title: FGF23-S129F mutant bypasses ER/Golgi to the circulation of hyperphosphatemic familial tumoral calcinosis patients.
abstract: FGF23 is essential for the homeostasis of phosphate, and vitamin D. Loss-of-function mutations in this hormone cause hyperphosphatemic familial tumoral calcinosis (HFTC). Earlier reports suggested that intact FGF23 from loss of function mutants such as FGF23/S129F (iFGF23/S129F) is retained intracellularly while the carboxy-terminal fragment is secreted. We sought to investigate the fate of iFGF23/S129F mutant hormone in vivo and in vitro. Five patients clinically diagnosed with HFTC and confirmed by DNA sequencing to carry the c.386 C>T; p.S129F mutation in the homozygous state were studied. Healthy and heterozygous individuals were used as controls in the study. Using ELISA assays, we showed that iFGF23/S129F was 2-5 folds higher in patients' plasma, compared to heterozygous or healthy controls. Importantly, the mutant hormone could not be detected in the patients' sera. However, using proteinase inhibition profiling, we found that a serum metalloproteinase degraded the iFGF23/S129F explaining our failure to detect it in sera. The serum metalloproteinase degrades the WT and the mutant at different rates. Also, confocal microscopy imaging using wild-type (WT) FGF23 or FGF23/S129F mutant in transiently transfected HEK293 and HeLa cells showed weak staining of the Golgi complex with some vesicular staining resembling the ER. Additionally, FGF23 variants (FGF23/WT, FGF23/S129F, FGF23/S71G, and FGF23/R176Q) from stably transfected HEK293 cells secreted high levels into a serum-free medium that can be detected by ELISA and Western blot. Our results suggest that iFGF23/S129F mutant bypasses the ER/Golgi quality control system to the circulation of HFTC patients by an unknown pathway. Finally, we hypothesize that either the mutant hormone is unable to bind α-Klotho-FGFR1c, or it binds the dyad receptor with low affinity and, therefore, incapable of initiating maximal intracellular signaling. Our findings raise the potential use of the WT hormone in therapies of some HFTC patients.
authors.affiliation: Life Sciences Department, Medical Biotechnology, Arabian Gulf University, Manama, Bahrain. Electronic address: saidms@agu.edu.bh.
authors.forename: Said M
authors.initials: SM
authors.lastname: Shawar
authors.affiliation: Life Sciences Department, Medical Biotechnology, Arabian Gulf University, Manama, Bahrain.
authors.forename: Ahmad R
authors.initials: AR
authors.lastname: Ramadan
authors.affiliation: Pathology Department, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Bassam R
authors.initials: BR
authors.lastname: Ali
authors.affiliation: Life Sciences Department, Medical Biotechnology, Arabian Gulf University, Manama, Bahrain.
authors.forename: Manal A
authors.initials: MA
authors.lastname: Alghamdi
authors.affiliation: Pathology Department, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Anne
authors.initials: A
authors.lastname: John
authors.affiliation: Life Sciences Department, Medical Biotechnology, Arabian Gulf University, Manama, Bahrain.
authors.forename: Ferial M
authors.initials: FM
authors.lastname: Hudaib
mesh_terms: D002114:Calcinosis / Q000097:blood / Q000235:genetics*; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D006056:Golgi Apparatus / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006720:Homozygote; D006801:Humans; D006958:Hyperostosis, Cortical, Congenital / Q000097:blood / Q000235:genetics*; D054559:Hyperphosphatemia / Q000097:blood / Q000235:genetics*; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D008954:Models, Biological; D050505:Mutant Proteins / Q000097:blood; D009154:Mutation / Q000235:genetics*; D021381:Protein Transport / Q000187:drug effects; D014162:Transfection
chemical_list: C000717427:FGF23 protein, human; D061965:Matrix Metalloproteinase Inhibitors; D050505:Mutant Proteins; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23
keywords: ER/Golgi bypass; FGF 23; FGF 23/S129F; Hormone therapy; Hyperphosphatemic familial tumoral calcinosis; Metalloproteinase; Mutant secretion