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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 5 2213-2222
Copyright © 2003 by The Endocrine Society

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

Yves Sabbagh, Guy Boileau, Marcelo Campos, Adriana K. Carmona and Harriet S. Tenenhouse

Departments of Biology (Y.S., H.S.T.), Pediatrics (H.S.T.), and Human Genetics (H.S.T.), McGill University, and The McGill University–Montreal Children’s Hospital Research Institute (Y.S., H.S.T.), Montreal, Quebec, Canada H3Z 2Z3; Department of Biochemistry (G.B.), Université de Montréal, Montréal, Quebec H3C 3J7, Canada; and Department of Biophysics (M.C., A.K.C.), Universidade Federal de São Paulo, Escola Paulista de Medicina, 04044-020, São Paulo, Brazil

Address all correspondence and requests for reprints to: Dr. Harriet S. Tenenhouse, Montreal Children’s Hospital Research Institute, 4060 Ste-Catherine Street West, Room 222, Montreal, Quebec, Canada H3Z 2Z3. E-mail: mdht{at}debelle.mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
X-LINKED HYPOPHOSPHATEMIA (XLH), a dominant disorder of phosphate homeostasis, is the most prevalent form of inherited rickets (1). The disease is characterized by growth retardation, defective bone mineralization, hypophosphatemia secondary to renal phosphate wasting, and an inappropriately normal serum concentration of 1,25-dihydroxyvitamin D. What distinguishes XLH from other inherited hypophosphatemias is its high prevalence (1 in 20,000), its X-linked dominant mode of inheritance, and the availability of two murine homologs, Hyp (2) and Gy (3), that have served as models to study the pathophysiology of the human disease (4).

The gene that is mutated in XLH patients was identified by positional cloning and designated PHEX (formerly PEX) to depict a phosphate-regulating gene with homology to endopeptidases on the X chromosome (5). The PHEX gene spans approximately 243 kb, comprises 22 exons, and encodes a protein of 749 amino acids (Fig. 1Go; Ref. 6). PHEX exhibits significant homology to members of the M13 family of zinc metallopeptidases, which includes among others neutral endopeptidase 24.11 (NEP; Ref. 7), endothelin-converting enzymes 1 (ECE-1; Ref. 8) and 2 (ECE-2; Ref. 9), and the KELL antigen (10). These enzymes are type II integral membrane glycoproteins characterized by a short NH2-terminal cytoplasmic domain, a single transmembrane hydrophobic region, and a large extracellular domain (Fig. 1Go; Ref. 11). The latter includes 10 highly conserved cysteine residues and a zinc-binding motif (Fig. 1Go), which in the case of NEP and ECE-1 are essential for conformational integrity and catalytic activity, respectively (11).



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  		Figure 1. Location of PHEX missense mutations investigated in the present study. Shown are the 22 exons of the PHEXcDNA (bottom) and the structural domains of the PHEX protein (top). These include the short N-terminal cytoplasmic region, the single transmembrane domain, and the large extracellular domain containing the zinc-binding motif (Zn) and conserved cysteine residues (c). Mutations examined in the present study all reside in the extracellular domain.

 
The M13 zinc metallopeptidases have diverse physiological functions. NEP plays a role in blood pressure regulation and pain control by inactivating several small peptides, including substance P, bradykinin, atrial natriuretic peptide, and enkephalins (7). NEP action in vivo has been attributed to both its tissue distribution and that of its substrates (7). In contrast, ECE-1 and ECE-2 play a role in the regulation of vascular tone and function by activating big endothelin-1 to endothelin-1 (8, 9, 11).

PHEX mRNA and protein are predominantly expressed in osteoblasts and odontoblasts, but not in kidney (12, 13, 14, 15, 16, 17). These data are consistent with studies in the murine Hyp homolog of XLH, in which both a primary mineralization defect (18) and a renal phosphate leak, attributed to a circulating factor (19, 20, 21), have been documented. Several studies have examined the developmental expression of Phex mRNA and protein. Phex mRNA expression was observed in osteoblasts in developing bone on embryonic day 15, just half a day after the onset of ossification, and in teeth on embryonic day 19 (14). In addition, Phex protein expression was demonstrated in osteogenic precursors in developing vertebral bodies and developing long bones on d 16 postcoitum and thereafter, and in osteoblasts from calvaria of d 18 postcoitum mice (17). On the basis of the tissue distribution of PHEX and the reported actions of NEP and ECE-1 and ECE-2 (7, 8, 9, 11), it was postulated that PHEX plays a role in the activation or inactivation of peptide factors that play a role in osteoblast differentiation and/or mineralization (4). In addition, these factors may also be involved in the regulation of renal phosphate transport and vitamin D metabolism (4). However, endogenous PHEX substrates have not yet been identified.

