1. Study of Protein Haptenation by Amoxicillin Through the Use of a Biotinylated Antibiotic
- Author
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Ezequiel Perez-Inestrosa, María José Torres, Maria I. Montañez, F. Javier Cañada, Dolores Pérez-Sala, Yolanda Vida, Adriana Ariza, Miguel Blanca, Daniel Collado, [Ariza,A, Cañada,FJ, Pérez-Sala,D] Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain. [Ariza,A, Torres,MJ] Research Laboratory Carlos Haya Hospital-IBIMA, Málaga, Spain. [Collado,D, VidaY, Pérez-Inestrosa,E] Department of Organic Chemistry, University of Málaga, Malaga, Spain. [Collado,D, Vida,Y, Montañez,MI, Pérez-Inestrosa,E] BIONAND-Andalusian Centre for Nanomedicine and Biotechnology, Parque Tecnológico de Andalucía, Málaga, Spain. [Blanca,M] Allergy Service, Hospital Carlos Haya, Málaga, Spain., and This work was supported by grants SAF2012-36519 from MINECO and RETIC RD07/0064/0007 and RD12/0013/0008 to DPS, Junta de Andalucía CTS- 6603 and ISCIII PS09/01768 and PI12/02529 to MJT, funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement nu 300230, to MIM. The stay of A.A. at CIB-CSIC was funded in part by a fellowship from ISCIII.
- Subjects
Haptenos ,Hipersensibilidad ,Proteomics ,Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Microscopy::Microscopy, Confocal [Medical Subject Headings] ,genetic structures ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Globulins::Serum Globulins::Immunoglobulins::Antibodies::Immunoglobulin Isotypes::Immunoglobulin E [Medical Subject Headings] ,Cell ,lcsh:Medicine ,Biochemistry ,Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings] ,Mice ,chemistry.chemical_compound ,0302 clinical medicine ,Biotin ,Molecular Cell Biology ,Organisms::Eukaryota::Animals [Medical Subject Headings] ,lcsh:Science ,Amoxicilina ,0303 health sciences ,Microscopy, Confocal ,Multidisciplinary ,Molecular Structure ,Allergy and Hypersensitivity ,Anatomy::Hemic and Immune Systems::Immune System::Phagocytes::Macrophages [Medical Subject Headings] ,Beta-lactamas ,Human serum albumin ,Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Binding, Competitive [Medical Subject Headings] ,Blood proteins ,Small molecule ,Anti-Bacterial Agents ,3. Good health ,Chemistry ,medicine.anatomical_structure ,Antibacterianos ,Inmunoglobulina E ,Biotinylation ,Medicine ,Cellular Types ,Antibody ,Chemicals and Drugs::Chemical Actions and Uses::Pharmacologic Actions::Therapeutic Uses::Anti-Infective Agents::Anti-Bacterial Agents [Medical Subject Headings] ,Research Article ,medicine.drug ,Biotinilación ,Drugs and Devices ,Drug Research and Development ,Immune Cells ,Immunology ,Diseases::Immune System Diseases::Hypersensitivity [Medical Subject Headings] ,Chemicals and Drugs::Biological Factors::Antigens::Epitopes::Haptens [Medical Subject Headings] ,Biology ,Butylamines ,beta-Lactams ,Binding, Competitive ,Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams::Penicillins::Penicillin G::Ampicillin::Amoxicillin [Medical Subject Headings] ,03 medical and health sciences ,Phenomena and Processes::Chemical Phenomena::Molecular Structure [Medical Subject Headings] ,Adverse Reactions ,Diagnostic Medicine ,Phenomena and Processes::Chemical Phenomena::Biochemical Phenomena::Biochemical Processes::Biotinylation [Medical Subject Headings] ,Chemical Biology ,Chemicals and Drugs::Organic Chemicals::Amides::Lactams::beta-Lactams [Medical Subject Headings] ,Hypersensitivity ,medicine ,Chemicals and Drugs::Amino Acids, Peptides, and Proteins::Proteins::Albumins::Serum Albumin [Medical Subject Headings] ,Animals ,Humans ,Macrófagos ,Serum Albumin ,030304 developmental biology ,Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Rodentia::Muridae::Murinae::Mice [Medical Subject Headings] ,Macrophages ,Estructura molecular ,lcsh:R ,Amoxicillin ,Proteins ,Immunoglobulin E ,Butilaminas ,chemistry ,Membrane protein ,Small Molecules ,Chemicals and Drugs::Organic Chemicals::Amines::Butylamines [Medical Subject Headings] ,Animales ,biology.protein ,Clinical Immunology ,lcsh:Q ,Haptens ,030215 immunology - Abstract
12 p.-9 fig.-2 tab., Allergic reactions towards b-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a b-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 b-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 b-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 b-lactams., This work was supported by grants SAF2012-36519 from MINECO and RETIC RD07/0064/0007 and RD12/0013/0008 to DPS, Junta de Andalucía CTS-6603 and ISCIII PS09/01768 and PI12/02529 to MJT, funding from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013) under REA grant agreement n° 300230, to MIM. The stay of A.A. at CIB-CSIC was funded in part by a fellowship from ISCIII.
- Published
- 2014