Defective O-glycosylation due to a novel homozygous S129P mutation is associated with lack of fibroblast growth factor 23 secretion and tumoral calcinosis

Abstract

Background: Homozygous mutations in fibroblast growth factor (FGF23) have recently been described as the genetic cause of one form of hyperphosphatemic tumoral calcinosis (HFTC). However, it remained unclear to date how these mutations lead to loss of biologically active FGF23 in the circulation.

Methods: We here report a novel homozygous mutation, c.385T>C in FGF23 exon 2, which changes codon 129 from serine to proline (S129P) in a previously described individual affected by HFTC. The S129P mutation as well as two known FGF23 mutations, S71G and S129F, were introduced into an expression vector encoding wild-type (wt) human (h) FGF23 to yield [P129]hFGF23, [F129]hFGF23, and [G71]hFGF23; whole lysates, glycoprotein fractions, and conditioned media from HEK293 and COS-7 cells expressing these constructs were subjected to Western blot analysis using affinity-purified goat anti-hFGF23(51-69) and anti-hFGF23(206-222) antibodies.

Results: We detected 25- and 32-kDa protein species in total lysates of HEK293 cells expressing wt-hFGF23. The 32-kDa band, representing O-glycosylated hFGF23, was not detectable in the glycoprotein fraction of lysates from HEK293 cells expressing [P129]hFGF23, and in comparison with wt-FGF23 only small amounts of [P129]hFGF23 were secreted into the medium. Similar results were obtained for cells expressing [G71]hFGF23 and [F129]hFGF23.

Conclusion: Our data for the first time directly show that FGF23 mutations associated with HFTC impair O-glycosylation in vitro resulting in poor secretion of the mutant hormone thereby explaining the characteristic hyperphosphatemic phenotype of homozygous carriers in vivo.

Figure 1

A, Primers flanking exons 1–3 of FGF23 were used to PCR amplify the entire coding sequence and exon-flanking intronic sequences using blood genomic DNA from the index case. B, Nucleotide sequence analysis revealed a homozygous transition C>T at nucleotide 385, leading to a serine to proline change at codon 129, which is highly conserved among the available species (C).

Figure 2

COS-7 cells were transiently transfected with [Q176] hFGF23(1-251)Stop, [G71]hFGF23(1-251)Stop, [P129]hFGF23(1-251)Stop, or [F129]hFGF23(1-251)Stop in pcDNA3.1V5His. After 2 d, cultures were switched to serum-free medium, and medium was harvested and cells were lysed on d 3. Lysates and medium were subjected to 15% SDS-PAGE, transferred to PVDF membranes, and probed with goat anti-hFGF23(51-69) (A) or goat anti-hFGF23 (206-222) antibodies (B). COS-7 cell lysates and media lacked the 32-kDa modified protein species (star) when expressing the FGF23 variants carrying the tumoral calcinosis mutations S71G, S129P, or S129F; in contrast, the 32-kDa species was observed when analyzing cells expressing the wt-FGF23. The 25-kDa protein species (arrow) was present in lysates for mutant and wt-hFGF23 but absent in the respective media.

Figure 3

COS-7 cells were transiently transfected with pcDNA3.1V5His plasmids encoding hFGF23(1-251)Stop comprising one of the following mutations: [Q176], [G71], [P129], [F129], [G71, Q176], [P129, Q176], [F129, or Q176]. After 2 d, cultures were switched to serum-free medium, and medium was harvested and cells lysed on d 3. Lysates and medium were subjected to 15% SDS-PAGE, transferred to PVDF membranes, and probed with goat anti-hFGF23(51-69) (A) or goat anti-hFGF23(206-222) antibodies (B). COS-7 cell lysates and media lacked the 32-kDa modified protein species (star) when expressing the FGF23 variants carrying the tumoral calcinosis mutations S71G, S129P, S129F, or double mutations; the 25-kDa protein species (arrow) is present in lysates expressing mutant and wt-hFGF23 but undetectable in the respective media. Note that a nonspecific protein band (arrowhead) was detected in medium but not in cell-lysates. Two novel 14-kDa N- and C-terminal protein species are observed for double-mutant FGF23. C, Intact and C-terminal FGF23 ELISA (Immutopics) of conditioned media from HEK293 cells expressing the above wild-type and mutant forms of hFGF23. C-terminal and intact FGF23 are similarly reduced when compared with wild-type. Q176 is unable to rescue secretion of hFGF23.

Figure 4

A, Enzymatic deglycosylation of recombinant FGF23: Ni-agarose chromatography was used to purify: recombinant (presumably glycosylated) [Q176]hFGF23(25-251)V5His in pcDNA3.1V5His from medium of Free-Style 293-F cells (Invitrogen) and recombinant (presumably nonglycosylated) [M24, Q176]hFGF23(24-251)V5His in pCRT7/CT-Topo from the BL21 TrxB bacterial strain. N-linked and O-linked carbohydrates were removed under denaturing conditions using approximately 1 μg of peptide and the E-DEGLY kit (Sigma). Expected glycan-free fragment lengths are: recombinant [Q176]hFGF23(25-251)V5His (45 additional amino acids) purified from Free-Style 293-F cells, 272 amino acids approximately 30 kDa; and recombinant [M24, Q176]hFGF23(24-251)V5His (30 additional amino acids) purified from the BL21 TrxB bacterial strain, 258 amino acids approximately 28 kDa. Immunoblot analysis was performed as described in Patients and Methods and in the previous figures. B, Glycoprotein purification of total lysates from HEK293 cells expressing [G71]hFGF23(1-251)Stop, [P129]hFGF23(1-251)Stop, [F129]hFGF23(1-251)Stop, or wt-hFGF23(1-251)Stop using WGL-agarose, followed by 10% SDS-PAGE analysis using a biotinylated goat anti-hFGF23(186-206) antibody. Whereas whole lysates of HEK293 cells expressing mutant and wt-hFGF23 showed equal expression of the 25-kDa protein species, only the wt-FGF23 32 kDa protein species was detected in the glycoprotein fraction after purification with wheat germ lectin agarose (star). C, A 32-kDa, presumably glycosylated, but not the more abundant intracellular 25-kDa protein species was detected after 50 times concentration of media conditions with HEK293 cells transfected with the tumoral calcinosis mutations of hFGF23 (star).

Figure 5

Working model to illustrate the role of O-glycosylation by GALNT3 for proper secretion of hFGF23 (A). When O-glycosylation is impaired by loss-of-function mutations in GALNT3 as in HFTC1 or mutations in FGF23 as in HFTC2, secretion of intact hFGF23 may be impaired. Nonglycosylated FGF23 may be targeted for degradation by subtilisin/furine-like endopeptidases. hFGF23 fragments may spill over or be actively secreted into the circulation, particularly if gene expression of hFGF23 is stimulated by feedback up-regulation (B). The ADHR mutation Q176 may protect glycosylated, partially, and nonglycosylated intact FGF23, leading to accumulation and excess secretion of intact FGF23. By protecting the subtilisin/furine-like cleavage site, the ADHR mutation may also lead to intracellular accumulation of otherwise unstable 25- and 14-kDa protein species and secretion of partially glycosylated 25-kDa protein species, which may have biological activity but escape negative feedback suppression of gene transcription (C).