To date, 171 distinct mutations in the PHEX gene have been reported in patients with XLH (http://www.phexdb.mcgill.ca; Ref. 22). The mutations are scattered throughout the gene, and most are consistent with loss of function of the PHEX protein (22). Missense mutations account for 22% of all PHEX mutations, and previous analysis of three disease-causing missense mutations in PHEX revealed endoplasmic reticulum (ER) sequestration of the mutant proteins in contrast to plasma membrane localization of the wild-type protein (23).

The present study was undertaken to examine the effect of nine PHEX missense mutations (Fig. 1Go) on cellular trafficking, glycosylation, endopeptidase activity, and conformation of the recombinant proteins. The mutations examined included C85R, D237G, Y317F, G579V, G579R, S711R, A720T, and F731Y, identified in XLH patients (http://www.phexdb.mcgill.ca), and E581V, previously engineered in NEP where it completely abrogated catalytic activity but did not interfere with cell-surface localization of the recombinant protein (24). Soluble and secreted forms of wild-type and mutant PHEX proteins were expressed in HEK (293) cells, and the recombinant proteins in cell culture media were assessed for catalytic activity. The rationale for this approach derives from our previous report that a soluble and secreted form of the wild-type PHEX protein (secPHEX) hydrolyzed PTHrP107–139 (25). In addition, genetically engineered soluble and secreted forms of NEP (26) and ECE-1 (27) exhibit catalytic activity and kinetic parameters that are indistinguishable from their respective membrane-associated counterparts. We also evaluated the effect of the mutations on protein conformation by examining the sensitivity of mutant secPHEX proteins to protease digestion.

We demonstrate that the C85R, G579V, G579R, S711R, and A720T mutations interfere with proper targeting of the recombinant protein and that low temperature is able to rescue the G579V, S711R, and A720T mutant proteins from the cell into the media. In addition, using an internally quenched fluorogenic peptide substrate, we show that the mutant D237G, Y317F, A720T, and F731Y proteins retain considerable endopeptidase activity, whereas mutant E581V and S711R proteins are devoid of catalytic activity. Finally, we suggest that differential sensitivity of wild-type and mutant D237G and F731Y proteins to protease digestion is indicative of conformational differences of the mutant proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Construction of cDNAs encoding secPHEX mutants

PHEX missense mutations, 253T>C (C85R), 710A>G (D237G), 950A>T (Y317F), 1735G>C (G579R), 1736G>T (G579V), 2133T>A (S711R), 2158G>A (A720T), and 2192T>A (F731Y) identified in XLH patients, and 1742T>A (E581V), investigated in NEP (24) and PHEX (23, 25), were generated by PCR-mutagenesis (28) of a cDNA encoding secPHEX (25) as previously described (23). Wild-type and mutant cDNAs were subcloned in plasmid pCDNA3 (Invitrogen, Carlsbad, CA) with the Rous sarcoma virus promoter for transfection in mammalian cells (29).

Cell transfection

HEK (293) cells (CRL-1573, ATCC, Manassas, VA) were grown in DMEM (Invitrogen, Burlington, Ontario, Canada) containing 10% fetal bovine serum at 37 C in 5% CO2 and 95% air. Transfection of cDNAs was accomplished using the calcium-phosphate coprecipitation method (29), as previously described (23).

SDS-PAGE and immunoblot analysis

Equivalent volumes of whole cell lysates or culture medium, concentrated using centriprep-50 columns according to the manufacturer’s recommendations (Millipore Corp., Bedford, MA), were suspended in 6x Laemmli sample buffer (30) and boiled for 3 min. Proteins were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes for 1 h. PHEX proteins were visualized by incubation with a mouse monoclonal anti-PHEX antibody (BioMep Inc., Montreal, Quebec, Canada; Ref. 25) at a 1:200 dilution as previously described (15, 23).

