Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23

Kenneth White, G. Carn, B. Lorenz-Depiereux, A. Benet-Pages, T. M. Strom, Michael Econs

Research output: Contribution to journalArticle

361 Citations (Scopus)

Abstract

Background. The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23. Methods. The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses. Results. Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin. Conclusions. FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.

Original languageEnglish
Pages (from-to)2079-2086
Number of pages8
JournalKidney International
Volume60
Issue number6
DOIs
StatePublished - 2001

Fingerprint

Mutation
Proteins
Fibroblast Growth Factors
HEK293 Cells
fibroblast growth factor 23
Autosomal Dominant Hypophosphatemic Rickets
Peptide Hydrolases
Phosphates
Proprotein Convertases
Antibodies
Missense Mutation
Conditioned Culture Medium
Site-Directed Mutagenesis
Proteolysis
Heparin
Complementary DNA
Clone Cells
Kidney
Peptides
Genes

Keywords

  • 12p13
  • Chromosome 12
  • FGF23
  • Fibroblast growth factor
  • Hypophosphatemia
  • Proprotein convertase
  • Renal phosphate wasting
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Nephrology

Cite this

Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. / White, Kenneth; Carn, G.; Lorenz-Depiereux, B.; Benet-Pages, A.; Strom, T. M.; Econs, Michael.

In: Kidney International, Vol. 60, No. 6, 2001, p. 2079-2086.

Research output: Contribution to journalArticle

White, Kenneth ; Carn, G. ; Lorenz-Depiereux, B. ; Benet-Pages, A. ; Strom, T. M. ; Econs, Michael. / Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. In: Kidney International. 2001 ; Vol. 60, No. 6. pp. 2079-2086.
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abstract = "Background. The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23. Methods. The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses. Results. Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin. Conclusions. FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.",
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T1 - Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23

AU - White, Kenneth

AU - Carn, G.

AU - Lorenz-Depiereux, B.

AU - Benet-Pages, A.

AU - Strom, T. M.

AU - Econs, Michael

PY - 2001

Y1 - 2001

N2 - Background. The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23. Methods. The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses. Results. Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin. Conclusions. FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.

AB - Background. The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23. Methods. The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses. Results. Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin. Conclusions. FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.

KW - 12p13

KW - Chromosome 12

KW - FGF23

KW - Fibroblast growth factor

KW - Hypophosphatemia

KW - Proprotein convertase

KW - Renal phosphate wasting

KW - Site-directed mutagenesis

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U2 - 10.1046/j.1523-1755.2001.00064.x

DO - 10.1046/j.1523-1755.2001.00064.x

M3 - Article

C2 - 11737582

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VL - 60

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JO - Kidney International

JF - Kidney International

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