Title: Regulation of Fibroblastic Growth Factor 23 Expression but Not Degradation by PHEX
Abstract: Inactivating mutations of Phex cause X-linked hypophosphatemia (XLH) by increasing levels of a circulating phosphaturic factor. FGF23 is a candidate for this phosphaturic factor. Elevated serum FGF23 levels correlate with the degree of hypophosphatemia in XLH, suggesting that loss of Phex function in this disorder results in either diminished degradation and/or increased biosynthesis of FGF23. To establish the mechanisms whereby Phex regulates FGF23, we assessed Phex-dependent hydrolysis of recombinant FGF23 in vitro and measured fgf23 message levels in the Hyp mouse homologue of XLH. In COS-7 cells, overexpression of FGF23 resulted in its degradation into N- and C-terminal fragments by an endogenous decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-sensitive furin-type convertase. Phex-dependent hydrolysis of full-length FGF23 or its N- and C-terminal fragments could not be demonstrated in the presence or absence of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone in COS-7 cells expressing Phex and FGF23. In a reticulolysate system, apparent cleavage of FGF23 occurred with wild-type Phex, the inactive Phex-3′M mutant, and vector controls, indicating nonspecific metabolism of FGF23 by contaminating enzymes. These findings suggest that FGF23 is not a direct Phex substrate. In contrast, by real-time reverse transcriptase PCR, the levels of fgf23 transcripts were highest in bone, the predominant site of Phex expression. In addition, Hyp mice displayed a bone-restricted increase in fgf23 transcripts in association with inactivating Phex mutations. Increased expression of fgf23 was also observed in Hyp-derived osteoblasts in culture. These findings suggest that Phex, possibly through the actions of unidentified Phex substrates or other downstream effectors, regulates fgf23 expression as part of a potential hormonal axis between bone and kidney that controls systemic phosphate homeostasis and mineralization. Inactivating mutations of Phex cause X-linked hypophosphatemia (XLH) by increasing levels of a circulating phosphaturic factor. FGF23 is a candidate for this phosphaturic factor. Elevated serum FGF23 levels correlate with the degree of hypophosphatemia in XLH, suggesting that loss of Phex function in this disorder results in either diminished degradation and/or increased biosynthesis of FGF23. To establish the mechanisms whereby Phex regulates FGF23, we assessed Phex-dependent hydrolysis of recombinant FGF23 in vitro and measured fgf23 message levels in the Hyp mouse homologue of XLH. In COS-7 cells, overexpression of FGF23 resulted in its degradation into N- and C-terminal fragments by an endogenous decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-sensitive furin-type convertase. Phex-dependent hydrolysis of full-length FGF23 or its N- and C-terminal fragments could not be demonstrated in the presence or absence of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone in COS-7 cells expressing Phex and FGF23. In a reticulolysate system, apparent cleavage of FGF23 occurred with wild-type Phex, the inactive Phex-3′M mutant, and vector controls, indicating nonspecific metabolism of FGF23 by contaminating enzymes. These findings suggest that FGF23 is not a direct Phex substrate. In contrast, by real-time reverse transcriptase PCR, the levels of fgf23 transcripts were highest in bone, the predominant site of Phex expression. In addition, Hyp mice displayed a bone-restricted increase in fgf23 transcripts in association with inactivating Phex mutations. Increased expression of fgf23 was also observed in Hyp-derived osteoblasts in culture. These findings suggest that Phex, possibly through the actions of