Fig 2: FAM20C can regulate GAG abundance.a Biosynthetic pathways for CS and HS GAG chains. Multiple glycosyltransferases and distinct kinase/phosphatase(s) contribute to the synthesis of the common tetrasaccharide linkage region and repeating disaccharide region characteristic of CS and HS chains. Transient phosphorylation of the Xyl residue by XYLKs, including FAM20B, enhances linkage tetrasaccharide synthesis. Before subsequent GAG assembly by distinct biosynthetic machinery for CS and HS chains, the Xyl residue is dephosphorylated by a 2-phosphoxylose phosphatase (PXYLP). This XYLK-enhanced GAG biosynthesis determines the number of CS and HS chains attached to core proteins. XylT xylosyltransferase, GalT-I ß1,4-galactosyltransferase-I, GalT-II ß1,3-galactosyltransferase-II, GlcAT-I ß1,3-glucuronyltransferase-I. b, c Constitutive overexpression (OE) of FAM20C in HeLa cells elevated the levels of (b) CS and (c) HS chains, whereas its knockdown (KD) abrogated both GAG levels (b, c) compared with the mock controls (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). d, e Gel filtration elution profiles of (d) CS and (e) HS chains from FAM20C-related stable HeLa cell lines. Numbered arrowheads (200 k, 65 k, 37 k, and 18 k) indicate the elution position of 200-, 65-, 37-, and 18-kDa saccharides derived from size-defined commercial dextran, respectively. Results represent the average of two series of independent experiments. f The 4S/6S ratio of CS chains from FAM20C-related stable HeLa cell lines (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in b, c, and f are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig 3: FAM20C mutants enhance biomineralization in human osteosarcoma cells.a, b Expression of FAM20C, FAM20B, and C4ST-1 in stable Saos-2 cell lines overexpressing individual FAM20 proteins at the a transcriptional and b translational levels. GAPDH was used as an internal standard in (a), and as a loading control in (b). Data are obtained from three independent experiments and representative images are shown. c The 4S/6S ratio of CS chains from the stable Saos-2 cell lines in (a) and (b) (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). d, e Biomineralization level of the 21-day cultures of the stable Saos-2 cell lines in (a) and (b) was assessed by Alizarin red staining (d), followed by colorimetric quantification of the dye extracts derived from the stained cultures (e, n = 4 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in c and e are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig 4: FAM20C-mediated control of CS biosynthesis is exerted by its physical interaction with C4ST-1.a Schematic diagram of sulfation pathways for the chondroitin backbone. Characteristic CS disaccharide units, including A, C, D, and E, are sequentially formed under the control of CS-specific sulfotransferases such as C4ST-1, C4ST-2, and C6ST-1. b Gel filtration elution profiles of CS chains from mouse fibroblastic L or sog9 cells overexpressing wild-type FAM20B or FAM20C (Data are derived from the single experiment). Numbered arrowheads (200 k, 65 k, 37 k, and 18 k) indicate the elution position of 200-, 65-, 37-, and 18-kDa saccharides derived from size-defined commercial dextran, respectively. c Pulldown (PD) and immunoblotting (IB) analysis of culture medium from COS-1 cells co-expressing soluble forms of His-tagged C4ST-1 and FLAG-tagged FAM20 proteins. Data are obtained from three independent experiments and representative images are shown. d Sulfotransferase activities of C4ST-1 alone (Mock) and that from cells co-transfected with the individual FAM20 proteins (n = 3 independent experiments, Dunnett’s multiple comparison test, two-sided). e The 4S/6S ratio of CS chains from HeLa cell lines overexpressing wild-type or mutant FAM20C proteins (n = 4 independent experiments, Dunnett’s multiple comparison test, two-sided). Data in d and e are represented as the mean ± s.d. Source data are provided as a Source Data file.

Fig 5: Furin inhibition reduces FGF23 cleavage in vivo and in vitro.Treatment with a furin inhibitor (A) reduces cFGF23, (B) increases iFGF23, and (C) reduces cFGF23/iFGF23 in the plasma of WT and Vdr–/– mice (n = 6–11 per group). (D) The furin inhibitor diminishes serum phosphate in WT mice. (E) OCY454 cells with a knockdown of Vdr (Vdr-KD OCY) exhibit a higher cFGF23/iFGF23 ratio than control cells (Cas9 OCY), and furin inhibition increases the relative abundance of iFGF23 in both Cas9 OCY and Vdr KD OCY cells. (F) Quantification of Western blot bands by ImageJ. (G) Vdr KD does not increase cFGF23 in OCY454 with GalnT3 overexpression (GalnT3 Tg) and Fam20C KD; β-actin blots are derived from the same samples run contemporaneously in parallel gels. (H) Quantification of Western blot bands by ImageJ (NIH). Data represent the mean ± SEM. P values by 2-tailed Student’s test. FI, furin inhibitor; Scr, scrambled.