA expression in GC-treated suckling mice (1.5-fold) and in rat osteogenic sarcoma (UMR-106) cells (2.5-fold). An increase was also seen in PHEX promoter activity in transiently transfected UMR-106 cells with GC treatment. Analysis of nested promoter deletions revealed that an atypical GC response element was located between -337 and -315 bp. Mutational analysis and electrophoretic mobility shift assays further identified -326 to -321 bp as a site involved in GC regulation. Supershift analyses and electrophoretic mobility shift assay competition studies indicated that the core binding factor alpha1-subunit transcription factor is able to bind to this region and may therefore play a role in the GC response of the murine PHEX gene.

Fibroblast growth factor-23 is the phosphaturic factor in tumor-induced osteomalacia and may be phosphatonin.

Three hypophosphatemic diseases, X-linked dominant hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and tumor-induced rickets/osteomalacia (TIO), show very similar clinical features including hypophosphatemia due to renal phosphate wasting. Because of some evidence that XLH and TIO are caused by a humoral mechanism, the presence of a phosphate-regulating hormone, phosphatonin, was hypothesized. The causative factor of TIO has been thought to be a strong candidate for phosphatonin. In this review, we summarize recent findings concerning a humoral factor which causes TIO, and discuss the nature of phosphatonin. The PHEX gene and fibroblast growth factor (FGF)-23 were identified as responsible genes for XLH and ADHR, respectively. In addition, FGF-23 was cloned as a gene abundantly expressed in a responsible tumor for TIO and was shown to reproduce almost all characteristics of TIO when overexpressed in mice. Furthermore, FGF-23 was proteolytically processed between Arg(179) and Ser(180), and all mutations found in ADHR existed in this proteolytic consensus site. Mutant FGF-23 proteins were resistant to the processing and seem to have somehow increased biological activity. There is not yet enough evidence that FGF-23 is phosphatonin, and the relation between PHEX and FGF-23 is unclear. FGF-23 plays important roles in the development of hypophosphatemic diseases. These findings will certainly contribute to the development of new diagnostic and therapeutic maneuvers for hypophosphatemic diseases.

The X chromosome deletion in Hyp mice extends into the intergenic region but does not include the Sat gene downstream from Phex

The murine Hyp mutation is a model for X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans. Although mutations in the murine Phex gene and the human PHEX gene have been identified in both murine and human disorders, the extent of the Hyp deletion on the mouse X chromosome has not been delineated. In the present study we demonstrate that the Hyp deletion starts in the middle of Phex intron 15 and includes ∼48 kb of the 3′ region of the Phex gene and ∼10 kb of intergenic sequence on the mouse X chromosome. In addition, we show that the Hyp deletion does not involve the downstream spermidine/spermine N1-acetyl transferase (Sat; formerly Ssat) gene and thus is not a contiguous gene deletion syndrome. Our data indicate that the Hyp mouse is a true homolog of XLH in humans and underscore the validity of this murine model in studies of XLH pathophysiology and for testing novel treatment modalities.

IGF-I and GH stimulate Phex mRNA expression in lungs and bones and 1,25-dihydroxyvitamin D(3) production in hypophysectomized rats.

X-linked hypophosphatemia, a renal phosphate (Pi)-wasting disorder with defective bone mineralization, is caused by mutations in the PHEX gene (a Pi-regulating gene with homology to endopeptidases on the X chromosome). We wondered whether changes in Phex and neprilysin (NEP) (another member of the family of zinc endopeptidases) mRNA expression could be observed in relation to vitamin D and Pi metabolism during GH- and IGF-I-stimulated growth of hypophysectomized rats. Animals were infused s.c. for 2 days with vehicle, 200 mU (67 microg) GH or 300 microg IGF-I/rat per 24 h. We determined serum osteocalcin and osteocalcin mRNA in bone, Phex mRNA in bone and lungs, serum 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and serum Pi levels, and renal expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), of 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) and of the Na-dependent Pi-cotransporter type I and II (Na(d)Pi-I and -II). As compared with vehicle-treated controls, body weight and tibial epiphyseal width significantly increased in GH- and IGF-I-treated animals. Serum osteocalcin and osteocalcin mRNA levels in bone, Phex mRNA in bone and lungs, serum 1,25-(OH)(2)D(3) and renal 1alpha-hydroxylase mRNA rose concomitantly, whereas expression of NEP in lungs was barely affected and renal 24-hydroxylase mRNA decreased. Na(d)Pi-I and -II gene expression in the kidney and serum Pi levels remained unchanged. Our findings suggest a coordinate regulation of Phex mRNA expression in lungs and bone and vitamin D metabolism during GH- and IGF-I-stimulated growth.