unidentified Phex substrates or other downstream effectors, regulates fgf23 expression as part of a potential hormonal axis between bone and kidney that controls systemic phosphate homeostasis and mineralization. X-linked hypophosphatemia (XLH) 1The abbreviations used are: XLH, X-linked hypophosphatemia; ADHR, autosomal dominant hypophosphatemic rickets; TIO, tumor-induced osteomalacia; PHEX, phosphate regulating endopeptidase on the X-chromosome; rPhex, recombinant Phex; Nl, normal; WT, wild-type; dec-RVKR-cmk, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone; ZAAL-pNA, Z-Ala-Ala-Leu-p-nitroanilide; MES, 4-morpholineethanesulfonic acid; FGF23, fibroblastic growth factor 23. is a disorder characterized by defective calcification of cartilage and bone, growth retardation, impaired renal tubular reabsorption of phosphate, aberrant regulation of 1,25(OH)2D3 production, and resistance to phosphorus and vitamin D therapy (1Rasmussen H. Anast C. Familial Hypophosphatemic Rickets and Vitamin D-dependent Rickets. McGraw-Hill Book Co., New York1983: 1743-1773Google Scholar). XLH is caused by inactivating mutations of PHEX (2The HYP ConsortiumNat. 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FGF23 is an ∼30-kDa (251 amino acids) protein with an N-terminal region containing the FGF homology domain and a novel 71-amino acid C terminus. Several studies have confirmed that full-length FGF23 is a phosphaturic hormone (25Bai X.Y. Miao D. Goltzman D. Karaplis A.C. J. Biol. Chem. 2003; 278: 9843-9849Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 26Saito H. Kusano K. Kinosaki M. Ito H. Hirata M. Segawa H. Miyamoto K.I. Fukushima N. J. Biol. Chem. 2003; 278: 2206-2211Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 27Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1223) Google Scholar) and that cleavage of FGF23 at the RXXR motif generates biologically inactive N- and C-terminal fragments (27Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1223) Google Scholar). In ADHR mutations of the 176RXXR179 site (R176Q, R179W, and R179Q) prevent cleavage and inactivation of FGF23 (24White K.E. Evans W.E O'Riordan J.L.H. Speer M.C. Econs M.J. Lorenz-Depiereux B. Grabowski M. Meitinger T. Strom T.M. Nat. Genet. 2000; 26: 345-348Crossref PubMed Scopus (1281) Google Scholar). Tumor-induced osteomalacia (TIO), also called oncogenic osteomalacia, is an acquired hypophosphatemic disorder with phenotypic features similar to ADHR and XLH (28Yamazaki Y. Okazaki R. Shibata M. Hasegawa Y. Satoh K. Tajima T. Takeuchi Y. Fujita T. Nakahara K. Yamashita T. Fukomoto S. J. Clin. Endocrinol. Metab. 2002; 87: 4957-4960Crossref PubMed Scopus (579) Google Scholar). FGF23 is also secreted from TIO tumors (27Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. 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Res. 2003; 18: 1227-1234Crossref PubMed Scopus (308) Google Scholar). In addition, FGF23 is elevated in some subjects with XLH (28Yamazaki Y. Okazaki R. Shibata M. Hasegawa Y. Satoh K. Tajima T. Takeuchi Y. Fujita T. Nakahara K. Yamashita T. Fukomoto S. J. Clin. Endocrinol. Metab. 2002; 87: 4957-4960Crossref PubMed Scopus (579) Google Scholar, 31Weber T. Liu S. Indridason O. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (308) Google Scholar, 32Jonsson K.B. Zahradnik R. Larsson T. White K.E. Sugimoto T. Imanishi Y. Yamamoto T. Hampson G. Koshiyama H. Ljunggren O. Oba K. Yang I.M. Miyauchi A. Econs M.J. Lavigne J. Juppner H. N. Engl. J. Med. 2003; 348: 1656-1663Crossref PubMed Scopus (769) Google Scholar). Serum phosphate concentrations are negatively correlated with circulating FGF23 levels in patients with XLH, suggesting that elevated FGF23 is causing the hypophosphatemia in this disorder as well (31Weber T. Liu S. Indridason O. