title: Molecular genetic and biochemical analyses of FGF23 mutations in familial tumoral calcinosis.
abstract: Fibroblast growth factor 23 (FGF23) is a hormone required for normal renal phosphate reabsorption. FGF23 gain-of-function mutations result in autosomal dominant hypophosphatemic rickets (ADHR), and FGF23 loss-of-function mutations cause familial hyperphosphatemic tumoral calcinosis (TC). In this study, we identified a novel recessive FGF23 TC mutation, a lysine (K) substitution for glutamine (Q) (160 C>A) at residue 54 (Q54K). To understand the molecular consequences of all known FGF23-TC mutants (H41Q, S71G, M96T, S129F, and Q54K), these proteins were stably expressed in vitro. Western analyses revealed minimal amounts of secreted intact protein for all mutants, and ELISA analyses demonstrated high levels of secreted COOH-terminal FGF23 fragments but low amounts of intact protein, consistent with TC patients' FGF23 serum profiles. Mutant protein function was tested and showed residual, yet decreased, bioactivity compared with wild-type protein. In examining the role of the FGF23 COOH-terminal tail (residues 180-251) in protein processing and activity, truncated mutants revealed that the majority of the residues downstream from the known FGF23 SPC protease site ((176)RXXR(179)/S(180)) were not required for protein secretion. However, residues adjacent to the RXXR site (between residues 188 and 202) were required for full bioactivity. In summary, we report a novel TC mutation and demonstrate a common defect of reduced FGF23 stability for all known FGF23-TC mutants. Finally, the majority of the COOH-terminal tail of FGF23 is not required for protein secretion but is required for full bioactivity.
authors.affiliation: Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Holly J
authors.initials: HJ
authors.lastname: Garringer
authors.forename: Mahdi
authors.initials: M
authors.lastname: Malekpour
authors.forename: Fatemehsadat
authors.initials: F
authors.lastname: Esteghamat
authors.forename: Seyed M J
authors.initials: SM
authors.lastname: Mortazavi
authors.forename: Siobhan I
authors.initials: SI
authors.lastname: Davis
authors.forename: Emily G
authors.initials: EG
authors.lastname: Farrow
authors.forename: Xijie
authors.initials: X
authors.lastname: Yu
authors.forename: Dan E
authors.initials: DE
authors.lastname: Arking
authors.forename: Harry C
authors.initials: HC
authors.lastname: Dietz
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: White
mesh_terms: D000328:Adult; D015153:Blotting, Western; D002114:Calcinosis / Q000235:genetics* / Q000378:metabolism / Q000601:surgery; D002117:Calcitriol / Q000097:blood; D002648:Child; D002675:Child, Preschool; D004252:DNA Mutational Analysis; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis / Q000235:genetics; D005091:Exons / Q000235:genetics; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D054559:Hyperphosphatemia / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008967:Molecular Biology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics; D010281:Parathyroid Hormone / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D002117:Calcitriol; D051766:Early Growth Response Protein 1; D048049:Extracellular Signal-Regulated MAP Kinases; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors; D017350:N-Acetylgalactosaminyltransferases; D010281:Parathyroid Hormone; D000097763:Polypeptide N-acetylgalactosaminyltransferase; C497325:EGR1 protein, human; C000717427:FGF23 protein, human