Abstract
Apolipoprotein A-I (apoAI), the major protein of high density lipoprotein, plays an important role in reverse cholesterol transport via its activity as an ABCA1-dependent acceptor of cellular cholesterol. We reported recently that myeloperoxidase (MPO) modification of apoAI inhibits its ABCA1-dependent cholesterol acceptor activity (Zheng, L., Nukuna, B., Brennan, M. L., Sun, M., Goormastic, M., Settle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P. L., Ischiropoulos, H., Smith, J. D., Kinter, M., and Hazen, S. L. (2004) J. Clin. Invest. 114, 529-541). We also reported that MPO-mediated chlorination preferentially modifies two of the seven tyrosines in apoAI, and loss of parent peptides containing these residues dose-dependently correlates with loss in ABCA1-mediated cholesterol acceptor activity (Zheng, L., Settle, M., Brubaker, G., Schmitt, D., Hazen, S. L., Smith, J. D., and Kinter, M. (2005) J. Biol. Chem. 280, 38-47). To determine whether oxidative modification of apoA-I tyrosine residues was responsible for the MPO-mediated inactivation of cholesterol acceptor activity, we made recombinant apoAI with site-specific substitutions of all seven tyrosine residues to phenylalanine. ApoAI and the tyrosine-free apoAI were equally susceptible to dose-dependent MPO-mediated loss of ABCA1-dependent cholesterol acceptor activity, as well as lipid binding activity. MPO modification altered the migration of apoAI on SDS gels and decreased its alpha-helix content. MPO-induced modification also targeted apoAI tryptophan and lysine residues. Specifically, we detected apoAI tryptophan oxidation to mono- and dihydroxytryptophan and apoAI lysine modification to chlorolysine and 2-aminoadipic acid. Thus, tyrosine modification of apoAI is not required for its MPO-mediated inhibition of cholesterol acceptor activity.
Highlights
Liver via the reverse cholesterol transport pathway
ApoAI tyrosine chlorination was associated with the MPOmediated loss of the cholesterol acceptor activity of Apolipoprotein A-I (apoAI), tyrosine chlorination might only serve as a molecular fingerprint for apoAI modification by MPO, and not the cause of apolipoprotein loss of cholesterol efflux function, as other changes in apoAI have been found after oxidative modification (25–30)
Tyrosine-free ApoAI Is Susceptible to MPO-induced Loss of Function —We prepared recombinant human His-tagged wild type apoA-I and a mutant form in which all seven tyrosine residues were substituted with phenylalanine residues, as described under “Experimental Procedures.”
Summary
Generation and Isolation of Recombinant Human ApoAI and ApoAI Mutants—cDNA encoding His6-tagged recombinant human apoAI (rh-apoAI) in the pET-20b bacterial expression vector was gift from Dr Oda (see Ref. 31). ApoAI Mass Spectrometry Analysis—Intact protein mass of unmodified and modified apoAI were determined by LC ESI-MS analysis with an LCQ Deca MS (Thermo, CA) equipped with a ultra plus II HPLC system (Micro-tech Scientific, Vista, CA) and a Vydac C4 column (5 m, 300 Å, 1 mm ϫ 5 cm). For these analyses, aqueous trifluoroacetic acid/acetonitrile solvents were used with a gradient of 30 –70% acetonitrile over 20 min and a flow rate of 30 l/min. Acetic acid treatment of 7 M MPO-modified apoAI was performed at room temperature at a 1 M HAc final concentration, with stock reagent added in three intervals over a 30-min period. Statistical comparison of three or more groups was performed by using analysis of variance and the Newman-Keuls multiple comparison post-test
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