Endoglycosidase digestion

Recombinant secPHEX proteins in whole cell extracts and in the media were boiled for 10 min in 10x denaturation buffer (5% SDS, 10% ß-mercaptoethanol) and incubated for 1 h at 37 C with endoglycosidase H (endo H), according to the manufacturer’s recommendations (New England Biolabs, Inc. Mississauga, Ontario, Canada) as previously described (23). Digestion products were fractionated on 10% SDS-PAGE and subjected to immunoblot analyses as described above.

Assay of secPHEX endopeptidase activity

secPHEX cleaved PTHrP107–139 at three different positions, all at the amino terminus of Asp residues (25). On the basis of this observation, we selected a peptide, ortho-aminobenzoic acid (Abz)-GFRDWK[2,4-dinitrophenyl(Dnp)]-OH, containing a putative secPHEX cleavage site, from a collection of intramolecularly quenched fluorogenic peptides synthesized by methods already described (31, 32), with the exception that Abz/Dnp was used as fluorescence donor/acceptor pair. The peptide was tested for cleavage by purified secPHEX in 50 mM 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.5) containing 150 mM NaCl and 0.001% BSA. The reaction was followed by measuring the fluorescence at {lambda}ex = 320 nm and {lambda}em = 420 nm in a Hitachi F-2000 spectrofluorometer (Hitachi Scientific Instruments, Inc., Tokyo, Japan). The slope was converted into micromoles of substrate hydrolyzed per minute, and kinetic parameters, Km and kcat, were calculated by the nonlinear regression data analysis Grafit program (33). To determine the position of cleavage in peptide Abz-GFRDWK(Dnp)-OH, the products resulting from hydrolysis by secPHEX were submitted to N-terminal amino acid sequencing in a PPSQ-23 protein sequencer (Shimadzu, Tokyo, Japan).

The same fluorogenic peptide was used to compare the endopeptidase activity of the secreted wild-type and mutant PHEX proteins. For those mutant proteins that were not secreted at 37 C, an attempt was made to rescue them by growing the transfected cells at 26 C, as previously described (23). The media from the transfected cells was collected and concentrated using centriprep-50 columns (Millipore Corp.), and the secPHEX protein concentration was determined by densitometry of Western blots using known amounts of purified secPHEX protein (25) as a standard. The hydrolysis of the fluorogenic peptide by wild-type and mutant secPHEX proteins was carried out in 50 mM MES buffer (pH 6.5) containing 150 mM NaCl and 0.001% BSA in the presence of 30 µM fluorogenic peptide, unless otherwise indicated. The reaction was monitored for 30 min by measuring the fluorescence at {lambda}ex = 320 nm and {lambda}em = 420 nm in a Shimadzu RF-1501 spectrofluorometer (Shimadzu Corp., Kyoto, Japan), and reaction rates were determined by measuring the slope of the linear portion of the curve. For inhibition studies, 5 mM EDTA or 10 mM phosphate was added to the reaction. For heat stability studies, secPHEX proteins were incubated at the different temperatures (25, 40, 45, 50, and 55 C) for 5 min, and the samples were placed on ice before assaying for activity as described above.

Polymorphism screen

To determine whether the F731Y (2192T>A) missense mutation is a disease-causing mutation rather than a neutral polymorphism, 120 normal chromosomes were analyzed for the mutation. The ethnic backgrounds of the 60 individuals were British/Irish (n = 20), Italian (n = 20), and French-Canadian (n = 20). Informed consent for DNA was obtained from the subjects, and approval for use of DNA was obtained from the local Institutional Review Board. The DNA was amplified by PCR using primers on either side of the mutation in exon 22: sense primer F22 (5'-CAGAACCTGTTGATGTGCAAGA-3'); antisense primer R22 (5'-GTCTCAGGATGCCATAAACCAGC-3'). The amplified products were digested with the restriction enzyme MseI for 2 h at 37 C and analyzed on 2% low melting point-agarose. Digestion of the 191-bp amplified product with MseI will generate fragments of 103 and 88 bp if the mutation is absent.

