Identification of Modified Tryptophan Residues in Apolipoprotein B-100 Derived from Copper Ion-Oxidized Low-Density Lipoprotein

Over the past several years, considerable evidence has been obtained in support of the hypothesis that oxidants generated by the heme enzyme myeloperoxidase (MPO, EC1.11.2.2) play a key role in oxidation reaction of the artery wall. The enzyme, abundantly present in neutrophils and, to a lesser extent, in monocytes, is released during infl ammatory activation of immune cells. MPO produces hypochlorous acid (HOCl) by the reaction of hydrogen Abstract Oxidation of LDL by the myeloperoxidase (MPO)-H 2 O 2 -chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modifi ed LDL and at revealing posttranslational modifi cations on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently refl ected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifi cations of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H 2 O 2 -chloride system or added as a reagent. A total of 97 peptides containing modifi ed residues could be identifi ed. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H 2 O 2 -chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confi rm that our in vitro fi ndings are also relevant in vivo. We show that several HOCl-mediated modifi cations of apoB-100 identifi ed in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modifi ed LDL. In conclusion, these data emphasize the specifi city of MPO to oxidize LDL. -Delporte,

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“…No Cl-Tyr formation was observed, while low levels of (di-)OxTrp may also occur, data that parallel previous fi ndings ( 37 ).…”

Section: Discussionsupporting

confidence: 90%

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Delporte

Boudjeltia

Noyon

et al. 2014

Journal of Lipid Research

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“…1B). Kynurenine has been identified previously in a low density lipoprotein (37). In that protein, cleavage of the indole ring is a metal-catalyzed oxidation reaction.…”

Section: Discussionmentioning

confidence: 97%

Posttranslational modifications in the CP43 subunit of photosystem II

Anderson

Maderia

Ouellette

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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, and ؉18 daltons. The masses of the modifications suggest that the tryptophan is modified to kynurenine (؉4), a keto-͞ amino-͞hydroxy-(؉16) derivative, and a dihydro-hydroxy-(؉18) derivative of the indole side chain. Peptide synthesis and MS͞MS confirmed the kynurenine assignment. The ؉16 and ؉18 tryptophan modifications may be intermediates formed during the oxidative cleavage of the indole ring to give kynurenine. The sitedirected mutations, W352C, W352L, and W352A, exhibit an increased rate of photoinhibition relative to wild type. We hypothesize that Trp-352 oxidative modifications are a byproduct of PSII water-splitting or electron transfer reactions and that these modifications target PSII for turnover. As a step toward understanding the tertiary structure of this CP43 peptide, structural modeling was performed by using molecular dynamics.mass spectrometry ͉ collision-induced dissociation ͉ tryptophan ͉ kynurenine ͉ photoinhibition P hotosystem II (PSII) is a protein-pigment complex located in thylakoid membranes of plants, eukaryotic algae, and cyanobacteria. PSII catalyzes the light-driven oxidation of water to O 2 , and the reduction of plastoquinone. PSII contains both intrinsic and extrinsic polypeptides. The intrinsic polypeptides include chlorophyll-binding proteins, CP47, CP43, and the D1 and D2 polypeptides (reviewed in ref. 1). The D2͞D1 heterodimer binds P 680 , pheophytin, and the quinone receptors, Q A and Q B (2). Three extrinsic subunits, the manganese stabilizing, 24-kDa, and 18-kDa proteins, are required for maximum oxygen evolution in plants (3, 4). Recently, a 3.8-Å structure of the cyanobacterial PSII reaction center has been reported (5, 6).The intrinsic PSII subunits, CP43 and CP47, function as light-harvesting proteins and play a role in PSII assembly and activity (7-11). CP47 and CP43 have similar tertiary and secondary structures (5). Each polypeptide has six membranespanning regions and a large luminal, hydrophilic loop (E) between helix V and VI (5, 7). In Synechocystis sp. PCC 6803, loop E of the CP43 subunit extends from residue Asn-280 to . Mutations or deletions in this loop inactivate or impair PSII activity in Synechocystis (9, 11-13).Posttranslational modifications can play important roles in the assembly, degradation, structure, and function of proteins. However, little is known about the roles of such modifications in membrane proteins. For example, in cytochrome c oxidase, a crosslinked tyrosine-histidine cofactor has been identified at the binuclear metal site (14); the function of this cofactor has not yet been definitively established. Recently, it has been suggested that posttranslationally modified amino acids, containing carbonyl groups, covalently bind hydrazines and amines at the catalytic site of PSII (10, 15). Because amines and hydrazines are inhibitors of photosynthetic water oxidation, it was suggested that these carbonyl-containing amino acids play roles in the structure, function, or assembly of PSII.To obtain more information about posttranslational mod...

