Cell-surface expression of an amino-terminal fragment of apolipoprotein B increases lipoprotein lipase binding to cells.
Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.
Pang
L
L
Division of Preventive Medicine and Nutrition, Department of Medicine, Columbia University, New York, New York 10032, USA.
Sivaram
P
P
Goldberg
I J
IJ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001055:Apolipoproteins B / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018958:CD55 Antigens / Q000235:genetics / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D019763:Heparin Lyase; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008071:Lipoprotein Lipase / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000378:metabolism; D011869:Radioligand Assay; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D001055:Apolipoproteins B; D018958:CD55 Antigens; D017261:Glycosylphosphatidylinositols; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D006493:Heparin; D008071:Lipoprotein Lipase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase; D043265:Phosphatidylinositol Diacylglycerol-Lyase