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 Goldbe rg, I. J. (1994) J. Biol. Chem. 269, 9409-9412) from this laboratory s howed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL), LPL binding to endothelial cells, we hyp othesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-su rface NTAB would increase the amount and affinity of LPL binding to ce lls, we produced stable Chinese hamster ovary cell Lilies that have NT AB 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 ac ids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment, The fused construct wa s 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-defi cient Chinese hamster ovary cells (745 cells), and stable cell lines m ere established, Expression of apoB17 on the cell surface was confirme d by the release of apoB17 by phosphatidylinositol-specific phospholip ase C. LPL binding to WT and apoB17-DAF-transfected cells was determin ed, Using 0.8-6 mu g of LPL, 1.3-2.2-foId more LPL associated with apo B17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells, After heparinase treatment, LPL binding to apoB 17-DAF cells was still greater than to treated WT cells, This increase d binding to apoB17-DAF cells was almost abolished by treatment of cel ls with phosphatidylinositol-specific phospholipase C or anti-apoB mon oclonal antibody, LPL dissociated from WT cells with k(-1) = 2.55 x 10 (-2) min(-1), whereas LPL dissociated more slowly from apoB17-DAF-cont aining cells with k(-1) = 1.08 x 10(-2) min(-1), Furthermore, almost 9 5% 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 co ncentration. Similarly, in high salt, more LPL remained associated wit h apoB17-DAF cells than with nontransfected 745 cells, These data show that NTAB on cell surfaces can function as a LPL binding protein, Mor eover, they demonstrate that LPL association with cells can be increas ed by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.