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The aim of the present study was to determine in vitro the effects of various purified apolipoproteins (apo) on the activity of the cholesteryl ester transfer protein (CETP). It appeared that the ability of apoA-I, A-II, and A-IV to modulate the CETP-mediated transfer of radiolabeled cholesteryl esters between low density lipoproteins (LDL) and high density lipoproteins (HDL) was markedly influenced by the final apolipoprotein:lipoprotein ratio in incubation mixtures. At low apolipoprotein:lipoprotein ratio, the rate of radiolabeled cholesteryl esters transferred from HDL3 to LDL was significantly increased in the presence of apoA-I and apoA-IV. Under similar conditions, the rate of radiolabeled cholesteryl esters transferred from LDL to HDL3 was increased in the presence of apoA-I while apoA-IV had no significant effects. At high apolipoprotein:lipoprotein ratio, the ability of apoA-I and apoA-IV to enhance the rate of radiolabeled cholesteryl esters transferred either from HDL3 to LDL or from LDL to HDL3 was considerably reduced. At the highest apolipoprotein:lipoprotein ratio studied, apoA-I and A-IV became inhibitors of the CETP-mediated transfer reaction. Interestingly, apoA-II differed markedly from other apolipoproteins as, even at a low apolipoprotein:lipoprotein ratio, it significantly inhibited CETP activity as measured either from HDL3 to LDL or from LDL to HDL3. The inhibition by apoA-II was concentration-dependent and, at the highest apolipoprotein:lipoprotein ratio studied, cholesteryl ester transfer activity was totally suppressed. The possibility of a direct interaction between CETP and the two major HDL apolipoproteins, apoA-I and apoA-II, was further investigated by combining crosslinking and immunoblotting techniques. Whereas CETP alone had an apparent molecular mass of 76,000 +/- 3,100 Da, crosslinking reactions in incubation mixtures containing CETP and either apoA-I or apoA-II revealed the appearance of additional protein bands with apparent molecular masses of 99,600 +/- 6,100 and 86,900 +/- 4,500 Da, respectively. These complexes corresponded to the association of one molecule of CETP with one molecule of apoA-I or apoA-II. Interestingly, the mass concentrations of apoA-II needed to produce visible CETP-apolipoprotein complexes appeared to be about ten times higher as compared with apoA-I, suggesting that CETP may have a lower affinity for apoA-II than for apoA-I. In conclusion, data from the present study indicate that apolipoproteins A-I, A-II, and A-IV could be potent modulators of the CETP-mediated transfer of cholesteryl esters between HDL and LDL fractions.(ABSTRACT TRUNCATED AT 400 WORDS)

The implication of cholesteryl ester transfer protein (CETP) in the high density lipoprotein (HDL) conversion process was studied by incubating HDL3 with a purified CETP preparation for 24 h at 37 degrees C. At a physiological plasma level, CETP induced a decrease of the HDL3 fraction (8.6 nm diameter) and the appearance of two new distinct particle subpopulations with mean diameters of 9.5 and 7.8 nm. To determine whether the effects of the CETP preparation could be assigned to CETP itself, the incubations were conducted either in the absence or in the presence of specific anti-CETP monoclonal antibodies. The HDL3 conversion process induced by the CETP preparation was totally blocked by addition to the incubation mixture of TP1 anti-CETP monoclonal antibody, known to completely inhibit the cholesteryl ester transfer activity in vitro. Moreover, the HDL conversion activity was retained, together with the CETP activity, on an anti-CETP affinity column and was insensitive to the presence of a lecithin:cholesterol acyltransferase inhibitor. Compared with incubations with CETP, incubations with CETP and apoA-IV increased the size range redistribution of the HDL3 particles, particularly by promoting the formation of very small-sized lipoprotein particles. The results of the study demonstrate thhat CETP can mediate an HDL size conversion even in the absence of lipid transfers between HDL and other lipoprotein fractions. They constitute a supplementary argument for a multipotential role of CETP in lipid transport.

Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central β-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxyterminal β-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies. [ABSTRACT FROM AUTHOR]