Cysteine mutants of human apolipoprotein A-I: a study of secondary structural and functional properties.
Apolipoprotein A-I(Milano) (A-I(M)) (R173C), a natural mutant of human apolipoprotein A-I (apoA-I), and five other cysteine variants of apoA-I at residues 52 (S52C), 74 (N74C), 107 (K107C), 129 (G129C), and 195 (K195C) were generated. Cysteine residues were incorporated in each of the various helices at the same helical wheel position as for the substitution in A-I(M). The secondary structural properties of the monomeric mutants, their abilities to bind lipid and to promote cholesterol efflux from THP-1 macrophages, and the possibility of antiperoxidation were investigated. Results showed that the alpha helical contents of all of the cysteine mutants were similar to that of wild-type apoA-I (wtapoA-I). The cysteine variant of A-I(M) at residue 173 [A-I(M)(R173C)] exhibited weakened structural stability, whereas A-I(G129C) a more stable structure than wtapoA-I. A-I(G129C) and A-I(K195C) exhibited significantly impaired capabilities to bind lipid compared with wtapoA-I. A-I(K107C) possessed a higher capacity to promote cholesterol efflux from macrophages than wtapoA-I, and A-I(M)(R173C) and A-I(K195C) exhibited an impaired efflux capability. Neither A-I(M)(R173C) nor any other cysteine mutant could resist oxidation against lipoxygenase. In summary, in spite of the similar mutant position on the helix, these variants exhibited different structural features or biological activities, suggesting the potential influence of the local environment of mutations on the whole polypeptide chain.
Zhu
Xuewei
X
Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 100005.
Wu
Gang
G
Zeng
Wuwei
W
Xue
Hong
H
Chen
Baosheng
B
D000975:Antioxidants / Q000494:pharmacology; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics*; D001053:Apolipoproteins / Q000737:chemistry; D002784:Cholesterol / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics*; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D015227:Lipid Peroxidation; D008084:Lipoxygenase / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013997:Time Factors
D000975:Antioxidants; D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D010455:Peptides; D011994:Recombinant Proteins; D002784:Cholesterol; D008084:Lipoxygenase; D003545:Cysteine; D004134:Dimyristoylphosphatidylcholine