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (308) Google Scholar). The phenotypic similarities among ADHR, XLH, and TIO form the basis for an unproven model to explain their common pathogenesis (33Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (72) Google Scholar). This model presumes that PHEX degrades active full-length FGF23 into inactive fragments (33Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (72) Google Scholar) and that ADHR, XLH, and TIO are caused by increased circulating levels of FGF23, which acts as a phosphaturic hormone to inhibit sodium-dependent phosphate uptake in the renal proximal tubule. In this model, FGF23 is increased in ADHR because of mutations in FGF23 that render it resistant to PHEX-dependent cleavage, in XLH because of inactivating mutations of PHEX that prevent the normal degradation of FGF23, and in TIO, because overproduction of FGF23 by the tumor over-whelms the degradation capacity of PHEX. Currently, three fundamental aspects of this model have been established, namely that mutations preventing the metabolism of FGF23 causes ADHR (33Strewler G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5945-5946Crossref PubMed Scopus (72) Google Scholar), FGF23 possess phosphaturic actions (26Saito H. Kusano K. Kinosaki M. Ito H. Hirata M. Segawa H. Miyamoto K.I. Fukushima N. J. Biol. Chem. 2003; 278: 2206-2211Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 27Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1223) Google Scholar), and serum concentrations of FGF23 are elevated in some subjects with TIO and XLH (28Yamazaki Y. Okazaki R. Shibata M. Hasegawa Y. Satoh K. Tajima T. Takeuchi Y. Fujita T. Nakahara K. Yamashita T. Fukomoto S. J. Clin. Endocrinol. Metab. 2002; 87: 4957-4960Crossref PubMed Scopus (579) Google Scholar, 31Weber T. Liu S. Indridason O. Quarles L.D. J. Bone Miner. Res. 2003; 18: 1227-1234Crossref PubMed Scopus (308) Google Scholar, 32Jonsson K.B. Zahradnik R. Larsson T. White K.E. Sugimoto T. Imanishi Y. Yamamoto T. Hampson G. Koshiyama H. Ljunggren O. Oba K. Yang I.M. Miyauchi A. Econs M.J. Lavigne J. Juppner H. N. Engl. J. Med. 2003; 348: 1656-1663Crossref PubMed Scopus (769) Google Scholar). The requirement that FGF23 is a substrate for PHEX, however, has not been tested rigorously. Indeed, the data regarding Phex metabolism of FGF23 are conflicting. One report suggests that recombinant Phex may cleave FGF23 at the RXXR motif or a nearby site (34Bowe A.E. Finnegan R. Jan de Beur S.M. Cho J. Levine M.A. Kumar R. Schaivi S.C. Biochem. Biophys. Res. Commun. 2001; 284: 977-981Crossref PubMed Scopus (298) Google Scholar), but other studies have failed to confirm Phex-dependent cleavage of FGF23 (35Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. Metab. 2001; 281: E837-E847Crossref PubMed Google Scholar). In addition, the RXXR motif is the consensus cleavage site for pro-protein convertases (36Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (702) Google Scholar), and all cell lines and expression systems tested to date for generating recombinant FGF23 contain enzymes capable of metabolizing FGF23 into its N- and C-terminal fragments (27Shimada T. Mizutani S. Muto T. Yoneya T. Hino R. Takeda S. Takeuchi Y. Fujita T. Fukumoto S. Yamashita T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6500-6505Crossref PubMed Scopus (1223) Google Scholar, 30White K.E. Jonsson K.B. Carn G. Hampson G. Spector T.D. Mannstadt M. Lorenz-Depiereux B. Miyauchi A. Yang I.M. Ljunggren O. Meitinger T. Strom T.M. Juppner H. Econs M.J. J. Clin. Endocrinol. Metab. 