Membrane anchoring of heparan sulfate proteoglycans by phosphatidylinositol and kinetics of synthesis of peripheral and detergent-solubilized proteoglycans in Schwann cells.
Previous studies have shown that Schwann cells synthesize both peripheral and integral hydrophobic cell surface heparan sulfate proteoglycans (HSPGs). The experiments reported here were undertaken to investigate the mode of attachment of these proteins to the cell surface and their potential interrelationship. The binding of the hydrophobic HSPGs to membranes appears to be via covalently linked phosphatidylinositol based on the observation that incubation of the detergent-solubilized protein with purified phosphatidylinositol-specific phospholipase C significantly reduces the ability of the HSPGs to associate with phospholipid vesicles in a reconstitution assay. The peripherally associated HSPGs were released from the cells by incubation in the presence of heparin (10 mg/ml), 10 mM phytic acid (inositol hexaphosphate), or 2 M NaCl. These treatments also solubilized basement membrane HSPGs synthesized by the Schwann cells. These data suggest that the peripheral HSPGs are bound to the surface by electrostatic interactions. The peripheral and hydrophobic HSPGs were identical in overall size, net charge, length of glycosaminoglycan chains, and patterns of N-sulfation. To determine whether the peripheral HSPGs were derived from the membrane-bound form by cleavage of the membrane anchor, we examined the kinetics of synthesis and degradation of the two forms of HSPGs. The results obtained indicated the existence of two pools of detergent-solubilized HSPG with fast (t1/2 = 6 h) and slow (t1/2 = 55 h) turnover kinetics. The data were consistent with a model in which the peripheral HSPGs were derived from the slowly turning over pool of detergent-solubilized HSPGs.
Carey
D J
DJ
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Evans
D M
DM
D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D003902:Detergents; D006025:Glycosaminoglycans / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis* / Q000378:metabolism*; D051381:Rats; D012583:Schwann Cells / Q000378:metabolism*; D012584:Sciatic Nerve / Q000378:metabolism; D012995:Solubility
D011508:Chondroitin Sulfate Proteoglycans; D003902:Detergents; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D010716:Phosphatidylinositols; D011509:Proteoglycans