Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: Lipid binding, displacement of ApoE, and inhibition oflipoprotein lipase

The physicochemical properties of recombinant wild type and three site-dire cted mutants of apolipoprotein C-III (apoC-III), designed by molecular mode ling to alter specific amino acid residues implicated in lipid binding (L9T /T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid b inding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT > K21A > F64A/W65A with an inverse correlation with size of the discoi dal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter an d more stable lipid complexes with phospholipids, while F64A/W65A associate s less tightly. Lipid displacement properties were tested by gel-filtrating apoE:dipalmitoylphosphatidylcholine (DPPC) discoidal complexes mixed with the various apoC-III variants. All apoC-III proteins bound to the apoE: DPP C complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL i n the presence or absence of apoC-II, with F64A/W65A displaying the most in hibition, suggesting that apoC-III inhibition of LPL is independent of lipi d binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipi d binding properties of apoC-III and that redistribution of the N-terminal helix of apoC-III (L9T/T20L) enhances the stability of the lipid-bound prot ein, while LPL inhibition by apoC-III is likely to be due to protein:protei n interactions.