Abstract
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 alpha-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, GlcUAbeta1-3Galbeta1-O-naphthalenemethanol and GlcUAbeta1-3Galbeta1-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).
Highlights
Plays an important role in chondroitin polymerization
Glycosyltransferase Activity of CSGlcA-T (ChSy-3)—Recent studies revealed that co-expression of any two of chondroitin synthase-1 (ChSy-1), Chains thase-2 (ChSy-2) (CSS3), and chondroitin-polymerizing factor (ChPF) augmented glycosyltransferase activities when compared with ChSy-1 or ChSy-2 expressed alone [14, 16]
We have demonstrated that CSGlcA-T (ChSy-3) exhibits chondroitin polymerization activity on ␣-TM bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF
Summary
Materials—UDP-[U-14C]GlcUA (285.2 mCi/mmol) and UDP-[3H]GalNAc (10 Ci/mmol) were purchased from PerkinElmer Life Sciences. The PCR fragment was subcloned into the BamHI site of pGIR201protA [20], resulting in the fusion of the insulin signal sequence and the protein A sequence present in the vector, as described previously [8, 14, 16]. Each DNA fragment was inserted into a pcDNA3Ins-His expression vector, resulting in the fusion of the protein with the insulin signal sequence and His sequence present in the vector Combinations of these constructs and the protein A-tagged expression vectors were transfected into COS-1 cells on 100-mm plates using FuGENETM 6 (Roche Applied Science) according to the manufacturer’s instructions. Each digest was derivatized with 2-aminobenzamide and analyzed by HPLC, as reported previously [29]
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