2001; 86: 497-500Crossref PubMed Scopus (253) Google Scholar), suggesting that the processing enzyme is widely expressed. Also, no studies have investigated the possibility that FGF23 biosynthesis might be increased in XLH/Hyp. In the current investigations we have generated recombinant FGF23 and Phex and tested whether Phex metabolizes FGF23. In addition, we have examined whether fgf23 expression is increased in the Hyp mouse homologue of XLH. Generation of FGF23 and Phex Constructs—We amplified human FGF23 cDNA coding sequence from human heart total RNA (Clontech, Palo Alto, CA) by reverse transcriptase PCR similar to previous reports (30White K.E. Jonsson K.B. Carn G. Hampson G. Spector T.D. Mannstadt M. Lorenz-Depiereux B. Miyauchi A. Yang I.M. Ljunggren O. Meitinger T. Strom T.M. Juppner H. Econs M.J. J. Clin. Endocrinol. Metab. 2001; 86: 497-500Crossref PubMed Scopus (253) Google Scholar). Briefly, 1 μg of total RNA was reverse transcribed using random primers, and PCR was performed with 5′-ACGATGTTGGGGGCCCG forward and 5′-GATGAACTTGGCGAAGGGG reverse primers. High fidelity platinum Pfx DNA polymerase (Invitrogen) was used in all PCR reactions. A 760-bp PCR product containing sequence from –3 to +756 was directly cloned into pcDNA3.1/V5-His vector (Invitrogen) to generate a C-terminal V5 and His-tagged FGF23 (pcDNA3.1-FGF23-V5-His). We created additional dual N-terminal FLAG and C-terminal His6-tagged FGF23 constructs in the pFLAG-CMV-3 expression vector (Sigma), including full-length FGF23 (pFLAG-FGF23-His), C-terminal FGF23 (pFLAG-C-FGF23-His), and N-terminal FGF23 (pFLAG-N-FGF23-His), using the pcDNA3.1-FGF23-V5-His as the template and the following primer sets: 5′-GGAATTCATATCCCAATGCCTCCCCA forward and 5′-CGGATCCTCAATGGTGATGGTGATGATGGATGAACTTGGCGAAGGGG reverse for full-length FGF23, 5′-GGAATTCATATCCCAATGCCTCCCCA forward and 5′-GGATCCTCAATGGTGATGGTGATGATGCCGGGTGTGCCGCCG reverse for N-terminal FGF23, and 5′-GGAATTCAAGCGCCGAGGACGACTCGGA forward and 5′-CGGATCCTCAATGGTGATGGTGATGATGGATGAACTTGGCGAAGGGG reverse for the C-terminal FGF23 fragment. Cleavage-resistant FGF23 R179Q mutation (25Bai X.Y. Miao D. Goltzman D. Karaplis A.C. J. Biol. Chem. 2003; 278: 9843-9849Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar) was created in the pcDNA3. 1-FGF23-V5-His and pFLAG-FGF23-His constructs using the QuikChange XL site-directed mutagenesis kit (Stratagene) and primers with desired point mutation (5′-CACGGCGGCACACCCAGAGCGCCGAGGACGAC forward and 5′-GTCGTCCCGGCGCTCTGGGTGTGCCGCCGTG reverse) according to the manufacturer's instructions. We also subcloned the mouse fgf23 construct into pcDNA3.1/V5-His using as a PCR template a mouse fgf23 cDNA construct obtained from T. Yamashita (37Yamashita T. Yoshioka M. Itoh N. Biochem. Biophys. Res. Commun. 2000; 277: 494-498Crossref PubMed Scopus (447) Google Scholar). All sequences were confirmed by DNA sequencing. The previously generated Phex full-length cDNA coding sequence (Phex-WT) and the Phex-3′ truncated mutant cDNAs in pBSK (3Guo R. Quarles L.D. J. Bone Miner. Res. 1997; 12: 1009-1017Crossref PubMed Scopus (107) Google Scholar) were subcloned in pcDNA3.1/V5-His vector by a PCR approach using the 5′-GTGATGGAAGCAGAAACAGGGAG forward and 5′-CCAGAGTCGGCAAGAATCTGCA reverse primers for the full-length Phex (pcDNA3.1-Phex-V5-His) and 5′-GTGATGGAAGCAGAAACAGGGAG forward and 5′-TTCCTTCTTATCCTCCTGGAAG reverse primers for the 3′ deletion mutant (3′M) Phex (pcDNA3.1-Phex3′M-V5-His). Epitope-tagged recombinant Phex proteins were also produced in Sf9 cells as described previously (35Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. Metab. 2001; 281: E837-E847Crossref PubMed Google Scholar). Briefly, the full-length mouse Phex cDNA (35Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. Metab. 