Abstract
We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an approximately 43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565-2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.
Highlights
We identified the gene encoding chondroitin-glucuronate C5epimerase (EC 5.1.3.19) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis
The enzymes involved in the biosynthesis of heparin/heparan sulfate and CS/DS have been cloned, all except the chondroitin-glucuronate C5-epimerase required for IdoUA formation in DS
Tosamine residues); CS, chondroitin sulfate; dK4, defructosylated K4; DS, dermatan sulfate; HexA, hexuronic acid; HS, heparan sulfate; IdoUA, L-iduronic acid; LC, liquid chromatography; MS, mass spectrometry; SART2, squamous cell carcinoma antigen recognized by T cell 2; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; DTT, dithiothreitol; FBS, fetal bovine serum; ConA, concanavalin A; PAPS, adenosine 3Ј-phosphate,5Ј-phosphosulfate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
Summary
We identified the gene encoding chondroitin-glucuronate C5epimerase (EC 5.1.3.19) that converts D-glucuronic acid to L-iduronic acid residues in dermatan sulfate biosynthesis. IdoUA units are consistently found adjacent to 4-O-sulfated GalNAc residues The extent of these modifications varies between tissues and seems to be influenced by the core protein structure (16). We purified this enzyme ϳ43,000-fold from bovine spleen microsomes and identified by mass spectrometry a candidate protein, SART2 (squamous cell carcinoma antigen recognized by T cells 2) that had previously been cloned but not assigned any specific function. Transgenic expression of this protein in mammalian cell lines yielded a product with DS epimerase activity, capable of inducing IdoUA formation in exogenous chondroitin substrate. Tosamine residues); CS, chondroitin sulfate; dK4, defructosylated K4; DS, dermatan sulfate; HexA, hexuronic acid; HS, heparan sulfate; IdoUA, L-iduronic acid; LC, liquid chromatography; MS, mass spectrometry; SART2, squamous cell carcinoma antigen recognized by T cell 2; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin; DTT, dithiothreitol; FBS, fetal bovine serum; ConA, concanavalin A; PAPS, adenosine 3Ј-phosphate,5Ј-phosphosulfate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