Endoproteolytic digestions

SecPHEX proteins were subjected to either trypsin (0, 80, 100, 200, 400, 600, and 800 ng) or endoproteinase Glu-c (0, 100, 200, 400, 600, 800, and 1000 ng) digestion (20 µl reaction volume) for 1 h at room temperature. Trypsin digestion was stopped by the addition of 2x Laemmli sample buffer (30) and boiling for 3 min. The endoproteinase Glu-c digestion was stopped by the addition phenylmethylsulfonyl fluoride (0.6 µg/µl final concentration). The digests were loaded on 10% SDS-PAGE and analyzed by Western blotting as described above. The autoradiographs were scanned, and the disappearance of the 97-kDa band was quantified by image analysis using the Quantity One V.4.2.1 program (Bio-Rad Laboratories, Inc., Philadelphia, PA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of wild-type and mutant secPHEX cDNAs

HEK (293) cells were transfected with wild-type or mutant secPHEX cDNAs, and media and cell lysates were examined by immunoblotting using a PHEX-specific monoclonal antibody. An approximately 97-kDa protein band was detected in media of cells expressing the wild-type and D237G, Y317F, E581V, and F731Y mutant proteins (Fig. 2AGo). In contrast, media from cells expressing C85R, G579R, G579V, S711R, and A720T mutants were devoid of secreted PHEX protein (Fig. 2AGo). Analysis of the corresponding cell lysates revealed a band at approximately 93 kDa that was sensitive to endo H digestion (Fig. 2AGo), indicating that the protein comprised the core-glycosylated form of secPHEX present in the ER. In contrast, proteins secreted at 37 C were resistant to endo H digestion, indicating that these secPHEX proteins are fully glycosylated and present in their mature forms (Fig. 2AGo).



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  		Figure 2. Expression and glycosylation of the recombinant wild-type (WT) and mutant secPHEX proteins in HEK (293) cells. A, Whole cell lysates and concentrated media from HEK (293) cells, transiently transfected with wild-type or mutant secPHEX cDNAs and grown at 37 C, were incubated with either buffer or endo H. B, Concentrated media from cells grown at 26 C were incubated with either buffer or endo H, as described in Materials and Methods. The samples were fractionated on 10% SDS-PAGE, and analyzed by immunoblotting with anti-PHEX antibodies. *, Exposure for 75 min using ECL Plus kit.

 
We previously reported that PHEX proteins with trafficking defects arising from missense mutations in the PHEX gene could be rescued from the ER to the plasma membrane by growing the transfected cells at 26 C (23). We thus attempted to rescue those secPHEX mutants that were trapped in the cell at 37 C by growing the cells at 26 C. Of the five mutant PHEX proteins retained intracellularly, only three, G579V, S711R, and A720T, were rescued, as evident by the appearance of an approximately 97-kDa band in the medium (Fig. 2BGo). Rescued G579V, S711R, and A720T secPHEX proteins acquired resistance to endo H digestion (Fig. 2BGo), confirming that these secreted proteins were terminally glycosylated. No evidence for rescue of the C85R and G579R mutant proteins was obtained under these conditions (data not shown). The amount of rescued G579V secPHEX protein was insufficient for further analysis.

Endopeptidase activity of secPHEX proteins

Amino-terminal sequencing of the product resulting from digestion of Abz-GFRDWK(Dnp)-OH by purified secPHEX revealed that this substrate was hydrolyzed at the N terminal of the Asp residue, consistent with secPHEX cleavage of PTHrP107–139 (25). Km and kcat values were found to be 19.1 µM and 0.85 sec-1, respectively. The utility of the fluorogenic peptide substrate was then tested with culture medium of cells transfected with wild-type secPHEX. Hydrolysis of the fluorogenic peptide by wild-type secPHEX present in the culture medium was dependent on time of incubation (Fig. 3AGo), substrate concentration (Fig. 3BGo), and amount of secPHEX protein in the culture medium (Fig. 3CGo). As expected for a zinc metallopeptidase, secPHEX cleavage of the fluorogenic peptide was completely inhibited by 5 mM EDTA (Fig. 3AGo). Moreover, secPHEX activity was inhibited by 10 mM phosphate (Fig. 3AGo) and osteocalcin (data not shown), in agreement with secPHEX hydrolysis of PTHrP107–139 (25). In contrast, media from mock-transfected cells failed to hydrolyze the fluorogenic substrate, thereby confirming that the endopeptidase activity was entirely attributable to secPHEX (data not shown).



