title: Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.
abstract: The bradykinin (BK) B1 receptor (B1R) is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP) (enhanced green FP [EGFP] or mCherry) prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively). The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy). Both assays indicated that the best design was FP-(Asn-Gly)n-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly)15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology).
authors: Centre de recherche en rhumatologie et immunologie, CHU de Québec and Department of Microbiology-Infectious Disease and Immunology, Université Laval, Québec, QC, G1V 4G2, Canada.
authors: Xavier
authors: X
authors: Charest-Morin
authors: Centre de recherche en rhumatologie et immunologie, CHU de Québec and Department of Microbiology-Infectious Disease and Immunology, Université Laval, Québec, QC, G1V 4G2, Canada.
authors: François
authors: F
authors: Marceau
mesh_terms: D015415:Biomarkers; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D043783:Receptor, Bradykinin B1 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins; D043783:Receptor, Bradykinin B1; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein