Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Disease-causing missense mutations in the PHEX gene interfere with membrane targeting of the recombinant protein. 1146 71

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of Phex, a phosphate-regulating endopeptidase. Further advances in our knowledge of the pathogenesis of XLH require identification of the biological function of Phex and its physiologically relevant substrates. We evaluated several potential substrates using mouse recombinant wild-type Phex proteins (rPhex-WT) and inactive mutant Phex proteins (rPhex-3'M) lacking the COOH-terminal catalytic domain as controls. By Western blot analysis, we demonstrated that Phex is a membrane-bound 100-kDa glycosylated monomer. Neither casein, a substrate for the related endopeptidase thermolysin, human stanniocalcin 1 (hSTC-1), an osteoblast-derived phosphate-regulating factor, nor FGF-23 peptide (amino acid 172-186), comprising the region mutated in autosomal dominant hypophosphatemia, was cleaved by rPhex-WT. In addition, membranes expressing rPhex-WT, rPhex-3'M, and the empty vector hydrolyzed parathyroid hormone-(1-34), indicating the lack of Phex-specific cleavage of parathyroid hormone. In contrast, rPhex-WT did display an EDTA-dependent cleavage of the neutral endopeptidase substrate [Leu]enkephalin. Further studies with wild-type and mutant rPhex proteins should permit the identification of physiologically relevant substrates involved in the pathogenesis of XLH.
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PMID:Analysis of recombinant Phex: an endopeptidase in search of a substrate. 1155 62

Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH.
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PMID:Overexpression of Phex in osteoblasts fails to rescue the Hyp mouse phenotype. 1171 45

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.
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PMID: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. 1267 20

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.
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PMID:Structure and function of disease-causing missense mutations in the PHEX gene. 1272 77

X-linked hypophosphatemia (XLH) and autosomal dominant hypophosphatemic rickets (ADHR) are characterized by renal phosphate wasting, rickets, and osteomalacia. ADHR is caused by gain of function mutations in the fibroblast growth factor 23 gene (FGF23). During secretion, FGF23 is processed at the C-terminus between amino acids 179 and 180. The cleavage site is mutated in ADHR, preventing processing of FGF23. Here, we show that FGF23 is likely to be cleaved by subtilisin-like proprotein convertases (SPC) as cleavage can be inhibited by a specific SPC inhibitor in HEK293 cells. SPCs, which are widely expressed, were demonstrated to be also present in HEK293 cells as well as in osteoblasts. XLH is caused by loss of function mutations in the putative endopeptidase PHEX. It was tempting to speculate that FGF23 is a substrate of PHEX, but studies have been inconclusive so far. Here, we used a secreted form of PHEX (secPHEX) and tagged and untagged FGF23 constructs for co-incubation experiments. These experiments provided evidence against cleavage of intact FGF23(25-251) as well as of N-terminal (FGF23(25-179)) and C-terminal (FGF23(180-251)) fragments by the endopeptidase PHEX.
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PMID:FGF23 is processed by proprotein convertases but not by PHEX. 1526 97

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency/platelet disease due to mutations of WASP, a cytoskeletal regulatory protein of blood cells. Patients exhibit a range of immune defects generally attributed to defective T-cell function, including poor response to immunization, skewed immunoglobulin isotypes, eczema, recurrent infections, autoimmune disease and increased frequency of malignancies. Here we show a deficit of total B-cells in WAS patients of various ages and identify phenotypic perturbations involving complement receptors and CD27. Whereas B-cells of normal healthy donors are overwhelmingly CD21/CD35-positive, B-cells expressing these receptors are significantly reduced in number in WAS patients, and their paucity may cause suboptimal antigen capture and presentation. The frequencies of IgD(-) and IgG(+) patient B-cells were not different from healthy donors (although absolute numbers were decreased), indicating that isotype switching is occurring. In contrast, the frequency of cells positive for CD27, the marker of post germinal centre B-cells, was significantly decreased even among isotype-switched cells, and B-cells resembling germinal centre progenitors (CD10(+)CD27(-)CD38(bright)) were more frequent in adult patients, suggesting impaired germinal centre maturation/differentiation. The documentation of these phenotypic perturbations and deficit of total cells suggest that defects intrinsic to B-cells contribute to the impaired humoral immunity that characterizes this disease.
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PMID:Phenotypic perturbation of B cells in the Wiskott-Aldrich syndrome. 1565 28

