title: Differential effects of brefeldin A on chondroitin sulfate and hyaluronan synthesis in rat chondrosarcoma cells.
abstract: Brefeldin A, a fungal metabolite, interferes with vesicular transport causing disassembly of the Golgi complex with redistribution of Golgi components to the endoplasmic reticulum, and isolation of the trans-Golgi cisternae from the trans-Golgi network. We examined the effects of brefeldin A on the synthesis of hyaluronan and chondroitin sulfate by chondrocytes from the Swarm rat chondrosarcoma. Hyaluronan synthesis continues at a constant rate in the presence of brefeldin A for at least 8 h, and is therefore independent of vesicular transport. By contrast, chondroitin sulfate synthesis is rapidly inhibited (to < 1% within 15 min) by brefeldin A indicating that addition of chondroitin sulfate chains to the aggrecan core protein precursor requires vesicular transport. Removal of brefeldin A rapidly restored chondroitin sulfate chain elongation and sulfation on the aggrecan core protein precursor reaching 100% of control in 2 h and consistently establishing a higher steady state rate (up to 120%) by 4 h. Addition of p-nitrophenyl-beta-D-xylopyranoside, an exogenous acceptor for the synthesis of chondroitin sulfate chains, does not reverse the brefeldin A block. This suggests that xyloside-initiated synthesis of chondroitin sulfate depends on transport vesicles as might occur if the enzymes for synthesizing the linkage tetrasaccharide (i.e. galactosyltransferases) reside in the Golgi, while those required to elongate the chains reside in the trans-Golgi network. Recovery of chondroitin sulfate synthesis from brefeldin A treatment occurred efficiently in the presence of cycloheximide, indicating that the machinery for chondroitin sulfate synthesis reassembles from previously existing proteins. The results are consistent with the current model that hyaluronan synthesis occurs at the plasma membrane and is independent of vesicular transport, and with the hypothesis that the enzyme complex for chondroitin sulfate elongation and sulfation residues within the trans-Golgi network, and therefore isolated from the aggrecan core protein precursor in the presence of brefeldin A.
authors.affiliation: Proteoglycan Chemistry Section, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: A
authors.initials: A
authors.lastname: Calabro
authors.forename: V C
authors.initials: VC
authors.lastname: Hascall
mesh_terms: D000818:Animals; D020126:Brefeldin A; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D002813:Chondrosarcoma / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003517:Cyclopentanes / Q000494:pharmacology*; D005944:Glucosamine / Q000378:metabolism; D006027:Glycosides / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D007700:Kinetics; D008297:Male; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011509:Proteoglycans / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013431:Sulfates / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D003517:Cyclopentanes; D006027:Glycosides; D011500:Protein Synthesis Inhibitors; D011509:Proteoglycans; D013431:Sulfates; C008646:4-nitrophenyl beta-D-xyloside; D020126:Brefeldin A; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D003513:Cycloheximide; D005944:Glucosamine