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Benet-Pagès, Anna (2006): Characterization of native FGF23 protein and mutant forms causing autosomal dominant hypophosphatemic rickets and familial tumoral calcinosis. Dissertation, LMU München: Faculty of Biology
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Abstract

The regulation of phosphate metabolism is a complex process that is still only partly understood. At the end of the eighties, studies in a mouse model for hypophosphatemic rickets provided evidence that phosphate wasting could not be explained by a primary defect of the kidney but rather by an unknown circulating factor with phosphaturic properties. X-linked hypophosphatemia (XLH), autosomal dominant hypophosphatemic rickets (ADHR), and tumor induced osteomalacia (TIO) are three well defined human disorders of isolated renal phosphate wasting. XLH and ADHR are mendelian diseases while TIO is caused by rare, mostly benign tumors. The opposite phenotype, hyperphosphatemia due to increased renal phosphate reabsorption is associated to the recessive disorder familial tumoral calcinosis (FTC). At the beginning of this work the genes mutated in XLH and ADHR were cloned. One gene codes for the endopeptidase PHEX, the other for the fibroblast growth factor FGF23. Both proteins are probably involved in a novel common pathway of the regulation of phosphate homeostasis. Missense mutations in FGF23 causing phosphate wasting in patients with ADHR, overexpression of FGF23 in tumors from patients with TIO, and the observation that FGF23 plasma levels are elevated in most patients with XLH provided strong evidence that FGF23 is a hormone with phosphaturic activity. However, the function of FGF23 in the regulation of phosphate metabolism is far from understood. The intention of this study was to investigate the molecular properties of native FGF23 and its mutant forms. I conducted protein expression experiments in HEK293 cells which showed that native FGF23 is a secreted protein partially processed into an N-terminal fragment and a C-terminal fragment. I provided evidence that this cleavage occurs during protein secretion and it is performed by subtilisin like-proprotein convertases (SPCs). In addition, I determined that native FGF23 undergoes O-linked glycosylation before secretion by using a deglycosylation assay. Further, RT-PCR analysis of human tissues showed FGF23 expression in whole fetus, heart, liver, thyroid/parathyroid, small intestine, testis, skeletal muscle, differentiated chondrocytes and TIO tumor tissue. In mouse, FGF23 was expressed in day 17 embryo and spleen. The FGF23 ADHR mutations replace arginine residues at the SPC cleavage site (RXXR motif). By expression of the FGF23-R176Q and –R179Q mutant proteins in HEK293 cells I showed that ADHR mutations prevent cleavage at the RXXR site and stabilize FGF23. This alteration in the FGF23 cleavage enhances FGF23 phosphaturic activity in ADHR. Familial tumoral calcinosis (FTC) with hyperphosphatemia is a disease considered the mirror image of the hypophosphatemic condition. It is known that FTC is caused by mutations in the GALNT3 gene. By performing mutation analysis in two families with FTC, I could show that FTC can also be caused by inactivating mutations in the FGF23 gene. To characterize the FGF23-S71G mutant protein I conducted in vitro expression assays, immunocytochemistry and ELISA to measure the FGF23 plasma levels in the patient with FTC. Taken together the results of these experiments showed that intact FGF23-S71G mutant protein remained inside the cells and only the C-terminal FGF23 fragment was secreted. These investigations demonstrate that FGF23 mutations in ADHR and FTC have opposite effects on phosphate homeostasis. There is evidence that the endopeptidase PHEX which is mutated in patients with XLH and FGF23 act in the same pathway. PHEX function resides upstream of FGF23 and may be involved in the degradation of FGF23 thereby regulating its phosphaturic activity. I designed an assay with a recombinant secreted form of PHEX (secPHEX) to prove whether FGF23 is a substrate of PHEX. Although secPHEX activity could be demonstrated by degradation of PTHrP107-139, secPHEX failed to degrade FGF23 in this assay. These results provided evidence against a direct interaction of PHEX and FGF23.