2-O-phosphorylation of xylose has been detected in the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans. Recent mutant analyses in zebrafish suggest that xylosyltransferase I and FAM20B, a protein of unknown function that shows weak similarity to a Golgi kinase encoded by four-jointed, operate in a linear pathway for proteoglycan production. In the present study, we identified FAM20B as a kinase that phosphorylates the xylose residue in the linkage region. Overexpression of FAM20B increased the amount of both chondroitin sulfate and heparan sulfate in HeLa cells, whereas the RNA interference of FAM20B resulted in a reduction of their amount in the cells. Gel-filtration analysis of the glycosaminoglycan chains synthesized in the overexpressing cells revealed that the glycosaminoglycan chains had a similar length to those in mock-transfected cells. These results suggest that FAM20B regulates the number of glycosaminoglycan chains by phosphorylating the xylose residue in the glycosaminoglycan-protein linkage region of proteoglycans.
Background/Aims: Calcium homeostasis requires regulated cellular and interstitial systems interacting to modulate the activity and movement of this ion. Disruption of these systems in the kidney results in nephrocalcinosis and nephrolithiasis, important medical problems whose pathogenesis is incompletely understood. Methods: We investigated 25 patients from 16 families with unexplained nephrocalcinosis and characteristic dental defects (amelogenesis imperfecta, gingival hyperplasia, impaired tooth eruption). To identify the causative gene, we performed genome-wide linkage analysis, exome capture, next-generation sequencing, and Sanger sequencing. Results: All patients had bi-allelic FAM20A mutations segregating with the disease; 20 different mutations were identified. Conclusions: This au-tosomal recessive disorder, also known as enamel renal syndrome, of FAM20A causes nephrocalcinosis and amelogenesis imperfecta. We speculate that all individuals with biallelic FAM20A mutations will eventually show nephrocalcinosis.
Recently, we demonstrated that chondroitin polymerization is achieved by any two combinations of human chondroitin synthase-1 (ChSy-1), ChSy-2 (chondroitin sulfate synthase 3, CSS3), and chondroitin-polymerizing factor (ChPF). Although an additional ChSy family member, called chondroitin sulfate glucuronyltransferase (CSGlcA-T), has been identified, its involvement in chondroitin polymerization remains unclear because it possesses only glucuronyltransferase II activity responsible for the elongation of chondroitin sulfate (CS) chains. Herein, we report that CSGlcA-T exhibits polymerization activity on ␣-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF, and the chain length of chondroitin formed by the co-expressed proteins in various combinations is different. In addition, ChSy family members co-expressed in various combinations exhibited distinct but overlapping acceptor substrate specificities toward the two synthetic acceptor substrates, GlcUA1-3Gal1-O-naphthalenemethanol and GlcUA1-3Gal1-O-C 2 H 4 NH-benzyloxycarbonyl, both of which share the disaccharide sequence with the glycosaminoglycan-protein linkage region tetrasaccharide. Moreover, overexpression of CSGlcA-T increased the amount of CS in HeLa cells, whereas the RNA interference of CSGlcA-T resulted in a reduction of the amount of CS in the cells. Furthermore, the analysis using the CSGlcA-T mutant that lacks any glycosyltransferase activity but interacts with other ChSy family members showed that the glycosyltransferase activity of CSGlcA-T plays an important role in chondroitin polymerization. Overall, these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, ChSy-2, CSGlcA-T, and ChPF and that each combination may play a unique role in the biosynthesis of CS. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3).
Background:The enzyme responsible for the dephosphorylation of xylose in glycosaminoglycans remains unknown. Results: A protein that removed the phosphate from the phosphorylated linkage trisaccharide and localized in the Golgi was identified. Inhibition of this protein phosphatase resulted in decreased glycosaminoglycan levels in cells. Conclusion:We identified the phosphoxylose phosphatase that regulates glycosaminoglycan synthesis. Significance: Transient phosphorylation of xylose residues controls glycosaminoglycan synthesis.
Previously, we demonstrated that sog9 cells, a murine L cell mutant, are deficient in the expression of C4ST (chondroitin 4-O-sulfotransferase)-1 and that they synthesize fewer and shorter CS (chondroitin sulfate) chains. These results suggested that C4ST-1 regulates not only 4-O-sulfation of CS, but also the length and amount of CS chains; however, the mechanism remains unclear. In the present study, we have demonstrated that C4ST-1 regulates the chain length and amount of CS in co-operation with ChGn-2 (chondroitin N-acetylgalactosaminyltransferase 2). Overexpression of ChGn-2 increased the length and amount of CS chains in L cells, but not in sog9 mutant cells. Knockdown of ChGn-2 resulted in a decrease in the amount of CS in L cells in a manner proportional to ChGn-2 expression levels, whereas the introduction of mutated C4ST-1 or ChGn-2 lacking enzyme activity failed to increase the amount of CS. Furthermore, the non-reducing terminal 4-O-sulfation of N-acetylgalactosamine residues facilitated the elongation of CS chains by chondroitin polymerase consisting of chondroitin synthase-1 and chondroitin-polymerizing factor. Overall, these results suggest that the chain length of CS is regulated by C4ST-1 and ChGn-2 and that the enzymatic activities of these proteins play a critical role in CS elongation.
Recently, it has been shown that a deficiency in ChGn-1 (chondroitin N-acetylgalactosaminyltransferase-1) reduced the numbers of CS (chondroitin sulfate) chains, leading to skeletal dysplasias in mice. Although these results indicate that ChGn-1 regulates the number of CS chains, the mechanism mediating this regulation is not clear. ChGn-1 is thought to initiate CS biosynthesis by transferring the first GalNAc (N-acetylgalactosamine) to the tetrasaccharide in the protein linkage region of CS. However, in vitro chondroitin polymerization does not occur on the non-reducing terminal GalNAc-linkage pentasaccharide structure. In the present study we show that several different heteromeric enzyme complexes composed of different combinations of four chondroitin synthase family members synthesized more CS chains when a GalNAc-linkage pentasaccharide structure with a non-reducing terminal 4-O-sulfation was the CS acceptor. In addition, C4ST-2 (chondroitin 4-O-sulfotransferase-2) efficiently transferred sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of non-reducing terminal GalNAc-linkage residues, and the number of CS chains was regulated by the expression levels of C4ST-2 and of ChGn-1. Taken together, the results of the present study indicate that C4ST-2 plays a key role in regulating levels of CS synthesized via ChGn-1.
Osteoporosis is an age-related disorder of bone remodeling in which bone resorption outstrips bone matrix deposition. Although anticatabolic agents are frequently used as first-line therapies for osteoporosis, alternative anabolic strategies that can enhance anabolic, osteogenic potential are actively sought. Sex steroid hormones, particularly estrogens, are bidirectional regulators for bone homeostasis; therefore, estrogen-mediated events are important potential targets for such anabolic therapies. Here, we show that estrogen-induced, osteoanabolic effects were mediated via enhanced production of chondroitin sulfate-E (CS-E), which could act as an osteogenic stimulant in our cell-based system. Conversely, estrogen deficiency caused reduced expression of CS-E-synthesizing enzymes, including GalNAc4S-6ST, and led to decreased CS-E production in cultures of bone marrow cells derived from ovariectomized mice. Moreover, Galnac4s6st-deficient mice had abnormally low bone mass that resulted from impaired osteoblast differentiation. These results indicated that strategies aimed at boosting CS-E biosynthesis are promising alternative therapies for osteoporosis.
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