title: Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.
abstract: Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various natural or designed ligands with these receptors. Using this site-specific labeling strategy, other functional probes, such as fluorescent dyes, biotin, or nanoparticles could also be introduced to the A-chain N-terminal of the recombinant human relaxin-3. Additionally, we improved the time-resolved fluorescence assay for the DOTA-bound europium ion which paves the way for the use of DOTA as a lanthanide chelator for protein and peptide labeling in future studies.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China.
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.forename: Xiao
authors.initials: X
authors.lastname: Luo
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.forename: John D
authors.initials: JD
authors.lastname: Wade
authors.forename: Ross A D
authors.initials: RA
authors.lastname: Bathgate
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry*; D057930:Click Chemistry; D056831:Coordination Complexes / Q000737:chemistry; D005063:Europium / Q000737:chemistry*; D057809:HEK293 Cells; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018000:Receptors, Peptide / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D012065:Relaxin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
chemical_list: D002614:Chelating Agents; D056831:Coordination Complexes; D006573:Heterocyclic Compounds, 1-Ring; C471213:His-His-His-His-His-His; D009842:Oligopeptides; C480238:RLN3 protein, human; C417543:RXFP1 protein, human; C410092:RXFP3 protein, human; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; D005063:Europium; D006639:Histidine; D012065:Relaxin; D000626:Aminopeptidases