Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A.

Abstract

Abstract. Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex . We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains . BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain . In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synxE biosynthesis of glycosaminoglycan (GAG)' carbohydrate side chains involves an extensive series of posttranslational enzymatic reactions that occur dur ing the intracellular transport of core proteins from the ER through the Golgi complex . These reactions use specific initiation, elongation, and sulfation enzymes to modify appropriate core proteins and assemble them into mature proteoglycan molecules . For chondroitin sulfate (CS) GAGS, the carbohydrate-protein linkage region is initiated by a xylosyltransferase that transfers xylose from UDP-xylose to the hydroxyl groups of specific serine residues within a core protein. Initiation is completed by the addition oftwo galactosyl and one glucuronosyl residue through the action of galactosyltransferase I, II, and glucuronosyltransferase I, respectively. Once initiated, the repeating disaccharide backbone ofglucuronosyl and N-acetylgalactosaminyl residues is elongated by specific glucuronosyl-and N-acetylgalactosaminyltransferases . Sulfation of the repeating disaccharides by the 4-and/or 6-sulfotransferases completes the synthesis of mono-or disulfated CS (27) (see Fig . 11 thesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to ß-elimination . The ability of cells to use an exogenous acceptor, p-nitro phenyl-ß-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA . The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events . Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment .It is generally accepted that GAG synthesis is a Golgi complex-related event. Early studies using electron microscopy and radioautography showed that the Golgi complex is the major site of GAG chain synthesis (21,24,25) . Golgi-enriched preparations isolated from some cell types have also been shown to contain GAG synthesizing activity (32)...

Secretion of chondroitin sulfate from embryonic epidermal cells in Xenopus laevis.

“…the chondroitin chain in the mature granule is mostly sulfated and the site of sulfation may be the immature granule. This is consistent with the results of biochemical studies concerning the formation of chondroitin sulfate (Spiro et al, 1991), although information on the localization of sulfotransferase is needed to clarify the sulfation site.…”

“…The site of core protein synthesis of PG is considered to be rough endoplasmic reticulum, and the protein is transported to the Golgi apparatus. Glycosylation and sulfation occur in the Golgi apparatus and/or the trans-Golgi network (Sugumaran et al, 1992;Blair et al, 1991;Spiro et al, 1991;Ratcliffe et al, 1985).…”

“…To address these questions, we investigated the locations of chondroitin sulfate and the secretory pathway with anti-GAG antibodies and immunoelectron microscopy. Brefeldin A (BFA), a fungal metabolite, is known to block transport from the endoplasmic reticulum to the Golgi apparatus (Orci et al, 1991;Spiro et al, 1991;Chege and Pfeffer, 1990;Donaldson et al, 1990;Lippincott-Schwartz et al, 1989. On the basis of inhibition experiment by BFA, the life of mucous granules in cytoplasm was 5-10 hr, indicating that secretion was routed through a regulated pathway.…”

Sulfation in the Golgi Lumen of Madin-Darby Canine Kidney Cells Is Inhibited by Brefeldin A and Depends on a Factor Present in the Cytoplasm and on Golgi Membranes

“…The results for the cell fractions were similar to those for the apical medium, whereas the basolateral medium contained essentially only HSPG (data not shown) (33). These results indicate that in MDCK cells, HSPG sulfation, which generally is completed before the trans-Golgi network, may operate at a lower cellular PAPS concentration than sulfation events (protein and CS) that take place in the transGolgi network (15)(16)(17)(18)(19) (Fig. 3).…”

“…In most cell types, secretory transport is inhibited by BFA because treatment with this drug induces retrograde transport of components of the cis-, medial, and trans-Golgi cisternae but not of the trans-Golgi network to the endoplasmic reticulum (11)(12)(13)(14). In several cell types, the synthesis of HSPGs may be completed in the presence of BFA although with significantly reduced efficiency, whereas CSPG synthesis is not detected, which indicates that separate enzyme systems in early and late subcompartments of the Golgi complex are involved in HSPG and CSPG synthesis, respectively (15)(16)(17)(18)(19).…”