Inactivation of the growth factor-regulated S6 kinase RSK2 causes Coffin-Lowry syndrome in humans, an X-linked mental retardation condition associated with progressive skeletal abnormalities. Here we show that mice lacking RSK2 develop a progressive skeletal disease, osteopenia due to impaired osteoblast function and normal osteoclast differentiation. The phenotype is associated with decreased expression of Phex, an endopeptidase regulating bone mineralization. This defect is probably not mediated by RSK2-dependent phosphorylation of c-Fos on serine 362 in the C-terminus. However, in the absence of RSK2, c-Fos-dependent osteosarcoma formation is impaired. The lack of c-Fos phosphorylation leads to reduced c-Fos protein levels, which are thought to be responsible for decreased proliferation and increased apoptosis of transformed osteoblasts. Therefore, RSK2-dependent stabilization of c-Fos is essential for osteosarcoma formation in mice and may also be important for human osteosarcomas.
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PMID:Essential role of RSK2 in c-Fos-dependent osteosarcoma development. 1571 69

Renal proximal tubular reabsorption of phosphate and intestinal absorption both regulate phosphate homeostasis. Brush-border membrane Npt2a cotransporter is the key element in proximal tubular P (i) reabsorption. Inactivating mutations of Npt2a cause bone demineralisation and urolithiasis. An excess of a phosphaturic factor, called "Phosphatonin", could modulate phosphate reabsorption by inhibition on Npt2a. Inactivating mutation of PHEX, an endopeptidase-membrane coding gene, is responsible for X-linked Hypophosphatemia (XLH), because of an impaired degradation of phosphatonine by PHEX product. Autosomic Dominant Hypophosphatemic Rickets (ADHR) is explained by a mutation preventing FGF23 (one of the best identified phosphatonines) from cleavage. According recent data, FGF23, MEPE (Matrix Extracellular Phosphoglycoprotein) et FRP4 (frizzled related protein-4) are 3 putative "phosphatonines".
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PMID:[Genetic hypophosphatemia: recent advances in physiopathogenic concept]. 1595 11

X-linked lymphoproliferative disease (XLP) is a severe immunodeficiency associated with a marked reduction in circulating memory B cells. Our investigation of the B cell compartment of XLP patients revealed an increase in the frequency of a population of B cells distinct from those previously defined. This population displayed increased expression of CD10, CD24, and CD38, indicating that it could consist of circulating immature/transitional B cells. Supporting this possibility, CD10+CD24highCD38high B cells displayed other immature characteristics, including unmutated Ig V genes and elevated levels of surface IgM; they also lacked expression of Bcl-2 and a panel of activation molecules. The capacity of CD24highCD38high B cells to proliferate, secrete Ig, and migrate in vitro was greatly reduced compared with mature B cell populations. Moreover, CD24highCD38high B cells were increased in the peripheral blood of neonates, patients with common variable immunodeficiency, and patients recovering from hemopoietic stem cell transplant. Thus, an expansion of functionally immature B cells may contribute to the humoral immunodeficient state that is characteristic of neonates, as well as patients with XLP or common variable immunodeficiency, and those recovering from a stem cell transplant. Further investigation of transitional B cells will improve our understanding of human B cell development and how alterations to this process may precipitate immunodeficiency or autoimmunity.
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PMID:Expansion of functionally immature transitional B cells is associated with human-immunodeficient states characterized by impaired humoral immunity. 1642 79


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