Monoclonal antibodies as probes of high-density lipoprotein structure: identification and localization of a lipid-dependent epitope

Abstract

Eight stable murine monoclonal antibodies (mabs) were raised against human high-density lipoproteins (HDL). Three different antibody reactivities were demonstrated by immunoblotting. A group of five antibodies were specific for apolipoprotein A-I (apoA-I) and bound to similar or overlapping epitopes. The second type of reactivity, shown by mab-32, was specific for apoA-II. In the third group, two antibodies showed high reactivity with apoA-II and slight cross-reactivity with apoA-I. The properties of two antibodies, mab M-30 specific for apoA-I and mab M-32 specific for apoAII, were characterized in detail as probes of HDL structure. The association of 125I-labeled HDL or synthetic complexes of apoA-I and phosphatidylcholine with mab M-30 was lipid dependent. Mab M-32 binding to apoA-II was independent of lipid. The lipid-dependent epitope bound by mab M-30 has been localized to an 18 amino acid synthetic apoA-I peptide. Moreover, studies with HDL2, HDL3, and immunoadsorbed HDL subfractions indicate that binding of mab M-30 to HDL is influenced by some component within the microenvironment individual HDL particles. These lines of evidence suggest that the molar ratio of apoA-I to apoA-II is the critical determinant. Binding of mab M-32 to HDL increased the reactivity of HDL to mab M-30 in a dose-dependent manner, indicating an unusual form of cooperativity between two mabs that recognize different proteins in HDL. These monoclonal antibodies will be valuable in studies of the metabolic significance of protein-protein and lipid-protein interactions in HDL.

Use of synthetic peptide analogues to localize lecithin:cholesterol acyltransferase activating domain in apolipoprotein A-I.

“…While the EE consensus dimer closely mimics apo A-l in artificial LCAT activation systems, apo A-l in the intact HDL particle very likely has interactions with other apolipoproteins, such as apo A-ll. 34 We have shown that half of apo A-l can be removed from HDL without noticeably affecting the ability of that HDL to act as an LCAT substrate. 16 The latter observation suggests that different microenvironments within an individual HDL particle, for example interactions with other apolipoproteins, change the abiity of apo A-1 to act as an activator of LCAT.…”

The role of apoproteins AI and AII in binding of high-density lipoprotein3 to membranes derived from bovine aortic endothelial cells

“…Alor AII-DMPC) which markedly differ from HDL3 in lipid composition, particle size and shape (Blanche et al, 1988;Nichols et al, 1983). Such differences have been shown to affect apoprotein conformation (Brouillette et al, 1984;Kunitake et al, 1985;Silberman et al, 1987), which in turn apparently affected their ability to activate lecithin-cholesterol acytransferase (Jonas et al, 1987(Jonas et al, , 1989. It is therefore reasonable to conclude that differences in conformation may also affect the ability of the apoprotein to recognize binding sites or receptors, and would thus affect measurement of binding parameters such as affinity (Kd) and capacity (Bmax ).…”