Characterization of recombinant wild type and site-directed mutations of apolipoprotein C-III: lipid binding, displacement of ApoE, and inhibition of lipoprotein lipase.
The physicochemical properties of recombinant wild type and three site-directed mutants of apolipoprotein C-III (apoC-III), designed by molecular modeling to alter specific amino acid residues implicated in lipid binding (L9T/T20L, F64A/W65A) or LPL inhibition (K21A), were compared. Relative lipid binding efficiencies to dimyristoylphosphatidylcholine (DMPC) were L9T/T20L > WT >K21A > F64A/W65A with an inverse correlation with size of the discoidal complexes formed. Physicochemical analysis (Trp fluorescence, circular dichroism, and GdnHCl denaturation) suggests that L9T/T20L forms tighter and more stable lipid complexes with phospholipids, while F64A/W65A associates 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:DPPC complexes; the amount of apoE displaced from the complex was dependent on the apoC-III lipid binding affinity. All apoC-III proteins inhibited LPL in the presence or absence of apoC-II, with F64A/W65A displaying the most inhibition, suggesting that apoC-III inhibition of LPL is independent of lipid binding and therefore of apoC-II displacement. Taken together. these data suggest that the hydrophobic residues F64 and W65 are crucial for the lipid 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 protein, while LPL inhibition by apoC-III is likely to be due to protein:protein interactions.
Centre for Cardiovascular Genetics, Department of Medicine, Royal Free and University College, London Medical School.
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H
Liu
P J
PJ
Talmud
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L
Lins
R
R
Brasseur
G
G
Olivecrona
F
F
Peelman
J
J
Vandekerckhove
M
M
Rosseneu
C
C
Labeur
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001057:Apolipoproteins E / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014644:Genetic Variation; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D050356:Lipid Metabolism; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D001057:Apolipoproteins E; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D015060:1,2-Dipalmitoylphosphatidylcholine; D008071:Lipoprotein Lipase