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2-Alkenal modification of hemoglobin: Identification of a novel hemoglobin-specific alkanoic acid-histidine adduct.

ABSTRACT: ?,?-Unsaturated aldehydes generated during lipid peroxidation, such as 2-alkenals, give rise to protein degeneration in a variety of pathological states. 2-Alkenals are highly reactive toward nucleophilic amino acid residues, such as histidine and lysine, to form Schiff base adducts or Michael addition adducts. In this study, upon the reaction of hemoglobin with 2-octenal, we unexpectedly detected a product corresponding to the reduced form of the 2-octenal-histidine Michael adduct plus 14 mass unit. Based on the LC-ESI-MS/MS analysis of synthetic adduct candidates, the adduct was identified to be N?-(1-carboxyheptan-2-yl)-histidine (CHH), a novel alkanoic acid-type histidine adduct. The alkanoic acid-histidine adducts were detected in the 2-alkenal-treated hemoglobin and myoglobin, but not in the 2-alkenal-treated cytochrome c and transferrin. The addition of hemin to the reaction mixture, containing a non-heme protein and 2-alkenals, resulted in the formation of the alkanoic acid-histidine adducts, suggesting that a heme iron may play a role in the oxidation of covalently modified proteins. Moreover, using the stable isotope dilution method, we showed evidence for the endogenous formation of CHH in red blood cells exposed to hydrogen peroxide. Thus, this study establishes a novel mechanism for covalent modification of proteins by 2-alkenals, in which heme iron is involved in the formation of the alkanoic acid-histidine adducts. The potential implications of this novel adduct are discussed.

SUBMITTER: Yoshitake J

PROVIDER: S-EPMC6859543 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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2-Alkenal modification of hemoglobin: Identification of a novel hemoglobin-specific alkanoic acid-histidine adduct.

Yoshitake Jun J Shibata Takahiro T Shimayama Chihiro C Uchida Koji K

Redox biology 20190222

α,β-Unsaturated aldehydes generated during lipid peroxidation, such as 2-alkenals, give rise to protein degeneration in a variety of pathological states. 2-Alkenals are highly reactive toward nucleophilic amino acid residues, such as histidine and lysine, to form Schiff base adducts or Michael addition adducts. In this study, upon the reaction of hemoglobin with 2-octenal, we unexpectedly detected a product corresponding to the reduced form of the 2-octenal-histidine Michael adduct plus 14 mass ...[more]

PMID: 30819615

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Project description:Dehaloperoxidase hemoglobin A (DHP A) is a multifunctional hemoglobin that appears to have evolved oxidative pathways for the degradation of xenobiotics as a protective function that complements the oxygen transport function. DHP A possesses at least two internal binding sites, one for substrates and one for inhibitors, which include various halogenated phenols and indoles. Herein, we report the X-ray crystallographic structure of the carbonmonoxy complex (DHPCO). Unlike other DHP structures with 6-coordinated heme, the conformation of the distal histidine (H55) in DHPCO is primarily external or solvent exposed, despite the fact that the heme Fe is 6-coordinated. As observed generally in globins, DHP exhibits two distal histidine conformations (one internal and one external). In previous structural studies, we have shown that the distribution of H55 conformations is weighted strongly toward the external position when the DHP heme Fe is 5-coordinated. The large population of the external conformation of the distal histidine observed in DHPCO crystals at pH 6.0 indicates that some structural factor in DHP must account for the difference from other globins, which exhibit a significant external conformation only when pH < 4.5. While the original hypothesis suggested that interaction with a heme-Fe-bound ligand was the determinant of H55 conformation, the current study forces a refinement of that hypothesis. The external or open conformation of H55 is observed to have interactions with two propionate groups in heme, at distances of 3.82 and 2.73 Å, respectively. A relatively weak hydrogen bonding interaction between H55 and CO, combined with strong interactions with heme propionate (position 6), is hypothesized to strengthen the external conformation of H55. Density function theory (DFT) calculations were conducted to test whether there is a weaker hydrogen bond interaction between H55 and heme bonded CO or O2. Molecular dynamics simulations were conducted to examine how the tautomeric forms of H55 affect the dynamic motions of the distal histidine that govern the switching between open and closed conformations. The calculations support the modified hypothesis suggesting a competition between the strength of interactions with heme ligand and the heme propionates as the factors that determine the conformation of the distal histidine.

Project description:Skin (contact) allergy, the most predominant form of immunotoxicity in humans, is caused by small electrophilic compounds (haptens) that modify endogenous proteins. Approximately 20% of the general population in the Western world is affected by contact allergy. Although the importance of the hapten-protein conjugates is well established in the initiation of the immunological reaction, not much progress has been made regarding identification of these conjugates in vivo or exploration of their potential as diagnostic tools. In this study, the human serum albumin (HSA) and human hemoglobin (Hb) adductome for three representative contact allergens with different chemical properties, 1-chloro-2,4-dinitrobenzene (DNCB), 1,2-epoxy-3-phenoxypropane (PGE), and 2-bromo-2-(bromomethyl)glutaronitrile (MDBGN), were studied. Plasma and red blood cell lysate were used as a source for HSA and Hb, respectively. The Direct Peptide Reactivity Assay was used to investigate adduct formation of MDBGN with nucleophilic moieties and revealed that MDGBN is converted to 2-methylenepentanedinitrile in the presence of sulfhydryl groups prior to adduct formation. Following incubation of HSA and Hb with haptens, an Orbitrap Q Exactive high-resolution mass spectrometer was used to perform an initial untargeted analysis to screen for adduct formation, followed by confirmation by targeted Parallel Reaction Monitoring analysis. Although a subset of adducted sites was confirmed by targeted analysis, only some of the adducted peptides showed an increase in the relative amount of the adducted peptide with an increased concentration of hapten. In total, seven adduct sites for HSA and eight for Hb were confirmed for DNCB and PGE. These sites are believed to be the most reactive. Further, three of the HSA sites (Cys34, Cys62, and Lys190) and six of the Hb sites (subunit α: Val1, His45, His72; subunit β: Cys93, His97, and Cys112) were haptenated already at the lowest level of hapten to protein molar ratio (0.1:1), indicating that these sites are the most likely to be modified in vivo. To the best of our knowledge, this is the first time that the adductome of Hb has been studied in the context of contact allergens. Identification of the most reactive sites of abundant proteins, such as HSA and Hb, is the first step toward identification of contact allergy biomarkers that can be used for biomonitoring and to develop better diagnostic tools based on a blood sample.

Dataset Information

2-Alkenal modification of hemoglobin: Identification of a novel hemoglobin-specific alkanoic acid-histidine adduct.

Publications

2-Alkenal modification of hemoglobin: Identification of a novel hemoglobin-specific alkanoic acid-histidine adduct.

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