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  		Figure 3. Fluorometric assay for secPHEX activity. A, Time dependence of endopeptidase activity in the absence (dark solid line) and presence of 5 mM EDTA (gray line) or 10 mM phosphate (broken line) measured with 200 ng of secPHEX protein and 30 µM fluorogenic peptide substrate. B, Dependence of endopeptidase activity on fluorogenic peptide substrate concentration measured in the presence of 200 ng wild-type secPHEX protein. C, Dependence of endopeptidase activity on secPHEX protein concentration measured in the presence of 30 µM fluorogenic peptide substrate. F.U., Fluorometric units; V, velocity of the reaction.

 
Endopeptidase activity of wild-type secPHEX and secreted and rescued mutant proteins, D237G, Y317F, E581V, S711R, A720T, and F731Y, was determined and corrected for the amount of secPHEX protein in the incubation mixture as described in Materials and Methods. The secreted D237G and Y317F mutant proteins exhibited approximately 50–60% of wild-type secPHEX activity, indicating that both missense mutations interfere with catalytic function (Fig. 4Go). The secreted E581V mutant was devoid of catalytic activity, consistent with previous studies in which this mutation was introduced in NEP (24) and secPHEX (25). No substrate hydrolysis was observed with the rescued S711R mutant protein (Fig. 4Go), suggesting that this mutation interferes with both cellular trafficking and catalytic activity. The rescued A720T mutant exhibited full catalytic activity (Fig. 4Go), suggesting that the mutation interferes with cellular trafficking but not endopeptidase activity. Of interest was the demonstration that the secreted F731Y mutant protein exhibited full activity (Fig. 4Go), an unexpected finding for a disease-causing mutation (34).



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  		Figure 4. Endopeptidase activity of wild-type (WT) and mutant secPHEX proteins. secPHEX proteins (200 ng) were incubated with the fluorogenic peptide substrate (30 µM), and fluorescence was recorded every minute for 30 min. WT and mutant secPHEX protein concentration was determined by densitometry of Western blots, using known amounts of purified secPHEX protein (25 ) as a standard. The velocity (V) was calculated from the linear portion of the curve, and V for WT secPHEX was set at 100%. Bars depict the mean ± SD of three assays for each secPHEX protein. *, P < 0.01, effect of missense mutation.

 
Heat stability of F731Y secPHEX

To test the hypothesis that the F731Y mutant protein was less stable than wild-type secPHEX, heat stability studies were undertaken. The data in Fig. 5Go demonstrate that both the wild-type and mutant proteins exhibited similar heat inactivation profiles.



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  		Figure 5. Heat stability profile of wild-type and F731Y secPHEX proteins. Wild-type (black bars) and F731Y mutant (white bars) proteins (200 ng) were incubated at 25, 40, 45, 50, and 55 C for 5 min and assayed for endopeptidase activity with the fluorogenic peptide substrate (30 µM). The fluorescence was recorded every minute for 30 min, and the velocity (V) was calculated from the linear portion of the curve. A representative of two similar experiments is shown.

 
Screening for F731Y (2192T>A) in a normal population

To determine whether the F731Y mutation is a normal variant rather than a disease-causing mutation, 120 normal chromosomes were analyzed using a PCR-based diagnostic test. The 191-bp PCR-amplified fragment from exon 22 was digested with MseI to distinguish between the wild-type (cleaved by MseI to 103-bp and 88-bp fragments) and mutant alleles (resistant to MseI cleavage). All exon 22 PCR products were sensitive to MseI digestion (see Fig. 6Go for representative digests). Thus, the F731Y (2192T>A) mutation is not found in the normal population and is most likely a disease-causing mutation.



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  		Figure 6. Screening for the F731Y mutation in a normal population. Genomic DNA from 60 normal individuals was PCR amplified using exon 22 specific primers as described in Materials and Methods. The amplicon was subsequently digested with MseI restriction enzyme and analyzed on 2% low melting point-agarose. The F731Y mutation eliminates the restriction site. In all representative samples, the 191-bp amplicon was cleaved to 103-bp and 88-bp fragments.

 
Proteolysis of secPHEX proteins

We also examined the structural consequences of those secPHEX mutations (D237G, Y317F, and F731Y) that do not interfere with cellular trafficking and fail to abrogate completely endopeptidase activity. This was accomplished by comparing the sensitivity of the mutant and wild-type secPHEX proteins to digestion with trypsin or endoproteinase Glu-c. Both the digestion patterns and the disappearance of intact 97-kDa protein as a function of protease concentration were taken into account.

