AN AMINO-TERMINAL FRAGMENT OF APOLIPOPROTEIN-B BINDS TO LIPOPROTEIN-LIPASE AND MAY FACILITATE ITS BINDING TO ENDOTHELIAL-CELLS

Lipoprotein lipase (LPL), the principal enzyme which hydrolyzes trigly cerides in circulating plasma lipoproteins, functions while bound to t he luminal surface of endothelial cells. LPL is a heparin-binding prot ein and has been assumed to associate with endothelial cell heparan su lfate proteoglycans (HSPG). Recently, using ligand blotting and affini ty chromatography we identified a 116-kDa heparin-releasable LPL-bindi ng protein (hrp-116) from endothelial cells which was not a HSPG (Siva ram, P., Klein, M. G., and Goldberg, I. J. (1992) J. Biol. Chem. 267,1 6517-16522). This suggested that, like a number of other heparin-bindi ng proteins, LPL binding to cells also involves non-HSPG proteins. Usi ng heparin-agarose affinity chromatography, a 116-kDa LPL-binding prot ein was purified from endothelial cell extracts. Microsequencing of pe ptides generated by Lys-C protease digestion revealed complete homolog y with four different regions in the NH2-terminal part of human apolip oprotein B (apoB). Western blots using anti-apoB monoclonal antibodies (mAb) that recognize the NH2-terminal region of apoB confirmed that a 116-kDa fragment of apoB was present on endothelial cell membranes. F urther evidence that LPL associates with the NH2-terminal region of ap oB was obtained by showing 1) that an NH2-terminal fragment of apoB ob tained from apoB-transfected CHO cells bound LPL on ligand blots and 2 ) that NH 2-terminal fragments of apoB generated by thrombin digestion of low density lipoprotein bind LPL. Evidence that the NH2-terminal r egion of apoB mediates LPL interaction with endothelial cells was obta ined using monoclonal antibodies. mAb3 and mAb19, which recognize epit opes near the NH2 terminus of apoB, inhibited I-125-LPL binding to cel ls by 60-65%. In contrast, mAb47, which has determinants at the COOH-t erminal end of apoB, inhibited LPL binding by only about 10%. The inhi bitory effects of mAb3 and mAb19 were abolished following treatment of cells with heparin, which removes the 116-kDa LPL-binding protein. Fu rthermore, incubation of I-125-LPL in medium containing an NH2-termina l apoB fragment reduced LPL binding to cells. These data suggest that an NH2-terminal fragment of apoB that binds to endothelial surfaces fa cilitates LPL binding to cells.