Fig 1: 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 2: Non-enzymatic functions of FAM20C in the control of CS biosynthesis and its involvement in Raine syndrome etiology.In physiological states (a), FAM20C can physically interact with C4ST-1 and augment the enzymatic activity of C4ST-1, leading to increased length of the individual CS chains via cooperative actions with glycosyltransferases (N-acetylgalactosaminyltransferases; ChGns)20,33 and to an elevated 4S/6S ratio. In contrast b, lethal Raine syndrome mutations in FAM20C cause functional uncoupling between FAM20C and C4ST-1, perturbing the C4ST-1-mediated chain elongation process and sulfation balance of CS chains. The consequent decrease in the 4S/6S ratio may be involved in the manifestation of chondroitin 6-sulfation-dependent skeletal phenotypes, which might be recapitulated by a gain-of-function of FAM20B.
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.
Supplier Page from Sino Biological, Inc. for Human CHST11 / C4ST-1 Protein (His Tag)