Apolipoprotein A-I tryptophan substitution leads to resistance to myeloperoxidase-mediated loss of function.
OBJECTIVE
Apolipoprotein A-I (apoAI) acts as an ABCA1-dependent acceptor of cellular phospholipids and cholesterol during the biogenesis of HDL, but this activity is susceptible to oxidative inactivation by myeloperoxidase. We tried to determine which residues mediated this inactivation and create an oxidant-resistant apoAI variant.


RESULTS
Mass spectrometry detected the presence of tryptophan, methionine, tyrosine, and lysine oxidation in apoAI recovered from human atheroma. We investigated the role of these residues in the myeloperoxidase-mediated loss of apoAI activity. Site-directed mutagenesis and chemical modification were used to create variants of apoAI which were tested for ABCA1-dependent cholesterol acceptor activity and oxidative inactivation. We previously reported that tyrosine modification is not required for myeloperoxidase-induced loss of apoAI function. Lysine methylation did not alter the sensitivity of apoAI to myeloperoxidase, whereas site-specific substitution of apoAI methionine to valine increased the sensitivity of apoAI to myeloperoxidase. ApoAI tryptophan residues were identified as essential in apoAI function and oxidant sensitivity as substitution of all four apoAI tryptophan residues to leucine led to loss of function, but the conservative substitution to phenylalanine retained full function and was resistant to oxidative inactivation.


CONCLUSIONS
Tryptophan modification of apoAI is primarily responsible for the myeloperoxidase-mediated loss of the cholesterol acceptor activity of apoAI.
Peng
Dao-Quan
DQ
Department of Cell Biology, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Brubaker
Gregory
G
Wu
Zhiping
Z
Zheng
Lemin
L
Willard
Belinda
B
Kinter
Michael
M
Hazen
Stanley L
SL
Smith
Jonathan D
JD
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D016632:Apolipoprotein A-I / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D050197:Atherosclerosis / Q000201:enzymology / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D008239:Lysine; D008264:Macrophages / Q000378:metabolism; D008715:Methionine; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014364:Tryptophan
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D014364:Tryptophan; D002784:Cholesterol; D008715:Methionine; D006861:Hydrogen Peroxide; D009195:Peroxidase; D008239:Lysine