Aplicación de la resonancia de plasmón superficial al estudio de la interacción CXCL12/CXCR4
AdvisorMellado García, José Mario
EntityUAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)
SubjectsQuimioquinas - Receptores - Tesis doctorales; Biología y Biomedicina / Biología
NoteTesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 11-11-2012
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Surface plasmon resonance (SPR)-based biosensors are established tools for measuring molecular interactions, and could provide an ideal method to evaluate G protein-coupled receptor (GPCR) binding interactions. All current SPR methods are based on capture of solubilized GPCR prior to their reconstitution in a lipid environment, however, and require the use of detergents and substantial protein purification prior to analysis. Based on a model using the chemokine receptor CXCR4 and its specific ligand, CXCL12, we outline a biosensor-based method to determine the binding parameters of this interaction. This highly reproducible technique circumvents the limitations of the direct immobilization approach, as well as the need for detergents. CXCR4 in the context of the cell membrane is incorporated into lentiviral particles, which approximates in vivo conditions. Virions prepared from transfected cells bearing the receptor are easily purified and attached to the biosensor surface. Binding of specific antibodies and of CXCL12 demonstrates the structural integrity of CXCR4 and the specificity of the interaction. Other chemokines do not interact, and CXCL12 binding is abrogated by incubation of the lentiviral particles with the CXCR4 antagonist AMD3100. The method shows a relative standard deviation of <10% and chip-to-chip variation <12%, with stability of the sensor response for more than 100 measurements in the same chip. The application of biosensor technology to clinical sample testing is nonetheless difficult, due mainly to non-specific interactions caused by the complex nature of biological fluids. Use of specific antibodies helps to minimize background signals and to increase assay sensitivity; detection nonetheless depends on epitope recognition by the antibody, which often does not distinguish active from inactive analytes. This problem is solved when the native receptor for a protein can be associated to a flow chamber surface. We developed this SPR system to detect CXCL12 in urine samples from rheumatoid arthritis (RA) patients, who have elevated CXCL12 levels in plasma and synovial fluid. The sample requires no pretreatment, and the method is sufficiently sensitive and reproducible to detect variations in protein concentration. The use of lentiviral particles allowed us to determine in real-time kinetic parameters for the CXCR4/ CXCL12 interaction, and to evaluate the effect of glycosaminoglycan/chemokine complexes on binding to the immobilized receptor. The data indicate that, at low concentrations, GAG modulates the CXCR4/CXCL12 interaction and in excess, blocks it. These observations suggest a role for GAG in modulating local chemokine availability as well as the receptor/ligand interaction. The use of lentiviral particles to present cell membrane proteins allows detailed study of ligand interactions using optical biosensors in conditions that approximate the in vivo context, including cell membrane lipid composition, receptor conformations, and the presence of other proteins that could modulate binding parameters. The approach has clear utility for drug discovery research, as it facilitates screening for agonist/antagonist effects and simplifies biomarker detection in biological fluids.
Google Scholar:Vega Blanco, Beatriz
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