Western blots of tryptic digests revealed that the wild-type, D237G, and Y317F proteins have similar banding patterns, with significant digestion of the 97-kDa protein and the appearance of a lower molecular mass band (93 kDa) evident with 400 ng trypsin (Fig. 7AGo). In contrast, with the F731Y protein two lower molecular mass bands (93 and 56 kDa) are apparent with 200 ng trypsin (Fig. 7AGo). The difference in banding patterns suggests that the F731Y mutant has a different folding conformation than the wild-type, D237G, and Y317F mutants. Densitometric analysis of the 97-kDa band also revealed that the wild-type, D237G, and Y317F proteins exhibit similar sensitivities to trypsin digestion, whereas the F731Y mutant is significantly more sensitive at 80 and 100 ng trypsin (Fig. 7BGo). These data again suggest that the conformation of the F731Y mutant differs from the wild-type, D237G, and Y317F proteins.



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  		Figure 7. Tryptic digests of secPHEX proteins. A, Secreted wild-type (WT) and mutant secPHEX proteins (D237G, Y317F, and F731Y) were treated with increasing amounts of trypsin for 1 h at room temperature. The proteins were fractionated on 10% SDS-PAGE and analyzed by immunoblotting with anti-PHEX antibodies. B, The disappearance of the high molecular mass band was analyzed by densitometry. Means and SD of three experiments are shown. Effect of F731Y mutation: #, P < 0.043; *, P < 0.021.

 
Additional studies with endoproteinase Glu-c also revealed differences in sensitivity of the wild-type and mutant secPHEX proteins (Fig. 8Go). With the wild-type protein, the appearance of a 93-kDa band at 200 ng protease and the disappearance of the 97-kDa band at 600 ng protease were evident. A similar pattern was observed with Y317F (Fig. 8AGo). Interestingly, D237G exhibited decreased sensitivity to protease digestion, with the disappearance of the 97-kDa band evident only at 1000 ng endoproteinase Glu-c (Fig. 8AGo). The F731Y mutant exhibited a different degradative profile, with no evidence of a lower molecular mass band (Fig. 8AGo). Densitometric analysis of the 97-kDa band revealed a significant decrease in the sensitivity of D237G and no significant difference in the degradation of the Y317F and F731Y proteins, relative to wild-type secPHEX (Fig. 8BGo). Taken together, the protease sensitivity results suggest that both the D237G and F731Y mutants may have a different conformation than wild-type secPHEX.



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  		Figure 8. Endoproteinase Glu-c digest of secPHEX proteins. A, Secreted wild-type (WT) and mutant secPHEX proteins (D237G, Y317F, and F731Y) were treated with increasing amounts of endoproteinase Glu-c for 1 h at room temperature. The proteins were fractionated on 10% SDS-PAGE and analyzed by immunoblotting with anti-PHEX antibodies. B, The disappearance of the high molecular mass band was analyzed by densitometry. Means and SD of three experiments are shown. Effect of D237G mutation: *, P < 0.0098; #, P < 0.0013; {ddagger}, P < 0.032; ¶, P < 0.030.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we examined the impact of disease-causing PHEX missense mutations on cellular processing, endopeptidase activity, and conformation of the recombinant proteins. To measure endopeptidase activity without interference by other peptidases that are abundant in the plasma membrane, the proteins were expressed in a soluble and secreted form. This approach was validated in previous studies in which it was demonstrated that a soluble and secreted form of NEP (26) and ECE-1 (27) retained full activity with similar kinetic parameters to the native enzyme. In addition, a soluble and secreted form of PHEX was genetically engineered by modifying the signal peptide/membrane anchor domain into a cleavage-competent signal sequence and was previously shown to have endopeptidase activity toward PTHrP107–139 (25). Finally, we report that a novel fluorogenic peptide is hydrolyzed by purified secPHEX and demonstrated its utility to assess the effect of PHEX missense mutations on endopeptidase activity.

