Biogenesis and speciation of nascent apoA-I-containing particles in various cell lines

.....Abstract It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; however, the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. Here, we show that incubation of exogenous apolipoprotein A-I (apoA-I) with fibroblasts, CaCo-2, or CHO-overexpressing ABCA1 cells generates only alpha-nascent apolipoprotein A-I-containing particles (alpha-LpA-I) with diameters of 8-20 nm, whereas human umbilical vein endothelial cells and ABCA1 mutant (Q597R) cells were unable to form such particles. Interestingly, incubation of exogenous apoA-I with either HepG2 or macrophages generates both (alpha-LpA-I) and pre alpha-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not pre alpha-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both pre beta(1)-LpA-I and LpA-I; by contrast, CaCo-2 cells secreted only beta(1)-LpA-I. To determine whether alpha-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated alpha-LpA-I as well as reconstituted HDL [ r( HDL)] was reacted with LCAT. Although both particles had similar V-max ( 8.4 vs. 8.2 nmol cholesteryl ester/ h/ mu g LCAT, respectively), the K m value was increased 2-fold for alpha-LpA-I compared with r( HDL) ( 1.2 vs. 0.7 mu M apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not pre beta-LpA-I; and alpha-LpA-I interacts efficiently with LCAT. Thus, our study provides direct evidence for a new link between specific cell lines and the speciation of nascent HDL that occurs by both ABCA1-dependent and -independent pathways.