A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction.
The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, (125)I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas (125)I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the alpha-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 Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [(3)H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [(3)H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes 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 demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.
Huang
W
W
Department of Internal Medicine, School of Medicine, Fukuoka, Japan.
Sasaki
J
J
Matsunaga
A
A
Han
H
H
Li
W
W
Koga
T
T
Kugi
M
M
Ando
S
S
Arakawa
K
K
D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001054:Apolipoproteins A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D050356:Lipid Metabolism; D008075:Lipoproteins, HDL / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D011498:Protein Precursors; D011994:Recombinant Proteins; C035250:preproapolipoprotein A-I; D004134:Dimyristoylphosphatidylcholine