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 prebeta1-LpA-I. Furthermore, glyburide inhibits almost completely the formation of alpha-LpA-I but not prebeta1-LpA-I. Similarly, endogenously secreted HepG2 apoA-I was found to be associated with both prebeta1-LpA-I and alpha-LpA-I; by contrast, CaCo-2 cells secreted only alpha-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/microg LCAT, respectively), the K(m) value was increased 2-fold for alpha-LpA-I compared with r(HDL) (1.2 vs. 0.7 microM apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of alpha-LpA-I but not prebeta1-LpA-I; and 2) 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.
Highlights
It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; the mechanisms governing HDL biogenesis and speciation are complex and poorly understood
Based on our previous studies demonstrating that the interaction of apolipoprotein A-I (apoA-I) with normal human fibroblasts generated only ␣-nascent apolipoprotein A-I-containing particle (LpA-I) [16, 20], the question was raised whether the formation of both ␣-LpA-I and pre1-LpA-I could be dependent on specific cell types
Because pre1LpA-I are electrophoretically indistinguishable from lipidfree apoA-I, and because of the difficulty of separating small amounts of apoA-I-containing particle subspecies by fast-protein liquid chromatography, we developed a rapid controlled technique to remove lipid-free apoA-I from the medium using a size-exclusion centrifugal filter (MWCO 50,000) combined with a dialysis membrane with a molecular weight cut off (MWCO) of 50,000 to remove any remaining lipid-free apoA-I, as described in Materials and Methods
Summary
It is generally thought that the large heterogeneity of human HDL confers antiatherogenic properties; the mechanisms governing HDL biogenesis and speciation are complex and poorly understood. To determine whether ␣-LpA-I generated by fibroblasts is a good substrate for LCAT, isolated ␣-LpA-I as well as reconstituted HDL [r(HDL)] was reacted with LCAT. Both particles had similar Vmax (8.4 vs 8.2 nmol cholesteryl ester/h/g LCAT, respectively), the Km value was increased 2-fold for ␣-LpA-I compared with r(HDL) (1.2 vs 0.7 M apoA-I). These results demonstrate that 1) ABCA1 is required for the formation of ␣-LpA-I but not pre1-LpA-I; and 2) ␣-LpA-I interacts efficiently with LCAT.
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