Biosynthesis of dermatan sulfate: chondroitin-glucuronate C5-epimerase is identical to SART2.

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.

Modulation of proteoglycan synthesis by bovine vascular smooth muscle cells during cellular proliferation and treatment with heparin.

Proliferating cultures of bovine vascular smooth muscle cells synthesized a variety of proteoglycans corresponding closely to those reported previously for monkey smooth muscle cells. These included a chondroitin sulfate proteoglycan (CSPG) (47%), a dermatan sulfate proteoglycan (DSPG) (22%), and a heparan sulfate proteoglycan (HSPG) (6%) which were secreted into the medium. Heparan sulfate proteoglycan (6%) and a second dermatan sulfate proteoglycan (14%) were also present in the cell layer. Confluent cultures synthesized a similar spectrum of proteoglycans although the medium CSPG and DSPG were of smaller hydrodynamic size. The cell layer HSPG was much reduced relative to DSPG in early proliferating cultures. Previous reports have shown that heparin inhibits vascular smooth muscle cell proliferation. Heparin had two effects on proteoglycan synthesis. In control cultures, 35S-Labeled proteoglycan synthesis doubled during the first 12 h after releasing cells from growth arrest, decreasing during the following 12 h during which time cell division occurred. Treatment with heparin delayed the onset of proliferation by 24 h and this was accompanied by a corresponding delay in the increase in 35S-labeled proteoglycan synthesis associated with the early phase of the cell cycle. Secondly, heparin treatment resulted in an increase in the anionic properties of heparan sulfate proteoglycan synthesized by the cells. This was independent of the proliferative state of the cultures. Pentosan polysulfate, semi-synthetic heparin, and a highly sulfated heparan sulfate modulated both cell proliferation and heparan sulfate proteoglycan synthesis in the same way as heparin.