Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A.
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 synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-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.
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Department of Immunology, Scripps Research Institute, La Jolla, California 92037.
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Sampath
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Garcia
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D000951:Antigens, Neoplasm / Q000378:metabolism; D001692:Biological Transport; D020126:Brefeldin A; D002809:Chondroitin Sulfates / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006027:Glycosides / Q000378:metabolism; D006801:Humans; D011509:Proteoglycans / Q000378:metabolism
D000951:Antigens, Neoplasm; D003517:Cyclopentanes; D006025:Glycosaminoglycans; D006027:Glycosides; D011509:Proteoglycans; C038142:chondroitin sulfate glycosaminoglycan; C069150:melanoma-associated proteoglycan antigen, human; C010807:xylosides; D020126:Brefeldin A; D002809:Chondroitin Sulfates