Hypophosphatemia and rickets/osteomalacia

It is important to know the pathophysiology of the inherited hypophosphatemic disorders for the research on the relationship between hypophosphatemia and rickets/osteomalacia. And the research on the pathogenesis of the X-linked hypophosphatemic vitamin D resistant rickets/osteomalacia has played central roles in this field. After the identification of PHEX gene for the responsible gene of the disorder, major interest of the research has shifted to the identification of phosphatonin, the natural substrate of PHEX and the novel phosphate-regulating hormone. Recent discovery of FGF23 for the responsible gene of oncogenic osteomalacia and autosomal dominant hypophosphatemic rickets may lead our knowledge on the phosphate regulation system to the new stage.

Cloning and characterization of the proximal murine Phex promoter.

Phex is an endopetidase that regulates systemic phosphate homeostasis. We investigated Phex gene transcription by cloning and performing functional analysis of the 2736 bp of the 5' flanking region of the mouse Phex gene containing its promoter. We identified a transcription start site, a consensus TATA-box, and multiple potential cis-acting regulator elements. To determine whether the promoter directs cell-type restricted Phex expression, we transfected full-length and 5'-deleted Phex luciferase reporter constructs into various cell lines. Phex-expressing C5.18 chondrocytes displayed the highest activity of the transfected Phex promoter constructs compared with non-Phex-expressing COS-7 cells, whereas promoter activity was intermediate in ROS 17/2.8 osteoblasts and maturation stage-dependent in MC3T3-E1 osteoblasts. Analysis of sequential 5'-deletion mutants of the Phex promoter in ROS 17/2.8 cells revealed bimodal activity, suggesting that both positive and negative cis-acting regions may be present. The chondrogenic factor SOX9 markedly stimulated Phex promoter activity, whereas Cbfa1, PTH, and 1,25(OH)(2)D(3) had no effect. Our findings are consistent with the predominant expression of Phex in bone and cartilage. Additional studies will be needed to confirm the regulatory regions in the Phex promoter that function in a cell-restricted manner.

Direct demonstration of humorally mediated inhibition of the transcription of phosphate transporter in XLH patients

Background. X-linked hypophosphatemia (XLH) is the most common form of familial hypophosphatemic rickets, which results from impaired renal phosphate (Pi) reabsorption, renal vitamin D metabolism, and skeletal mineralization. PHEX the gene responsible for XLH, is homologous to members of the neutral endopeptidase family. The role that the PHEX gene plays in Pi transport is not yet known. Methods. In the present study, we investigated the molecular mechanisms of the disorder of renal Pi transport in XLH in seven individuals from Japanese XLH families. Also, to characterize the effect of XLH serum from these patients on the expression of a renal phosphate transporter gene, we performed a functional analysis of the human type II Na/Pi cotransporter (NPT2) gene promoter in a kidney cell line, OK cells (named OK-B2400), which had stable expression of the luciferase reporter vector p3P2400 with 2462 base pairs (nucleotides −2409 to +53) of the NPT2 gene. Results. XLH serum significantly decreased the transcriptional activity of the NPT2 promoter. The mutations identified in our patients included three large deletions, frame shift mutations, and a stop mutation in the PHEX gene, which suggest that the gene is nonfunctional in affected individuals. Conclusion. The present study demonstrated that the serum of XLH patients with PHEX mutations contains inhibitory factor(s) for the transcriptional activity of the NPT2 gene. In addition, the transcription assay of the NPT2 promoter may provide a useful tool for the characterization of the putative PHEX protein substrate.