We show that, like wild-type secPHEX, the XLH mutant proteins D237G, Y317F, and F731Y and the control mutant E581V are normally secreted from the cells at 37 C and are fully glycosylated, as assessed by their resistance to endo H digestion (Fig. 2AGo). These data suggest that loss of PHEX function in these XLH mutants cannot be ascribed to a cellular trafficking defect. In contrast, the C85R, G579R, G579V, S711R, and A720T mutant proteins are sequestered intracellularly and are sensitive to endo H digestion (Fig. 2AGo), which would explain the loss of PHEX function in patients harboring these mutations. It is important to note that our findings are consistent with a previous study in which we reported that the native, nonsecreted forms of the mutant C85R, G579R, and S711R proteins were also retained in the ER, whereas the corresponding E581V protein was normally transported to the plasma membrane (23), in agreement with studies of this mutation in NEP (24). Our results thus indicate that cellular trafficking of the engineered soluble and secreted PHEX proteins is similar to that of the native membrane-bound forms.

The function of the XLH mutant proteins that were normally transported to the cell surface, i.e. D237G, Y317F, and F731Y, was assessed using a fluorogenic enzymatic assay to compare their endopeptidase activity to that of wild-type secPHEX. All three mutant proteins showed significant enzymatic activity. The demonstration of 50% residual activity for D237G and Y317F is consistent with the dominant mode of inheritance of XLH (1) and the report that heterozygous females exhibit the full disease phenotype despite the presence of one normal allele (35). Similarly, mutant heterozygous Hyp female mice are as severely affected as mutant hemizygous males and mutant homozygous females (36). The absence of a gene dose effect suggests that a threshold of PHEX activity must be reached to maintain normal osteoblast function. A dominant negative effect of the mutant membrane protein is unlikely in heterozygous female patients and mice because the process of X-inactivation is random (37), and each cell would thus express either the normal or the mutant allele.

Mutant F731Y was normally secreted at 37 C and exhibited approximately 90% of wild-type activity. These results were unexpected because this mutation was identified in a patient with XLH (34). Moreover, our failure to detect the mutation in the normal population strongly suggests that F731Y is truly responsible for XLH. One interpretation for our findings is that the synthetic fluorogenic peptide substrate used in our assay might not reflect the interaction of the mutant protein with its endogenous substrates, which have not yet been identified. Indeed, studies of NEP hydrolysis of different fluorogenic substrates have shown that the kinetic parameters are dependent on the environment and conformation of the amino acid residues around the cleavage site (38). We demonstrated that secPHEX has a relatively low affinity for the fluorogenic substrate (19.1 µM), and this may not permit the detection of subtle differences between wild-type and some mutant PHEX proteins. Indeed, synthetic substrates used for diagnosis of Tay-Sachs disease do not distinguish between heterozygotes for the pseudodeficiency allele and Tay-Sachs disease carriers (39), and the natural substrate remains the most specific method for determining Hex A activity (40). In addition, a study of mutations in the tissue-nonspecific alkaline phosphatase gene responsible for hypophosphatasia reported that catalytic activity of mutant proteins depends on the substrate used (41). For example, one mutant protein, A160T, exhibited decreased catalytic efficiency toward the artificial substrate, p-nitrophenylphosphate, and normal or increased activity, respectively, with the natural substrates inorganic pyrophosphate and pyridoxal-5'-phosphate (41). Thus, further work is necessary to test the activity of the F731Y PHEX mutant with endogenous PHEX substrate(s) as well as other artificial substrates.

We also assessed the effect of the D237G, Y317F, and F731Y mutations on protein conformation by subjecting the proteins to limited protease digestion with trypsin or endoproteinase Glu-c. Our data demonstrate that the wild-type, D237G, and Y317F proteins exhibit similar sensitivity to trypsin proteolysis, whereas F731Y is significantly more sensitive to proteolytic digestion. Furthermore, the banding patterns obtained with the F731Y mutant and wild-type secPHEX proteins are different, suggesting that the mutation may alter the conformation of the protein. In contrast, the sensitivity of D237G to endoproteinase Glu-c was decreased when compared with wild-type PHEX. These results are difficult to explain at the molecular level in the absence of a PHEX crystal structure. However, one can speculate that the mutations interfere with interactions essential to maintain the integrity of the protein structure and as such result in altered sensitivity to protease digestion and catalytic activity. In this regard, it is of interest that mutations in the calcium binding domains of fibrillin-1, which are responsible for Marfan syndrome, alter the sensitivity of the protein to protease digestion, thereby providing a mechanism for the disease phenotype (42). Alternatively, conformational changes in D237G and F731Y mutants detected by endoprotease digestion may prevent interaction of the PHEX ectodomain with putative partners at the cell surface. In this regard, it is of interest that KELL, another member of the M13 family of zinc metallopeptidase, has been shown to be associated with a membrane protein XK, which is mutated in patients with McLeod syndrome, a rare X-linked disorder (43, 44). The functional role of this association, however, remains to be determined.

