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 ∼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.

The ability of chondroitin/dermatan sulfate (CS/DS) to convey biological information is enriched by the presence of iduronic acid. DS-epimerases 1 and 2 (DS-epi1 and 2), in conjunction with DS-4-O-sulfotransferase 1, are the enzymes responsible for iduronic acid biosynthesis and will be the major focus of this review. CS/DS proteoglycans (CS/DS-PGs) are ubiquitously found in connective tissues, basement membranes, and cell surfaces or are stored intracellularly. Such wide distribution reflects the variety of biological roles in which they are involved, from extracellular matrix organization to regulation of processes such as proliferation, migration, adhesion, and differentiation. They play roles in inflammation, angiogenesis, coagulation, immunity, and wound healing. Such versatility is achieved thanks to their variable composition, both in terms of protein core and the fine structure of the CS/DS chains. Excellent reviews have been published on the collective and individual functions of each CS/DS-PG. This short review presents the biosynthesis and functions of iduronic acid-containing structures, also as revealed by the analysis of the DS-epi1- and 2-deficient mouse models.

Dermatan sulfate epimerase 1 (DS-epi1) and DS-epi2 convert glucuronic acid to iduronic acid in chondroitin/dermatan sulfate biosynthesis. Here we report on the generation of DS-epi1-null mice and the resulting alterations in the chondroitin/dermatan polysaccharide chains. The numbers of long blocks of adjacent iduronic acids are greatly decreased in skin decorin and biglycan chondroitin/dermatan sulfate, along with a parallel decrease in iduronic-2-O-sulfated-galactosamine-4-O-sulfated structures. Both iduronic acid blocks and iduronic acids surrounded by glucuronic acids are also decreased in versican-derived chains. DS-epi1-deficient mice are smaller than their wild-type littermates but otherwise have no gross macroscopic alterations. The lack of DS-epi1 affects the chondroitin/dermatan sulfate in many proteoglycans, and the consequences for skin collagen structure were initially analyzed. We found that the skin collagen architecture was altered, and electron microscopy showed that the DS-epi1-null fibrils have a larger diameter than the wild-type fibrils. The altered chondroitin/dermatan sulfate chains carried by decorin in skin are likely to affect collagen fibril formation and reduce the tensile strength of DS-epi1-null skin.

A second dermatan sulfate epimerase (DS-epi2) was identified as a homolog of the first epimerase (DS-epi1), which was previously described by our group. DS-epi2 is 1,222 amino acids long and has an approximately 700-amino acid N-terminal epimerase domain that is highly conserved between the two enzymes. In addition, the C-terminal portion is predicted to be an O-sulfotransferase domain. In this study we found that DS-epi2 has epimerase activity, which involves conversion of d-glucuronic acid to l-iduronic acid (EC 5.1.3.19), but no O-sulfotransferase activity was detected. In dermatan sulfate, iduronic acid residues are either clustered together in blocks or alternating with glucuronic acid, forming hybrid structures. By using a short interfering RNA approach, we found that DS-epi2 and DS-epi1 are both involved in the biosynthesis of the iduronic acid blocks in fibroblasts and that DS-epi2 can also synthesize the hybrid structures. Both iduronic acid-containing domains have been shown to bind to several growth factors, many of which have biological roles in brain development. DS-epi2 has been genetically linked to bipolar disorder, which suggests that the dermatan sulfate domains generated by a defective enzyme may be involved in the etiology of the disease.

The epimerization of glucuronic acid into iduronic acid adds structural variability to chondroitin/dermatan sulfate polysaccharides. Iduronic acid-containing domains play essential roles in processes such as coagulation, chemokine and morphogen modulation, collagen maturation, and neurite sprouting. Therefore, we generated and characterized, for the first time, mice deficient in dermatan sulfate epimerase 1 and 2, two enzymes uniquely involved in dermatan sulfate biosynthesis. The resulting mice, termed DKO mice, were completely devoid of iduronic acid, and the resulting chondroitin sulfate chains were structurally different from the wild type chains, from which a different protein binding specificity can be expected. As a consequence, a vast majority of the DKO mice died perinatally, with greatly variable phenotypes at birth or late embryological stages such as umbilical hernia, exencephaly and a kinked tail. However, a minority of embryos were histologically unaffected, with apparently normal lung and bone/cartilage features. Interestingly, the binding of the chemokine CXCL13, an important modulator of lymphoid organogenesis, to mouse DKO embryonic fibroblasts was impaired. Nevertheless, the development of the secondary lymphoid organs, including the lymph nodes and spleen, was normal. Altogether, our results indicate an important role of dermatan sulfate in embryological development and perinatal survival.

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid–containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid–forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.