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Characterization of non-covalent oligomers of proteins treated with hypochlorous acid.

ABSTRACT: Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

SUBMITTER: Chapman AL

PROVIDER: S-EPMC1223668 | biostudies-other | 2003 Oct

REPOSITORIES: biostudies-other

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Project description:Hypochlorous acid (HOCl)-treated whole tumor cell lysates (Ox-L) have been shown to be more immunogenic when used as an antigen source for therapeutic dendritic cell (DC)-based vaccines, improving downstream immune responses both in vitro and in vivo. However, the mechanisms behind the improved immunogenicity are still elusive. To address this question, we conducted a proteomic and immunopeptidomics analyses to map modifications and alterations introduced by HOCl treatment using a human melanoma cell line as a model system. First, we show that one-hour HOCl incubation readily induces extensive protein oxidation, mitochondrial biogenesis, and increased expression of chaperones and antioxidant proteins, all features indicative of an activation of oxidative stress-response pathways. Characterization of the DC proteome after loading with HOCl treated tumor lysate (Ox-L) showed no significant difference compared to loading with untreated whole tumor lysate (FT-L). On the other hand, detailed immunopeptidomic analyses on monocyte-derived DCs (mo-DCs) revealed a great increase in human leukocyte antigen class II (HLA-II) presentation in mo-DCs loaded with Ox-L compared to the FT-L control. Further, 2026 HLA-II ligands uniquely presented on Ox-L-loaded mo-DCs were identified. In comparison, identities and intensities of HLA class I (HLA-I) ligands were overall comparable. We found that HLA-II ligands uniquely presented by DCs loaded with Ox-L were more solvent exposed in the structures of their source proteins, contrary to what has been hypothesized so far. Analyses from a phase I clinical trial showed that vaccinating patients using autologous Ox-L as an antigen source efficiently induces polyfunctional vaccine-specific CD4+ T cell responses. Hence, these results suggest that the increased immunogenicity of Ox-L is, at least in part, due to qualitative and quantitative changes in the HLA-II ligandome, potentially leading to an increased HLA-II dependent stimulation of the T cell compartment (i.e., CD4+ T cell responses). These results further contribute to the development of more effective and immunogenic DC-based vaccines and to the molecular understanding of the mechanism behind HOCl adjuvant properties.

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Characterization of non-covalent oligomers of proteins treated with hypochlorous acid.

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