title: Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model.
abstract: Peptide hormones perform important biological functions by binding specific cell membrane receptors. For hormone-receptor interaction studies, receptor-binding assays are widely used. However, conventional receptor-binding assays rely on radioactive tracers that have drawbacks. In recent studies, we established novel non-radioactive receptor-binding assays for some recombinant protein hormones based on the ultrasensitive bioluminescence of a newly developed nanoluciferase (NanoLuc) reporter. In the present work, we extended the novel bioluminescent receptor-binding assay to peptide hormones that have small size and can be conveniently prepared by chemical synthesis. Human ghrelin, a 28-amino acid peptide hormone carrying a special O-fatty acid modification, was used as a model. To prepare a bioluminescent ghrelin tracer, a chemically synthesized ghrelin analog with a unique cysteine residue at the C-terminus was site-specifically conjugated with an engineered NanoLuc with a unique exposed cysteine residue at the C-terminus via a reversible disulfide linkage. The NanoLuc-conjugated ghrelin retained high binding affinity with the ghrelin receptor GHSR1a (K d = 1.14 ± 0.13 nM, n = 3) and was able to sensitively monitor the receptor-binding of various GHSR1a ligands. The novel bioluminescent receptor-binding assay will facilitate the interaction studies of ghrelin with its receptor. We also proposed general procedures for convenient conjugation of other peptide hormones with NanoLuc for novel bioluminescent receptor-binding assays.
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Song
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China.
authors.forename: Zeng-Guang
authors.initials: ZG
authors.lastname: Xu
authors.affiliation: Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China. zhan-yun.guo@tongji.edu.cn.
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D001681:Biological Assay / Q000379:methods*; D054439:Ghrelin / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D049329:Nanostructures / Q000737:chemistry; D011485:Protein Binding; D054440:Receptors, Ghrelin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
chemical_list: D054439:Ghrelin; D054440:Receptors, Ghrelin; D011993:Recombinant Fusion Proteins; D008156:Luciferases
keywords: Bioluminescence; Ghrelin; NanoLuc; Peptide hormone; Receptor-binding