Abstract
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.
Highlights
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)
The importance of iduronic acid (IdoA) is clearly demonstrated in patients diagnosed with EhlersDanlos syndrome caused by loss-of-function mutations in DSE, encoding DS-epi1, or dermatan 4-O-sulfotransferase 1 (D4ST1) who suffer from multiple organ disorders (6 –8)
DS-epi1 epimerase activity is enhanced when co-incubated with D4ST1
Summary
The necessity of both DS-epi and D4ST1 for the formation of long IdoA blocks is well established [16, 17]. To analyze the spatial distribution of the IdoA–GalNAc– 4S moieties in the polymer, products of D4ST1/DS-epi co-incubations were degraded with chondroitinase AC-I and -II, which cleave only GalNAc–GlcA linkages, allowing the analysis of the untouched IdoA-containing structures. The colocalization of DS-epi1/ D4ST1 was observed with the overexpressed FRET constructs in COS-7 cells (Fig. 4B). To determine whether these three enzymes interact and form heteromeric and functionally relevant complexes in live cells, we first confirmed the correct localization of the enzymes in the Golgi of COS-7 cells. FRET, immunoprecipitation, and cross-linking data indicate the presence of homocomplexes and heterocomplexes of the three enzymes, given the capability of DS-epi to interact with DS-epi and D4ST1. SiRNA-mediated reduction of DS-epi in MCF 10a cells resulted in a marked reduction of the two epimerases and IdoA biosynthesis
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