Enzyme interactions in heparan sulfate biosynthesis: Uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo

The formation of heparan sulfate occurs within the lumen of the endoplasmic reticulum-Golgi complex-trans-Golgi network by the concerted action of sev eral glycosyltransferases, an epimerase, and multiple sulfotransferases. In this report, we have examined the location and interaction of tagged forms of five of the biosynthetic enzymes: galactosyltransferase I and glucurono syltransferase 1, required for the formation of the linkage region, and Glc NAc N-deacetylase/N-sulfotransferase 1, uronosyl 5-epimerase, and uronosyl 2-O-sulfotransferase, the first three enzymes involved in the modification of the chains. All of the enzymes colocalized with the medial-Golgi marker ce-mannosidase II. To study whether any of these enzymes interacted with ea ch other, they were relocated to the endoplasmic reticulum (ER) by replacin g their cytoplasmic N-terminal tails with an ER retention signal derived fr om the cytoplasmic domain of human invariant chain (p33). Relocating either galactosyltransferase I or glucuronosyltransferase I had no effect on the other's location or activity. However, relocating the epimerase to the ER c aused a parallel redistribution of the 2-O-sulfotransferase. Transfected ep imerase was also located in the ER in a cell mutant lacking the 2-O-sulfotr ansferase, but moved to the Golgi when the cells were transfected with 2-O- sulfotransferase cDNA. Epimerase activity was depressed in the mutant, but increased upon restoration of 2-O-sulfotransferase, suggesting that their p hysical association was required for both epimerase stability and transloca tion to the Golgi. These findings provide in vivo evidence for the formatio n of complexes among enzymes involved in heparan sulfate biosynthesis. The functional significance of these complexes may relate to the rapidity of he paran sulfate formation.