The glycosaminoglycan components of proteoglycans are biosynthesized and modified in the golgi apparatus by highly organized carbohydrate transfer enzymes and sulfotransferases. The purpose of this project is to investigate the functional organization and subcellular localization of these enzyme complexes. Brefeldin A (BFA) is a chemical which specifically blocks anterograde protein transport within the golgi apparatus. It was used to disrupt the normal biosynthetic processes for adding glycosaminoglycan chains onto proteoglycans. We examined the effects of BFA on the synthesis of hyaluronan (HA) and aggrecan, two of the major extracellular matrix molecules, in rat chondrosarcoma cells. Biosynthesis of chondroitin sulfate (CS) associated with aggrecan was rapidly inhibited to >1% of the control, while that of HA continued at the normal level. This result was consistent with the current model that the biosynthesis of CS requires the transport of the core protein through the Golgi apparatus, while that of HA occurs at the plasma membrane and therefore is independent of the vesicular transport. When ovarian granulosa cells were treated with BFA, dermatan sulfate proteoglycan synthesis was abolished whereas heparan sulfate proteoglycan synthesis was only partially inhibited, suggesting that dermatan sulfate and heparan sulfate assembly on proteoglycans occurs in different subcellular compartments in these cells. The finding that only normal heparan sulfate protein core proteins were substituted with heparan sulfate chains in the presence of the drug indicated that glycosylation enzymes are highly specific to core proteins. Topics of present interest include elucidation of core protein structure which determines highly specific glycosylation enzymes and the effect of brefeldin A on endocytotic mechanism involving cell surface proteoglycans.
