title: Analysis of recombinant Phex: an endopeptidase in search of a substrate.
abstract: 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.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: R
authors.initials: R
authors.lastname: Guo
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: R F
authors.initials: RF
authors.lastname: Spurney
authors.forename: L D
authors.initials: LD
authors.lastname: Quarles
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D000089703:Fibroblast Growth Factor-23; D015723:Gene Library; D006801:Humans; D017674:Hypophosphatemia / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010006:Osteoblasts / Q000378:metabolism; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; D010281:Parathyroid Hormone / Q000378:metabolism / Q000494:pharmacology; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D018411:Spodoptera; D013379:Substrate Specificity; D013820:Thermolysin; D014162:Transfection
chemical_list: D017931:DNA Primers; D000939:Epitopes; C000717427:FGF23 protein, human; C000717428:Fgf23 protein, mouse; D010281:Parathyroid Hormone; D011506:Proteins; D011994:Recombinant Proteins; D000089703:Fibroblast Growth Factor-23; D000242:Cyclic AMP; D010450:Endopeptidases; D053402:PHEX Phosphate Regulating Neutral Endopeptidase; C506231:PHEX protein, human; C506232:Phex protein, mouse; D013820:Thermolysin