Amoxicillin haptenates intracellular proteins that can be transported in exosomes to target cells.
BACKGROUND
Allergic reactions to β-lactams are among the most frequent causes of drug allergy and constitute an important clinical problem. Drug covalent binding to endogenous proteins (haptenation) is thought to be required for activation of the immune system. Nevertheless, neither the nature nor the role of the drug protein targets involved in this process is fully understood. Here, we aim to identify novel intracellular targets for haptenation by amoxicillin (AX) and their cellular fate.


METHODS
We have treated B lymphocytes with either AX or a biotinylated analog (AX-B). The identification of protein targets for haptenation by AX has been approached by mass spectrometry and immunoaffinity techniques. In addition, intercellular communication mediated by the delivery of vesicles loaded with AX-B-protein adducts has been explored by microscopy techniques.


RESULTS
We have observed a complex pattern of AX-haptenated proteins. Several novel targets for haptenation by AX in B lymphocytes have been identified. AX-haptenated proteins were detected in cell lysates and extracellularly, either as soluble proteins or in lymphocyte-derived extracellular vesicles. Interestingly, exosomes from AX-B-treated cells showed a positive biotin signal in electron microscopy. Moreover, they were internalized by endothelial cells, thus supporting their involvement in intercellular transfer of haptenated proteins.


CONCLUSIONS
These results represent the first identification of AX-mediated haptenation of intracellular proteins. Moreover, they show that exosomes can constitute a novel vehicle for haptenated proteins, and raise the hypothesis that they could provide antigens for activation of the immune system during the allergic response.
Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
F J
FJ
Sánchez-Gómez
Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
J M
JM
González-Morena
Department of Organic Chemistry, University of Málaga, IBIMA, Málaga, Spain.|Andalusian Center for Nanomedicine and Biotechnology-BIONAND, Parque Tecnológico de Andalucía, Málaga, Spain.
Y
Y
Vida
Department of Organic Chemistry, University of Málaga, IBIMA, Málaga, Spain.|Andalusian Center for Nanomedicine and Biotechnology-BIONAND, Parque Tecnológico de Andalucía, Málaga, Spain.
E
E
Pérez-Inestrosa
Allergy Unit, IBIMA-Regional University Hospital of Málaga, UMA, Málaga, Spain.
M
M
Blanca
Allergy Unit, IBIMA-Regional University Hospital of Málaga, UMA, Málaga, Spain.
M J
MJ
Torres
Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
D
D
Pérez-Sala
D000658:Amoxicillin / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D055354:Exosomes / Q000276:immunology / Q000378:metabolism*; D006241:Haptens / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000276:immunology* / Q000378:metabolism*; D020543:Proteome; D040901:Proteomics / Q000379:methods; D047090:beta-Lactams / Q000276:immunology / Q000378:metabolism
D006241:Haptens; D011506:Proteins; D020543:Proteome; D047090:beta-Lactams; D000658:Amoxicillin
amoxicillin; cellular protein targets; drug adducts; exosomes; haptenation