2001; 281: E837-E847Crossref PubMed Google Scholar) was subcloned into the NotI and ApaI sites of pMT/V5-His vector (Invitrogen) to create a cassette containing Phex in-frame with V5 and histidine epitope tags at its C-terminal end (pMT-Phex-WT-V5-His), and Phex-WT-V5-His was subcloned in pFASTBac1 vector. Preparation of Phex-expressing Membrane Protein—We confirmed the activity of Phex against oligopeptide substrates using recombinant Phex expressed in Sf9 membranes. Crude Sf9 membranes expressing Phex were solubilized with 1% n-dodecyl-β-d-maltoside, collected after centrifugation at 10,000 × g for 15 min and stored at –70 °C in multiple aliquots. The protein content of each sample was determined by the NanoOrange™ protein quantitation kit (Molecular Probes, Eugene, OR). Cell Culture and Transfections—We evaluated Phex-dependent hydrolysis of recombinant FGF23 in co-transfection and co-culture models. COS-7 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. We transiently transfected pFLAG-FGF23-His, pFLAG-FGF23R179Q-His, pFLAG-C-FGF23-His, pFLAG-N-FGF23-His, pcDNA3.1-Phex-V5-His, or corresponding vector control plasmids in COS-7 cells. All transfections were performed with the FuGENE 6 transfection reagent (Roche Applied Science) following the manufacturer's protocols. In addition, COS-7 cells were co-transfected with FG23 expression constructs (1 μg) and either pcDNA3.1-Phex-V5-His or pcDNA3.1/V5-His vector (1 μg). A FuGENE 6 to DNA ratio of 3 μl:1 μg was maintained for all experiments. The cells and conditioned media were harvested 48 h post-transfection. For the generation of stable cell lines overexpressing FGF23, COS-7 cells were transfected with pFLAG-FGF23-His and cultured in the presence of G418 (500 μg/ml) for 14 days. TMOb-Nl and TMOb-Hyp immortalized cells derived from normal and Hyp mice calvaria (38Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar) were grown for periods of up to 14 days in α-minimum essential medium containing 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO) containing 5 mm β-glycerophosphate and 25 μg/ml ascorbic acid as reported previously (38Xiao Z.S. Crenshaw M. Guo R. Nesbitt T. Drezner M.K. Quarles L.D. Am. J. Physiol. 1998; 275: E700-E708Crossref PubMed Google Scholar). Assessment of FGF23 Hydrolysis in Culture—To assess endogenous FGF23 hydrolysis in the absence of Phex, we collected total cell lysates and conditioned media from COS-7 cells after transient and stable transfection with FGF23 expression constructs. To assess Phex-dependent hydrolysis of FGF23, we collected conditioned media from COS-7 cells following co-transfected with pFLAG-FGF23-His and pcDNA3.1-Phex-V5-His constructs. In addition, we plated COS-7 cells producing FGF23 (1 × 105 cells/well) with COS-7 cells expressing Phex (1 × 105 cells/well) or cells transfected with the control vector and incubated for 24 h in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The co-cultures were washed with Hanks' balanced salt solution, and conditioned media were collected after incubation for 48 h in serum-free Dulbecco's modified Eagle's medium. The conditioned medium was centrifuged at 7000 × g for 10 min to remove cells and cellular debris, and FGF23 hydrolysis was monitored by Western blot analysis (see below). For the inhibitor studies, COS-7 cells stably transfected with pFLAG-FGF23-His were seeded in 6-well plates. After 48 h the medium was replaced with serum-free media containing different concentrations of the furin inhibitor dec-RVKR-cmk (39Leitlin J. Aulwurn S. Waltereit R. Naumann U. Wagenknecht B. Garten W. Weller M. Plannen M. Am. Assoc. Immunol. 