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 Lilies 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 mere 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 mu g of LPL, 1.3-2.2-foId 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 nr 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.