A single amino acid deletion in the carboxy terminal of apolipoprotein A-Iimpairs lipid binding and cellular interaction

The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by m utagenesis or synthetic peptides to play an important role in lipid binding . However, the precise functional domain of the C-terminal remains to be de fined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I v ariant with a deletion of glutamic acid 235, was expressed in Escherichia c oli to examine the effect of this mutation on the functional domain of apoA -I for lipid binding and related consequences, A dimyristoyl phosphatidylch oline binding study with recombinant (r-) proapoA-I Nichinan showed a signi ficantly slow initial rate of lipid binding. On preincubation with human pl asma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, I-125 -labeled normal r-proapoA-I was chromatographed as a single peak at the hig h density lipoprotein (HDL) fraction, whereas I-125-labeled r-proapoA-I Nic hinan was chromatographed into the HDL fraction as well as the free r-proap oA-I fraction (23% of radioactivity). Circular dichroism measurements showe d that the cu-helix content of Lipid-bound r-proapoA-I Nichinan was reduced , being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I N ichinan and normal r-proapoA-I showed similar particle size. To study chole sterol efflux, human skin fibroblasts were labeled with [H-3]cholesterol. f ollowed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA- I complex. Fractional cholesterol efflux from [H-3]cholesterol-labeled fibr oblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan com plexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demons trate that the deletion of glutamic acid 235 at the C-terminus substantiall y reduces the lipid-binding properties of r-proapoA-I Nichinan, which may c ause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.