2001; 166: 7238-7243Google Scholar, 40Ntayi C. Lorimier S. Berthier-Vergnes O. Horneback W. Bernard P. Exp. Cell Res. 2001; 270: 110-118Crossref PubMed Scopus (39) Google Scholar) (BIOMOL Research Laboratories, Inc., Plymouth Meeting, PA) or equivalent amounts of Me2SO vehicle. The cells were cultured for an additional 24 h and then the conditioned medium was collected for Western blot analysis. FGF23 Cleavage by Rabbit Reticulocyte Lysates—To evaluate Phex-dependent hydrolysis of FGF23 in vitro, we generated recombinant proteins using the rabbit reticulocyte lysate system. For these studies, pcDNA3.1-Phex-V5-His and pcDNA3.1-Phex-3′M-V5-His, pcDNA3.1-FGF23-V5-His, and pcDNA3.1-FGF23R179Q-V5-His were transcribed in vitro and translated in rabbit reticulocyte lysates using the TnT T7 Quick Coupled Transcription/Translation System (Promega) following the manufacturer's instructions. Aliquots (5 μl) of the reaction from the in vitro translated pcDNA 3.1/V5-His vector, full-length, or 3′ mutant Phex were mixed 1:1 with human wild-type or R179Q mutant FGF23-V5-His epitope-tagged proteins and incubated for 30 min at 37 °C. The incubation mixtures were subsequently analyzed by immunoblot using anti-V5 antibody conjugated to horseradish peroxidase (Invitrogen) and visualized by enhanced chemiluminescence (Pierce). Assessing Phex Enzyme Activity in Vitro—We assessed Phex activity using either the Z-Ala-Ala-Leu-p-nitroanilide (ZAAL-pNA) chromogenic substrate (35Guo R. Liu S. Spurney R.F. Quarles L.D. Am. J. Physiol. Endocrinol. Metab. 2001; 281: E837-E847Crossref PubMed Google Scholar) or a fAFF1-TC-LIB phage clone containing the VPQSDS peptide sequence cloned between an epitope (YGGFL) tether and a truncated form of M13 gene (40Ntayi C. Lorimier S. Berthier-Vergnes O. Horneback W. Bernard P. Exp. Cell Res. 2001; 270: 110-118Crossref PubMed Scopus (39) Google Scholar, 41Quarles L.D. Guo R. J. Am. Soc. Nephrol. 2000; 11 (A3002): 569AGoogle Scholar). ZAAL-pNA was purchased from Bachem Biosciences, Inc. (King of Prussia, PA). Leucine aminopeptidase was purchased from Sigma-Aldrich. We used membrane fractions from the Sf9 cells (50 μg) expressing vector or the V5-His epitope-tagged Phex constructs. Protein samples were incubated with 50 μm ZAAL-pNA in 100 μl of 100 mm MES, pH 6.5, for 1 h at 37 °C. After completion of the initial incubation, the reaction mixture was further incubated with 0.4 milliunits of leucine aminopeptidase for 20 min at 37 °C. The reaction was stopped by the addition of 100 mm EDTA, and the absorbency was measured at 405 nm after centrifugation. In some studies membrane fractions were preincubated with inhibitors EDTA, phosphoramidon, or dec-RVKR-cmk at the stated concentrations for 30 min before the addition of ZAAL-pNA. For dot blot analysis of Phex activity, we used a phage clone containing the hexapeptide VPQSDS sequences demonstrated previously (41Quarles L.D. Guo R. J. Am. Soc. Nephrol. 2000; 11 (A3002): 569AGoogle Scholar, 42Smith M.M. Shi L. Navre M. J. Biol. Chem. 1995; 270: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 43Campos M. Couture C. Hirata I.Y. Juliano M.A. Loisel T.P. Crine P. Juliano L. Boileau G. Carmona A.K. Biochem. J. 2003; 373: 271-279Crossref PubMed Scopus (95) Google Scholar) to be optimal for Phex-dependent hydrolysis. The VPQSDS-expressing phage was amplified overnight in Escherichia coli K91 cells and precipitated by adding 20 μl of 20% polyethylene glycol in 2.5 m NaCl to 100 μl of phage supernatant as described previously (42Smith M.M. Shi L. Navre M. J. Biol. Chem. 1995; 270: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). After incubating on ice for 30 min, the precipitated phage were microfuged for 5 min and resuspended in 10 μl of TBS (50 mm Tris-HCl, pH 7.4, 150 mm NaCl). The phage suspension was incubated with Sf9 membranes (30 μg) expressing Phex-WT or vector control membranes at 37 °C in a total volume of 100 μl. At various time points, 30-μl aliquots of the reaction was removed and stopped by adding TBS buffer containing 5 mm EDTA. Aliquots were transferred on n