LIMITED PROTEOLYSIS OF HIGH-DENSITY-LIPOPROTEIN ABOLISHES ITS INTERACTION WITH CELL-SURFACE BINDING-SITES THAT PROMOTE CHOLESTEROL EFFLUX

High-density lipoprotein (HDL) components remove cholesterol from cell s by two independent mechanisms. Whereas HDL phospholipids pick up cho lesterol that desorbs from the plasma membranes, HDL apolipoproteins a ppear to interact with cell-surface binding sites that target for remo val pools of cellular cholesterol that feed into the cholesteryl ester cycle. Here we show that mild trypsin treatment of HDL almost complet ely abolishes this apolipoprotein-mediated cholesterol removal process . When HDL was treated with trypsin for various periods of time and th en incubated with cholesterol-loaded fibroblasts, treatment for only 5 min reduced the ability of HDL to remove excess cholesterol from cell ular pools that were accessible to esterification by the enzyme acyl C oA:cholesterol acyltransferase. This mild treatment digested less than 20% of HDL apolipoproteins and did not alter the lipid composition, s ize distribution, or electrophoretic mobility of the particles, Trypsi n treatment of HDL for up to 1 h caused no further reduction in its ab ility to remove cellular cholesterol despite a greater than 2-fold inc rease in apolipoprotein digestion, Trypsin treatment of HDL also reduc ed its ability to deplete the cholesteryl ester content of sterol-lade n macrophages. Promotion of cholesterol efflux from the plasma membran e by HDL phospholipids was unaffected by even extensive proteolysis. I n parallel to the loss of cholesterol transport-stimulating activity, trypsin treatment of HDL for only 5 min nearly abolished its interacti on with high-affinity binding sites on cholesterol-loaded fibroblasts. Reconstitution of trypsin-modified HDL with isolated apo A-I or apo A -II restored the cholesterol transport-stimulating activity of the par ticles. Thus a minor trypsin-labile fraction of HDL apolipoproteins is almost exclusively responsible for the apolipoprotein-dependent compo nent of cholesterol efflux mediated by HDL particles. (C) 1997 Elsevie r Science B.V.