Mutational Analysis and Genotype-Phenotype Correlation of the PHEX Gene in X-Linked Hypophosphatemic Rickets

PHEX is the gene defective in X-linked hypophosphatemic rickets. In this study, analysis of PHEX revealed mutations in 22 hypophosphatemic rickets patients, including 16 of 28 patients in whom all 22 PHEX exons were studied. In 13 patients, in whom no PHEX mutation had been previously detected in 17 exons, the remaining 5 PHEX exons were analyzed and mutations found in 6 patients. Twenty different mutations were identified, including 16 mutations predicted to truncate PHEX and 4 missense mutations. Phenotype analysis was performed on 31 hypophosphatemic rickets patients with PHEX mutations, including the 22 patients identified in this study, 9 patients previously identified, and affected family members. No correlation was found between the severity of disease and the type or location of the mutation. However, among patients with a family history of hypophosphatemic rickets, there was a trend toward more severe skeletal disease in patients with truncating mutations. Family members in more recent generations had a milder phenotype. Postpubertal males had a more severe dental phenotype. In conclusion, although identifying mutations in PHEX may have limited prognostic value, genetic testing may be useful for the early identification and treatment of affected individuals. Furthermore, this study suggests that other genes and environmental factors affect the severity of hypophosphatemic rickets.

X-linked hypophosphatemia attributable to pseudoexons of the PHEX gene.

X-linked hypophosphatemia is commonly caused by mutations of the coding region of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). However, such PHEX mutations are not detected in approximately one third of X-linked hypophosphatemia patients who may harbor defects in the noncoding or intronic regions. We have therefore investigated 11 unrelated X-linked hypophosphatemia patients in whom coding region mutations had been excluded, for intronic mutations that may lead to mRNA splicing abnormalities, by the use of lymphoblastoid RNA and RT-PCRs. One X-linked hypophosphatemia patient was found to have 3 abnormally large transcripts, resulting from 51-bp, 100-bp, and 170-bp insertions, all of which would lead to missense peptides and premature termination codons. The origin of these transcripts was a mutation (g to t) at position +1268 of intron 7, which resulted in the occurrence of a high quality novel donor splice site (ggaagg to gtaagg). Splicing between this novel donor splice site and 3 preexisting, but normally silent, acceptor splice sites within intron 7 resulted in the occurrences of the 3 pseudoexons. This represents the first report of PHEX pseudoexons and reveals further the diversity of genetic abnormalities causing X-linked hypophosphatemia.

X-Linked Hypophosphatemia Attributable to Pseudoexons of the PHEX Gene

X-linked hypophosphatemia is commonly caused by mutations of the coding region of PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). However, such PHEX mutations are not detected in approximately one third of X-linked hypophosphatemia patients who may harbor defects in the noncoding or intronic regions. We have therefore investigated 11 unrelated X-linked hypophosphatemia patients in whom coding region mutations had been excluded, for intronic mutations that may lead to mRNA splicing abnormalities, by the use of lymphoblastoid RNA and RT-PCRs. One X-linked hypophosphatemia patient was found to have 3 abnormally large transcripts, resulting from 51-bp, 100-bp, and 170-bp insertions, all of which would lead to missense peptides and premature termination codons. The origin of these transcripts was a mutation (g to t) at position+ 1268 of intron 7, which resulted in the occurrence of a high quality novel donor splice site (ggaagg to gtaagg). Splicing between this novel donor splice site and 3 preexisting, but normally silent, acceptor splice sites within intron 7 resulted in the occurrences of the 3 pseudoexons. This represents the first report of PHEX pseudoexons and reveals further the diversity of genetic abnormalities causing X-linked hypophosphatemia.

Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets.

PHEX is the gene defective in X-linked hypophosphatemic rickets. In this study, analysis of PHEX revealed mutations in 22 hypophosphatemic rickets patients, including 16 of 28 patients in whom all 22 PHEX exons were studied. In 13 patients, in whom no PHEX mutation had been previously detected in 17 exons, the remaining 5 PHEX exons were analyzed and mutations found in 6 patients. Twenty different mutations were identified, including 16 mutations predicted to truncate PHEX and 4 missense mutations. Phenotype analysis was performed on 31 hypophosphatemic rickets patients with PHEX mutations, including the 22 patients identified in this study, 9 patients previously identified, and affected family members. No correlation was found between the severity of disease and the type or location of the mutation. However, among patients with a family history of hypophosphatemic rickets, there was a trend toward more severe skeletal disease in patients with truncating mutations. Family members in more recent generations had a milder phenotype. Postpubertal males had a more severe dental phenotype. In conclusion, although identifying mutations in PHEX may have limited prognostic value, genetic testing may be useful for the early identification and treatment of affected individuals. Furthermore, this study suggests that other genes and environmental factors affect the severity of hypophosphatemic rickets.

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 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 degrees 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.