Cell-surface Expression of an Amino-terminal Fragment of Apolipoprotein B Increases Lipoprotein Lipase Binding to Cells

Abstract

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 NH 2 -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. Lipoprotein lipase (LPL)1 hydrolyzes triglycerides in the circulating plasma lipoproteins, chylomicrons, and very low density lipoproteins (1). Although LPL is synthesized by adipocytes, myocytes, and neurons, its primary site of action is at the luminal side of the endothelium. It is widely believed that LPL binds to endothelial cells via an electrostatic interaction with heparan sulfate proteoglycans (HSPG) (2-5). Earlier studies from this laboratory suggested that LPL binding to endothelial cells also involves a 116-kDa non-proteoglycan LPL-binding protein (6, 7). The 116-kDa LPL-binding protein has homology to the NH 2 -terminal region of apoB (NTAB) (8), the major structural protein of LDL, very low density lipoproteins, and chylomicrons (9). Furthermore, we have recently demonstrated that endothelial cells synthesize a full-length apoB protein and process it to generate the 116-kDa LPL-binding fragment (10).Several lines of evidence suggested that LPL interacts with NTAB. Ligand blotting of apoB fragments generated by thrombin digestion of LDL showed that labeled LPL bound specifically to NTAB. In addition, LPL bound to NH 2 -terminal fragments obtained from apoB-transfected CHO cells (8). 125I-LPL binding to endothelial cells was inhibited by antibodies to NH 2 -terminal regions of apoB, but not by antibodies that recognize epitopes closer to the carboxyl terminus of apoB. Based on these data, it was hypothesized that NTAB mediates LPL binding to cells and may confer specificity to LPL binding to endothelial cells.The amino-terminal region of apoB is relatively hydrophilic. Models of LDL suggest that NTAB extends away from the lipid core (9). Similarly, NTAB on the surface of a cell would not be expected to be buried within the cellular phospholipid bilayer and thus should be available to interact with LPL. Cells that overexpress truncated apoB fragments have been generated by other investigators. These cells, however, either secrete the small NH 2 -terminal fragments of apoB or degrade them intracellularly (11-13). Thus, these cells cannot be used to assess cell-surface actions of NTAB. Decay-accelerating factor (DAF) is a 70-kDa protein that contains sequences for glycosylphosphatidylinositol (GPI)...

Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B

“…Our data seemingly contradict previous findings of Goldberg and co-workers (17,18), who have suggested that proteinprotein interaction between LPL and LDL, especially the amino-terminal region of apoB, is more important than proteinlipid interaction. Recently, these authors (55) concluded that the high affinity binding between LPL and LDL involves multiple ionic and hydrophobic interactions and that the interaction is inhibited by heparin.…”

Modified LDL–Mediated Increases in Endothelial Layer Permeability Are Attenuated With 17β-Estradiol

“…Previous studies showed highavidity binding of constituents of apoB (eg, apoB17) with matrix molecules of the artery wall, such as heparan sulfate proteoglycans. 41 Extensive oxidation of LDL (ie, OX-LDL) may eliminate the exposed portions of apoB on LDL. 27 These studies imply that constituents of apoB are important or necessary for the interaction of LDL with the artery wall.…”

Differences between group X and group V secretory phospholipase A2 in lipolytic modification of lipoproteins

“…Boren et al [54] concluded that the LPL-apoB interaction is dependent on lipids but not on apoB because chemical modification of apoB did not abolish the interaction, and partial delipidation of LDL markedly decreased the binding to LDL. In contrast, Goldberg et al proposed that protein-protein interaction between LPL and apoB is more important than the interaction between LPL and LDL lipids [55][56][57][58][59] using mutant apoB proteins. Thus, whether apoB or lipids contribute to the interaction with LPL has been controversial.…”