Selective modification of apoB-100 in the oxidation of low density lipoproteins by myeloperoxidase in vitro

Abstract

Oxidative modification of LDL may be important in the initiation and/or progression of atherosclerosis, but the precise mechanisms through which low density lipoprotein (LDL) is oxidized are unknown. Recently, evidence for the existence of HOCl-oxidized LDL in human atherosclerotic lesions has been reported, and myeloperoxidase (MPO), which is thought to act through production of HOCl, has been identified in human atherosclerotic lesions. In the present report we describe the formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive modifications in the apolipoprotein (apo) by exposure of LDL to myeloperoxidase in vitro. In contrast with the complex mixture of peptides from oxidation of LDL with reagent HOCl, oxidation with MPO in vitro produced a major tryptic peptide showing absorbance at 365 nm. This peptide was isolated and characterized as VELEVPQL(*C)SFILK . . . , corresponding to amino acid residues 53-66 . . . on apoB-100. Mass spectrometric analyses of two tryptic peptides from oxidation of LDL by HOCl indicated formation of the corresponding methionine sulfoxide (M O), cysteinyl azo (*C), RS-N N-DNP, derivatives of EEL(*C)T(M O)FIR and LNDLNS VLV(M O)PTFHVPFTDLQVPS(*C)K, which suggest oxidation to the corresponding sulfinic acids (RSO 2 H) by HOCl.The present results demonstrate that DNPH-reactive modifications other than aldehydes and ketones can be formed in the oxidation of proteins and illustrate how characterization of specific products of protein oxidation can be useful in assessing the relative contributions of different and unexpected mechanisms to the oxidation of LDL and other target substrates. The data also suggest a direct interaction of the LDL particle with the active site on myeloperoxidase and indicate that effects of the protein microenvironment can greatly influence product formation and stability. -Yang, C-y.,

Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100

“…Although the oxidation of biological molecules by MPO is commonly attributed to generation of HOCl (5,(12)(13)(14)(15)(16)(17), we observed that MPO-catalyzed oxidation of LDL in vitro produced a much different pattern of DNPH-reactive products than was observed from reaction of LDL with reagent HOCl (9,10). Oxidation of LDL with HOCl produced a diverse array of DNPH-reactive peptides, with little indication of selectivity.…”

“…Similar oxidations were conducted at 37 Њ C in the presence or absence of MPO by addition of 16, 40, 160, 400, 1,000, or 4,000 nmol of HOCl, either added at 1.5 min intervals in 10 separate aliquots or as a single bolus dose, corresponding to an HOCl:apoB-100 molar ratio of 10, 25, 100, 250, 625, and 2,500, respectively. Fifteen minutes after initiation of the respective oxidations, the samples were reacted with DNPH, the protein was precipitated, delipidated, and digested with trypsin, and the tryptic peptides were analyzed by HPLC, as we have described previously (9).…”

“…In contrast, MPO-mediated oxidation of LDL in vitro, derivatization with DNPH, and tryptic digestion produced a dominant peptide detected by absorbance at 365 nm (9). Formation of this peptide required MPO, H 2 O 2 , and Cl Ϫ .…”

“…In our initial studies of the oxidation of LDL with reagent HOCl, followed by treatment with 2,4-dinitrophenylhydrazine (DNPH), intending to convert the presumed aldehydes and ketones formed by HOCl-driven oxidation of the protein, we instead found that most of the 14 products we characterized indicated modifications of cysteine or tryptophan residues (10). Subsequent studies indicated that the DNPH-reactive products of oxidation of the cysteine residues were best interpreted as the respective sulfinic acids (9). Additional studies of Cu 2 ϩ -catalyzed oxidation of LDL revealed tryptic peptide products of oxidation of the apolipoprotein that absorbed at 365 nm, but in a manner not dependent on derivatization with DNPH (11).…”

“…However, most of these studies have used total hydrolysis procedures that do not distinguish residues by position in the primary structure, as will be required for product identification to address important questions regarding the effects of the intact LDL particle on its interaction with specific oxidants. We have been working to characterize the specific products of oxidative modification of the LDL apolipoprotein, apoB-100, in order to distinguish among the mechanisms whereby LDL is oxidized in vivo (9)(10)(11). In our initial studies of the oxidation of LDL with reagent HOCl, followed by treatment with 2,4-dinitrophenylhydrazine (DNPH), intending to convert the presumed aldehydes and ketones formed by HOCl-driven oxidation of the protein, we instead found that most of the 14 products we characterized indicated modifications of cysteine or tryptophan residues (10).…”