title: Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.
abstract: Allergic reactions towards β-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a β-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by β-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards β-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards β-lactams.
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain; Research Laboratory Carlos Haya Hospital-IBIMA, Málaga, Spain.
authors.forename: Adriana
authors.initials: A
authors.lastname: Ariza
authors.affiliation: Department of Organic Chemistry, University of Málaga, Malaga, Spain; BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Collado
authors.affiliation: Department of Organic Chemistry, University of Málaga, Malaga, Spain; BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Vida
authors.affiliation: Research Laboratory Carlos Haya Hospital-IBIMA, Málaga, Spain; BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain.
authors.forename: María I
authors.initials: MI
authors.lastname: Montañez
authors.affiliation: Department of Organic Chemistry, University of Málaga, Malaga, Spain; BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain.
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Pérez-Inestrosa
authors.affiliation: Allergy Service, Hospital Carlos Haya, Málaga, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Blanca
authors.affiliation: Research Laboratory Carlos Haya Hospital-IBIMA, Málaga, Spain.
authors.forename: María José
authors.initials: MJ
authors.lastname: Torres
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Cañada
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Dolores
authors.initials: D
authors.lastname: Pérez-Sala
mesh_terms: D000658:Amoxicillin / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001667:Binding, Competitive / Q000276:immunology; D019921:Biotinylation / Q000379:methods; D002082:Butylamines; D006241:Haptens / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood; D008264:Macrophages; D051379:Mice; D018613:Microscopy, Confocal; D015394:Molecular Structure; D012709:Serum Albumin / Q000378:metabolism; D047090:beta-Lactams / Q000276:immunology / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D002082:Butylamines; D006241:Haptens; D012709:Serum Albumin; D047090:beta-Lactams; D007073:Immunoglobulin E; D000658:Amoxicillin