Amoxicillin haptenation of α-enolase is modulated by active site occupancy and acetylation

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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.
This work was supported by grants from the Ministerio de Ciencia e Innovación cofunded by ERDF (SAF2015-68590-R and RTI2018-097624-B-I00 to DPS and PCI2019-111825-2 Proyectos de I+D+I “Programación Conjunta Internacional” EuroNanoMed 2019 and PID2019-104293GB-I00 to EI), the Instituto de Salud Carlos III ERDF (RETIC ARADyAL RD16/0006/0021 to DPS, RETIC ARADyAL RD16/0006/0001 to MS and MIM, CPII20/00028 to MIM and RETIC ARADyAL RD16/0006/0012 to EPI, and CPII20/00028 to MIM), Junta de Andalucía and Universidad de Málaga (UMA18-FEDERJA-007 to EPI), Consejería de Transformación Económica, Industria, Conocimiento y Universidades of Junta de Andalucía (PY20_00384 to EPI). The funders had no role in study design, analysis of the data or decission to publish. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).