Mouse mastocytoma cells synthesize undersulfated heparin and chondroitin sulfate in the presence of brefeldin A

In order to study the subcellular localization and organization of the enzymes involved in the glycosylation of the hybrid proteoglycan serglycin, mouse mastocytoma cells were metabolically labeled with [S-35]sulfate or [H-3]glucosamine in the absence or presence of brefeldin A. This drug is known to induce a disassembly of the proximal part of the Golgi complex, resulting in a redistribution of cis-, medial-, and trans-Golgi resident enzymes back to the endoplasmic reticulum, and to block the anterograde transport of proteins to the trans-Golgi network. Although the total incorporation of [H-3]glucosamine into glycosaminoglycan chains was reduced to about 25% in brefeldin A-treated cells compared to control cells, both control cells and cells treated with brefeldin A synthesized heparin as well as chondroitin sulfate chains. Therefore, enzymes involved ill the biosynthesis of both types of glycosaminoglycan chains seem to be present proximal to the trans-Golgi network in these cells. Chondroitin sulfate and heparin synthesized in cells exposed to brefeldin A were undersulfated, as demonstrated by ion-exchange chromatography, compositional analyses of disaccharides, as well as by a lower [S-35]sulfate/[H-3]glucosamine ratio compared to controls. In heparin biosynthesis, both N- and O-sulfation reactions were impaired, with a larger relative decrease in 2-O-sulfation than in 6-O-sulfation. Despite undersulfation, the heparin chains synthesized in the presence of brefeldin A were larger (30 kDa) than the heparin synthesized by control cells (20 kDa), The reduced [H-3]glucosamine incorporation hi brefeldin A-treated cells was partly due to decreased number of glycosaminoglycan chains synthesized, but also to the biosynthesis of chondroitin sulfate chains of smaller molecular size (8 versus 15 kDa in control cells). Brefeldin A had no effect on the glycosaminoglycan synthesis when used in a cell-free, microsomal fraction, indicating that brefeldin A does not interfere directly with the enzymes involved in the biosynthesis of glycosaminoglycans.