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“…Tryptophan oxidation has been assumed to be an early event, possibly an elementary reaction, in the initiation of LDL oxidation (32)(33)(34)(35)(36)(37)(38). Oxidation may affect the structure and the biological properties of the tryptophans, and it is reasonable to speculate that oxidation of tryptophan 4369 could disrupt the binding of LDL to its receptor.…”

Section: The Molecular Mechanism For Fdbmentioning

confidence: 99%

The Molecular Mechanism for the Genetic Disorder Familial Defective Apolipoprotein B100

Borén

Ekström

Agren

et al. 2001

Journal of Biological Chemistry

136

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Familial defective apolipoprotein B100 (FDB) is a genetic disorder in which low density lipoproteins (LDL) bind defectively to the LDL receptor, resulting in hypercholesterolemia and premature atherosclerosis. FDB is caused by a mutation (R3500Q) that changes the conformation of apolipoprotein (apo) B100 near the receptorbinding site. We previously showed that arginine, not simply a positive charge, at residue 3500 is essential for normal receptor binding and that the carboxyl terminus of apoB100 is necessary for mutations affecting arginine 3500 to disrupt LDL receptor binding. Thus, normal receptor binding involves an interaction between arginine 3500 and tryptophan 4369 in the carboxyl tail of apoB100. W4369Y LDL and R3500Q LDL isolated from transgenic mice had identically defective LDL binding and a higher affinity for the monoclonal antibody MB47, which has an epitope flanking residue 3500. We conclude that arginine 3500 interacts with tryptophan 4369 and facilitates the conformation of apoB100 required for normal receptor binding of LDL. From our findings, we developed a model that explains how the carboxyl terminus of apoB100 interacts with the backbone of apoB100 that enwraps the LDL particle. Our model also explains how all known ligand-defective mutations in apoB100, including a newly discovered R3480W mutation in apoB100, cause defective receptor binding. The interaction between low density lipoprotein (LDL)1 and the LDL receptor is fundamental for the regulation of plasma cholesterol in humans (1). The sole protein component of LDL is apolipoprotein (apo) B100, which serves as the ligand for the LDL receptor (2). We recently identified the sequence in apoB100 that interacts with the LDL receptor (3). However, the ability of apoB100 to interact with the LDL receptor depends not only on sequence, but also on conformation, because apoB100 binds to the LDL receptor only after the hydrolysis of triglyceride-rich very low density lipoproteins (VLDL) to smaller cholesterol-rich LDL (1).Familial defective apoB100 (FDB) is a genetic disorder of LDL metabolism characterized by hypercholesterolemia and premature atherosclerosis (4, 5). Estimated to occur in 1/500 to 1/700 people in several Caucasian populations in North America and Europe, FDB is one of the most common single-gene defects known to cause an inherited abnormality (6, 7). Almost everyone with FDB is of European descent; in most cases, the CGG-to-CAG mutation in the codon for amino acid 3500 is on a chromosome with a rare haplotype at the apoB locus, suggesting that most probands descended from a common ancestor who lived in Europe about 6,750 years ago (8). The mutation substitutes glutamine for the normally occurring arginine at position 3500 (4). Except for a few cases in which tryptophan substitutes for arginine 3500 (9) or cysteine for arginine 3531 (10), leading to a minor decrease in LDL receptor binding, extensive searches have not revealed any other apoB100 mutations that cause defective receptor binding of LDL (10). Both of these mutation...

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Identification of Modified Tryptophan Residues in Apolipoprotein B-100 Derived from Copper Ion-Oxidized Low-Density Lipoprotein

Cited by 37 publications

References 23 publications

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Posttranslational modifications in the CP43 subunit of photosystem II

The Molecular Mechanism for the Genetic Disorder Familial Defective Apolipoprotein B100

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