Original ContributionImmunogenicity of a brominated protein and successive establishment of a monoclonal antibody to dihalogenated tyrosine
Introduction
Inflammation, to a greater or lesser degree, is a frequent event in the human body. Among the immune cells, neutrophils are some of the most important cells in first defense against a microbial invasion. Eosinophils also help protect against parasitic infection. These cells have granules containing peroxidases, such as myeloperoxidase (MPO) and eosinophil peroxidase (EPO). These enzymes can catalyze the formation of hypohalous acids, hypochlorous acid (HOCl) and/or hypobromous acid (HOBr), using hydrogen peroxide and Br−/Cl−. The active halogenating species modify various biomolecules such as nucleic acids [1], [2], [3], [4], proteins [5], [6], and lipids [7], [8]. By use of MPO and EPO knockout mice, it has already been shown that these peroxidases have an important role in the generation of reactive chlorinating and brominating species in vivo [9], [10]. Modification of the protein by the reactive intermediates causes protein tyrosine halogenation, forming chlorotyrosine (ClY) and bromotyrosine (BrY). These halotyrosines have been detected in cystic fibrosis [11], atherosclerosis [12], the sepsis model [13], and asthma [14], [15] using gas chromatography–mass spectrometry. BrY was shown to be increased in the airway proteins of a patient with asthmaticus [16]. During halogenation, the incorporation of two halogens into one tyrosine moiety also occurs and, as a consequence, 3,5-dichlorotyrosine (DiClY) and/or 3,5-dibromotyrosine (DiBrY) are generated [17], [18], [19]. A reactive intermediate, chloramine, is also formed by the reaction of an amino moiety and HOCl. The chloramine is considered to be a precursor of an aldehyde [20], [21]. The chloramine may be one of the reactive chlorinating species in vivo.
To detect the halogenation of proteins, antibodies specific to the HOCl-modified proteins have already been reported [22]. These antibodies react with atherosclerotic plaque, kidney, and placental tissues [23], [24], [25], [26]. It has been reported that the HOP-1 monoclonal antibody against HOCl-LDL also recognizes brominated proteins [18]. However, the detailed epitopes of HOP-1 and other antibodies to HOCl-modified proteins remain obscure [22], [24]. In our study, the immunogenicity of a brominated protein was initially examined. Next, we obtained and characterized a monoclonal antibody to dihalogenated tyrosine, and then used the antibody in immunohistochemical analysis.
Section snippets
Materials
Bovine serum albumin (BSA), N-acetyltyrosine (NAY), 3-chlorotyrosine (ClY), 3-aminotyrosine, 3,5-diiodotyrosine (DiIY), N-bromosuccinimide (NBS), cytochrome c, β-lactoglobulin, hemoglobin, lens protein, 5-bromouracil, and 8-bromoguanosine were purchased from Sigma. Sodium hypochlorite (NaOCl), 3-iodotyrosine (IY), and l-tyrosine (Y) were from Wako Pure Chemicals, Inc. 3,5-Dibromohydroxybenzoic acid (DiBrHBz), 3,5-dichlorohydroxybenzoic acid (DiClHBz), and 5-bromouridine were obtained from
Immunogenicity of brominated protein
The treatment of protein with HOBr, NBS, and N-bromamine causes the formation of BrY and DiBrY [17]. In this study, we obtained the brominated protein by exposure to NBS. To check the amount of tyrosine bromination, the obtained immunogen, a brominated KLH (NBS–KLH), was hydrolyzed and analyzed by LC/MS/MS. Brominated KLH contained 0.10 BrY and 0.14 DiBrY mol/mol of tyrosine. After immunization of mice, the reactivity of the antiserum with various proteins was examined by indirect
Discussion
Among the oxidative modifications of amino acid residues, tyrosine modification is quite unique because the profiles of the products differ depending on the generating active oxygen species. For example, 3-nitrotyrosine is considered to be a fingerprint of nitrating species such as peroxynitrite, NO2, and other nitrating species (such as NO2Cl). On the other hand, 3-ClY is one of the specific markers for the chlorination of a protein by HOCl and other chlorinating species (like Cl2). Similarly,
Acknowledgments
We thank Asako Komori and Shihoko Kanjya for their helpful technical assistance. We thank Dr. Anthony J. Kettle for his helpful advice on preparation of antibody to 3-chlorotyrosine provided at the 10th Biennial Meeting of the Society for Free Radical Research International (SFRRI) 2000 Conference (Tokyo).
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