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 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.
Sivaram
P
P
Department of Medicine, Columbia University, New York, New York 10032.
Choi
S Y
SY
Curtiss
L K
LK
Goldberg
I J
IJ
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
D001055:Apolipoproteins B; C484074:LPL binding proteins, human; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D008071:Lipoprotein Lipase