title: An amino-terminal fragment of apolipoprotein B binds to lipoprotein lipase and may facilitate its binding to endothelial cells.
abstract: Lipoprotein lipase (LPL), the principal enzyme which hydrolyzes triglycerides in circulating plasma lipoproteins, functions while bound to the luminal surface of endothelial cells. LPL is a heparin-binding protein and has been assumed to associate with endothelial cell heparan sulfate proteoglycans (HSPG). Recently, using ligand blotting and affinity chromatography we identified a 116-kDa heparin-releasable LPL-binding protein (hrp-116) from endothelial cells which was not a HSPG (Sivaram, P., Klein, M. G., and Goldberg, I. J. (1992) J. Biol. Chem. 267, 16517-16522). This suggested that, like a number of other heparin-binding proteins, LPL binding to cells also involves non-HSPG proteins. Using heparin-agarose affinity chromatography, a 116-kDa LPL-binding protein was purified from endothelial cell extracts. Microsequencing of peptides generated by Lys-C protease digestion revealed complete homology with four different regions in the NH2-terminal part of human apolipoprotein 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. Further evidence that LPL associates with the NH2-terminal region of apoB was obtained by showing 1) that an NH2-terminal fragment of apoB obtained from apoB-transfected CHO cells bound LPL on ligand blots and 2) that NH2-terminal fragments of apoB generated by thrombin digestion of low density lipoprotein bind LPL. Evidence that the NH2-terminal region of apoB mediates LPL interaction with endothelial cells was obtained using monoclonal antibodies. mAb3 and mAb19, which recognize epitopes near the NH2 terminus of apoB, inhibited 125I-LPL binding to cells by 60-65%. In contrast, mAb47, which has determinants at the COOH-terminal end of apoB, inhibited LPL binding by only about 10%. The inhibitory effects of mAb3 and mAb19 were abolished following treatment of cells with heparin, which removes the 116-kDa LPL-binding protein. Furthermore, incubation of 125I-LPL in medium containing an NH2-terminal apoB fragment reduced LPL binding to cells. These data suggest that an NH2-terminal fragment of apoB that binds to endothelial surfaces facilitates LPL binding to cells.
authors.affiliation: Department of Medicine, Columbia University, New York, New York 10032.
authors.forename: P
authors.initials: P
authors.lastname: Sivaram
authors.forename: S Y
authors.initials: SY
authors.lastname: Choi
authors.forename: L K
authors.initials: LK
authors.lastname: Curtiss
authors.forename: I J
authors.initials: IJ
authors.lastname: Goldberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta; D001055:Apolipoproteins B / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D008071:Lipoprotein Lipase / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D001055:Apolipoproteins B; C484074:LPL binding proteins, human; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D008071:Lipoprotein Lipase