We previously demonstrated that mutant PHEX proteins sequestered in the ER could be partially rescued by growing the corresponding transfected cells at 26 C (23). In the present study, we show that three of the trapped secPHEX mutants, G579V, S711R, and A720T, were rescued out of the cells and into the medium at 26 C and achieved endo H resistance (Fig. 2BGo). The extent of rescue of S711R and A720T was comparable, and the yield of secreted protein was sufficient to assess endopeptidase activity. However, in the case of the G579V mutant protein, only partial rescue was achieved, and the level of expression in the media was too low for further analysis. It is of interest that when the same residue is mutated to an Arg (G579R), no rescue was achieved at 26 C. These data suggest that the efficiency of processing, folding and rescue at 26 C is highly dependent on the nature of the substituted amino acid. Our results are consistent with those reported for ER-trapped cystic fibrosis transmembrane regulator mutant proteins, where rescue of mutant proteins was also dependent on the nature of the substituted amino acid (45). These data also confirm that the misfolding of mutant proteins cannot be attributed to a general disruption of the ER quality control machinery.

Although the G579V mutant protein was not assayed for endopeptidase activity, we predict that the mutation is likely to interfere with catalytic activity because it is adjacent to His580, which in NEP was shown to be one of the histidine residues involved in zinc coordination (46). It has been proposed that proper orientation of these histidine residues is essential for zinc binding (47). One can speculate that replacement of the small glycine side chain (hydrogen atom) with the bulky valine hydrophobic side chain may change the orientation of His580 imidazole group and prevent zinc binding. The observation that the mutant protein is retained in the ER at 37 C is in agreement with the structural changes in PHEX expected from the replacement of glycine by valine. Similar effects on enzyme structure and activity are predicted from the replacement of Gly579 by Arg in mutant G579R.

The S711 residue involves the HSP (His-Ser-Pro) consensus sequence, which is highly conserved in the zinc metallopeptidase family. Using our fluorometric assay, we demonstrate that the S711R mutant protein is completely inactive after rescue at 26 C (Fig. 4Go). Mutations in the adjacent histidine residue, when studied in NEP, resulted in an inactive enzyme with similar binding parameters as the native protein. The perturbation in catalytic activity suggested that the histidine residue is involved in the stabilization of the transition state of the reaction by forming a hydrogen bond with the oxyanion of the tetrahedral intermediate (48). Therefore, it is not surprising that the S711R mutation has an effect on both the structure and function of secPHEX.

In contrast to S711R, we show that the rescued A720T mutant protein exhibits full endopeptidase activity. These data suggest that the A720T mutation alters the folding pattern of the protein at 37 C. However, when correct folding is achieved at 26 C, the enzyme is catalytically active. Thus, this mutation may be a target for novel therapeutic approaches.

In summary, this study represents the first characterization of the effect of PHEX missense mutations on endopeptidase activity using a novel fluorogenic peptide substrate. We demonstrate that some mutations in secPHEX abrogate catalytic activity, whereas others alter the trafficking and conformation of the protein. We thus provide a mechanism whereby missense mutations result in loss of function of the PHEX protein. The identification of endogenous PHEX substrate(s) is necessary to define the physiologically relevant kinetic parameters of wild-type and mutant proteins.


   Acknowledgments
 
We thank BioMep Inc. for the mouse monoclonal anti-PHEX antibody and Drs. Thomas Loisel and Isabelle Lemire and Constance Couture of BioMep Inc. for technical help in the fluorometric assay.


   Footnotes
 
This work was supported by grants from the Canadian Institutes of Health Research (MT-14107 to H.S.T. and MT-13052 to G.B.). Yves Sabbagh is the recipient of Studentship Awards from the Canadian Institutes of Health Research and FRSQ-FCAR-Santé.

Abbreviations: Abz, Aminobenzoic acid; Dnp, dinitrophenyl; ECE, endothelin-converting enzyme; endo H, endoglycosidase H; ER, endoplasmic reticulum; NEP, neutral endopeptidase 24.11; secPHEX, secreted form of the wild-type PHEX protein; XLH, X-linked hypophosphatemia.

Received November 18, 2002.

Accepted February 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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