β2-Glycoprotein i is a major protein associated with very rapidly cleared liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles

Abstract

Liposomes recovered from the blood of liposome-treated CD1 mice were previously reported to have a complex protein profile associated with their membranes (Chonn, A., Semple, S. C., and Cullis, P. R. (1992) J. Biol. Chem. 267, 18759-18765). In this study, we have further characterized and identified the major proteins associated with very rapidly cleared large unilamellar vesicles. These liposomes contained phosphatidylcholine, cholesterol, and anionic phospholipids (phosphatidylserine, phosphatidic acid, or cardiolipin) that dramatically enhance the clearance rate of liposomes from the circulation. These anionic phospholipids are normally found exclusively in the interior of cells but become expressed when cells undergo apoptosis or programmed cell death, and thus, they are believed to be markers of cell senescence. Analysis of the proteins associated with these liposomes by SDS-polyacrylamide gel electrophoresis revealed that two of the major proteins associated with the liposome membranes are proteins with electrophoretic mobilities corresponding to Mr of 66,000 and 50,000-55,000. The 66-kDa protein was identified to be serum albumin by immunoblot analysis. Using various biochemical and immunological methods, we have identified the 50-55-kDa protein as the murine equivalent of human β2-glycoprotein I. β2-glycoprotein I has a strong affinity for phosphatidylserine, phosphatidic acid, and cardiolipin inasmuch as the levels of β2-glycoprotein I associated with these anionic liposomes approach or even exceed those of serum albumin, which is present in serum at a concentration 200-fold greater than β2-glycoprotein I. Further, we demonstrate that the amount of β2-glycoprotein I associated with liposomes, as quantitated by an enzyme-linked immunosorbent assay, is correlated with their clearance rates; moreover, the circulation residency time of cardiolipin-containing liposomes is extended in mice pretreated with anti-β2-glycoprotein I antibodies. These findings strongly suggest that β2-glycoprotein I plays a primary role in mediating the clearance of liposomes and, by extension, senescent cells and foreign particles.