Generation of Pre-β 1 -HDL and Conversion Into α-HDL

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Abstract
HDL encompasses several apoA-I–containing particles that differ by size and show pre-β- or α-mobility on agarose gel electrophoresis: pre-β 1 -LpA-I, pre-β 2 -LpA-I, pre-β 3 -LpA-I, α-LpA-I 2 , and α-LpA-I 3 . The quantitatively minor subclass pre-β 1 -LpA-I serves as an initial acceptor of cell-derived cholesterol. In this study, we generated a pre-β 1 -LpA-I–like particle in vitro by the incubation of biotinylated apoA-I with cholesterol-loaded macrophages. Both native pre-β 1 -LpA-I and in vitro–generated pre-β 1 -LpA-I were indistinguishable from lipid-free apoA-I by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis but exhibited a different size upon gel filtration. In vitro–generated biotin–pre-β 1 -LpA-I took up twofold to threefold more [ 3 H]cholesterol from labeled fibroblasts during a 1-minute pulse incubation than lipid-free apoA-I. The in vitro conversion of biotin–pre-β 1 -LpA-I was investigated in the presence of plasmas of healthy probands and patients with Tangier disease, with apoA-I deficiency, and with lecithin-cholesterol acyltransferase (LCAT) deficiency. Incubation of biotin–pre-β 1 -LpA-I with plasmas either from normoalphalipoproteinemic probands or from a patient with apoA-I deficiency generated a biotinylated particle with the size and electrophoretic mobility of α-LpA-I 2 . This conversion was sensitive to heating at 56°C but not to the removal of calcium. Inhibition of LCAT by dithiobisnitrobenzoic acid led to the formation of α-LpA-I 3 instead of α-LpA-I 2 . Incubation of biotin–pre-β 1 -LpA-I with the plasma of an LCAT-deficient patient also led to the generation of biotin–α-LpA-I 3 instead of α-LpA-I 2. By contrast, incubation of biotin–pre-β 1 -LpA-I with plasma of patients with Tangier disease did not cause the disappearance of biotin–pre-β 1 -LpA-I and the formation of biotin–α-LpA-I. However, co-incubation of Tangier disease plasma or of pre-β 1 -LpA-I isolated from Tangier disease plasma with apoA-I–deficient plasma generated α-LpA-I 2 . In conclusion, our data indicate that (1) pre-β 1 -LpA-I can be formed in vitro by the interaction of free apoA-I with cholesterol-loaded macrophages, (2) both normal and apoA-I–deficient plasmas contain a factor that converts pre-β 1 -LpA-I into α-LpA-I, and (3) this factor is absent in the plasma of patients with Tangier disease.

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