Submitted on May 21, 2008
Revised on July 16, 2008
Accepted on August 3, 2008
Molecular Genetic and Biochemical Analyses of FGF23 Mutations in Familial Tumoral Calcinosis
Holly Garringer, Mahdi Malekpour, Fatemehsadat Esteghamat, Seyed Mortazavi, Siobhan Davis, Emily Farrow, Xijie Yu, D.E. Arking1, Harry C. Dietz2, and Kenneth E. White3*
1 Johns Hopkins University
2 Johns Hopkins University School of Medicine
3 Indiana Univ. Sch. of Med.
* To whom correspondence should be addressed. E-mail: kenewhit{at}iupui.edu.
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) (160C>A) at residue 54 (Q54K). To understand the molecular consequences of all known FGF23-TC mutants (H41Q, S71G, M96T, S129F, 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 C-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 to wild type protein. In examining the role of the FGF23 C-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 (176RXXR179/S180) were not required for protein secretion. However, residues adjacent to the RXXR site (between residues 188-202) were required for full bioactivity. In sum, 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 C-terminal tail of FGF23 is not required for protein secretion, but is required for full bioactivity.
