Differential effect of brefeldin A on the biosynthesis of heparan sulfate and chondroitin/dermatan sulfate proteoglycans in rat ovarian granulosa cells in culture.
The subcellular localization of the enzymes involved in the glycosylation of proteoglycans was studied in rat ovarian granulosa cells by interfering with the normal traffic in the Golgi apparatus using brefeldin A. Cell cultures were metabolically labeled with [35S] sulfate and [3H]glucosamine, and the radiolabeled macromolecules were analyzed by ion-exchange and gel chromatography in combination with chondroitinase or heparitinase treatment. In the absence of brefeldin A, the cells synthesized both dermatan sulfate proteoglycans (DSPGs) and heparan sulfate proteoglycans (HSPGs) which were isolated from the culture medium, the plasma membrane, and intracellular compartments. However, in the presence of brefeldin A, the synthesized proteoglycans were almost exclusively HSPGs and were found only in the intracellular compartment. Analyses of HSPGs synthesized in the presence of brefeldin A indicated that: (i) the HS chains are synthesized on the same core protein as for the normal HSPGs, (ii) the chains are two to three times the normal molecular size; and (iii) a significant proportion of the HS chains are normally sulfated. Brefeldin A induces 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 (ER), and blocks the transport of proteins to the trans-Golgi network. Our results indicate that the complete set of enzymes involved in the biosynthesis of HS chains are localized in the ER/proximal part of the Golgi complex, whereas the enzymes involved in the elongation/sulfation of DS chains are exclusively located in the trans-Golgi network. Furthermore, our results indicate that the enzymes involved in the biosynthesis of HS chains are specific to HS core proteins, since no DS core proteins were substituted with HS chains in the presence of brefeldin A.
Uhlin-Hansen
L
L
Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Yanagishita
M
M
D000818:Animals; D020126:Brefeldin A; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003517:Cyclopentanes / Q000494:pharmacology*; D005260:Female; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D006497:Heparan Sulfate / Q000096:biosynthesis*; D007700:Kinetics; D051381:Rats; D017207:Rats, Sprague-Dawley
D020126:Brefeldin A; D011508:Chondroitin Sulfate Proteoglycans; D003517:Cyclopentanes; D006497:Heparan Sulfate