Abstract
Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.
Highlights
Adverse and hypersensitivity reactions to drugs used in clinical treatments constitute a severe clinical problem and may result in patient’s death
These results suggested either that no additional modification sites are exposed in the denatured protein or that exposition and modification of new haptenation sites occurred at the expenses of sites that were modified in the native protein
Haptenation of proteins by AX can determine their recognition by the immune system as foreign antigens and the trigger of an allergic response mediated by IgE that occurs within 1 h of administration (Antúnez et al, 2006; Torres et al, 2011)
Summary
Adverse and hypersensitivity reactions to drugs used in clinical treatments constitute a severe clinical problem and may result in patient’s death. Another important epitope is located on peptide D53-Q87 and recognized by both autoantibodies of cancer associated retinopathy and endometriosis patients (Walter et al, 1995) Allergen databases such as the AllFam database, created to identify common structural features of allergens (Radauer et al, 2008), include enolases of different species. This analysis identified TIM barrels (αβ) as the most frequent domain type (Radauer et al, 2008), which is the topology found in the αenolase C-terminal domain (PF00113) (Kang et al, 2008) This information on the role of α-enolase as an allergen and the epitopes related to allergic reactions highlight the importance of PTMs in its antigenicity. We have carried out an in depth in vitro characterization of α-enolase haptenation and identification of the AX-modified residues, as well as an analysis of its putative interplay or interference with additional PTMs targeting similar residues
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