PATHWAY-SPECIFIC REGULATION OF THE SYNTHESIS OF ANTICOAGULANTLY ACTIVE HEPARAN-SULFATE

L cells and endothelial cells synthesize a heparan sulfate (HS) subpop ulation, HSact, that exhibits anticoagulant activity due to a specific monosaccharide sequence; the remaining heparan sulfate, HSinact, lack s this region of defined structure and is anticoagulantly inactive. HS act biosynthesis was examined in these two cell types by stably expres sing epitope-tagged rat ryudocan (ryudocan(12CA5)), which possesses th ree glycosaminoglycan (GAG) acceptor sites. Both HSact and HSinact wer e present on ryudocan(12CA5) isolated from L cells and endothelial cel ls; thus, a core protein with a unique primary sequence initiates the synthesis of both GAGs. The expression in L cells of ryudocan(12CA5) v ariants containing a single functional GAG acceptor site demonstrated that each of the three acceptor regions initiates the synthesis of bot h types of GAGs to a similar extent. Most importantly, in both cell ty pes total HSact generation declined as a function of ryudocan(12CA5) o verexpression even though HSinact production increased linearly as a f unction of this variable. This discordant relationship is a general pr operty of the biosynthetic machinery since in both cell types HSact pr oduction was reduced to an equal extent on protein cores of either exo genous or endogenous origins. The suppression of HSact generation was also observed with a secreted form of core protein lacking transmembra ne and cytoplasmic domains or by a GAG acceptor site mutated form of c ore protein incapable of augmenting GAG synthesis. These results sugge st that elevated intracellular levels of core protein saturate the cap acity of a critical component of the HSact biosynthetic machinery. Thi s critical component is not a member of the common set of biosynthetic enzymes involved in the production of HSact and HSinact since no stru ctural changes were observed in either GAG during overexpression of co re protein. Based upon the above data, we conclude that increased intr acellular levels of ryudocan probably act by saturating the capacity o f components which regulate HSact production by coordinating the funct ion of biosynthetic enzymes.