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/Eu3+-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.
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Abbreviations
- BSA:
-
Bovine serum albumin
- DOTA:
-
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
- DMSO:
-
Dimethyl sulfoxide
- HPLC:
-
High performance liquid chromatography
- NHS:
-
N-hydroxysuccinimide
- PCR:
-
Polymerase chain reaction
- PEG:
-
Polyethylene glycol
- TBTA:
-
Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
- t-BuOH:
-
tert-Butyl alcohol
- TFA:
-
Trifluoroacetic acid
- TRF:
-
Time-resolved fluorescence
- UV:
-
Ultra-violet
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (30970609) and the National Basic Research Program of China (973 Program, no. 2010CB912604). Part of this research was funded by NHMRC (Australia) project Grants 350284, 508995 to JDW and RADB. We are grateful to Tania Ferraro & Sharon Layfield for assistance with biochemical assays. The studies at the FNI were supported by the Victorian Government’s Operational Infrastructure Support Program.
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W.–J. Zhang and X. Luo contributed equally to this work.
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Zhang, WJ., Luo, X., Liu, YL. et al. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies. Amino Acids 43, 983–992 (2012). https://doi.org/10.1007/s00726-011-1164-z
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DOI: https://doi.org/10.1007/s00726-011-1164-z