Novel bioluminescent receptor-binding assays for peptide hormones: using ghrelin as a model

详细信息    查看全文

• 作者：Yu Liu ; Xiao-Xia Shao ; Lei Zhang ; Ge Song ; Ya-Li Liu ; Zeng-Guang Xu…
• 关键词：Peptide hormone ; Receptor ; binding ; Ghrelin ; Bioluminescence ; NanoLuc
• 刊名：Amino Acids
• 出版年：2015
• 出版时间：October 2015
• 年：2015
• 卷：47
• 期：10
• 页码：2237-2243
• 全文大小：750 KB
• 参考文献：Bednarek MA, Feighner SD, Pong SS, McKee KK, Hreniuk DL, Silva MV, Warren VA, Howard AD, Van Der Ploeg LH, Heck JV (2000) Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 43:4370-376CrossRef PubMed
  Delporte C (2013) Structure and physiological actions of ghrelin. Scientifica (Cairo) 2013:518909
  Gahete MD, Rincón-Fernández D, Villa-Osaba A, Hormaechea-Agulla D, Ibá?ez-Costa A, Martínez-Fuentes AJ, Gracia-Navarro F, Casta?o JP, Luque RM (2013) Ghrelin gene products, receptors, and GOAT enzyme: biological and pathophysiological insight. J Endocrinol 220:R1–R24CrossRef PubMed
  Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV (2012) Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7:1848-857PubMed Central CrossRef PubMed
  He SX, Song G, Shi JP, Guo YQ, Guo ZY (2014) Nanoluciferase as a novel quantitative protein fusion tag: application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor. Biochimie 106:140-48CrossRef PubMed
  Heppner KM, Tong J (2014) Mechanisms in endocrinology: regulation of glucose metabolism by the ghrelin system: multiple players and multiple actions. Eur J Endocrinol 171:R21–R32CrossRef PubMed
  Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI, Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK, McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M, Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR, DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH (1996) A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 273:974-77CrossRef PubMed
  Katugampola SD, Pallikaros Z, Davenport AP (2001) [125I-His(9)]-ghrelin, a novel radioligand for localizing GHS orphan receptors in human and rat tissue: up-regulation of receptors with athersclerosis. Br J Pharmacol 134:143-49PubMed Central CrossRef PubMed
  Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K (1999) Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 402:656-60CrossRef PubMed
  Labarthe A, Fiquet O, Hassouna R, Zizzari P, Lanfumey L, Ramoz N, Grouselle D, Epelbaum J, Tolle V (2014) Ghrelin-derived peptides: a link between appetite/reward, GH Axis, and psychiatric disorders? Front Endocrinol (Lausanne) 5:163
  Matsumoto M, Hosoda H, Kitajima Y, Morozumi N, Minamitake Y, Tanaka S, Matsuo H, Kojima M, Hayashi Y, Kangawa K (2001) Structure-activity relationship of ghrelin: pharmacological study of ghrelin peptides. Biochem Biophys Res Commun 287:142-46CrossRef PubMed
  Muccioli G, Papotti M, Locatelli V, Ghigo E, Deghenghi R (2001) Binding of 125I-labeled ghrelin to membranes from human hypothalamus and pituitary gland. J Endocrinol Invest 24:RC7–RC9CrossRef PubMed
  Pradhan G, Samson SL, Sun Y (2013) Ghrelin: much more than a hunger hormone. Curr Opin Clin Nutr Metab Care 16:619-24PubMed Central CrossRef PubMed
  Rak-Mardyla A (2013) Ghrelin role in hypothalamus-pituitary-ovarian axis. J Physiol Pharmacol 64:695-04PubMed
  Zhang L, Song G, Xu T, Wu QP, Shao XX, Liu YL, Xu ZG, Guo ZY (2013) A novel ultrasensitive bioluminescent receptor-binding assay of INSL3 through chemical conjugation with nanoluciferase. Biochimie 95:2454-459CrossRef PubMed
  
• 作者单位：Yu Liu (1)
  Xiao-Xia Shao (1)
  Lei Zhang (1)
  Ge Song (1)
  Ya-Li Liu (1)
  Zeng-Guang Xu (1)
  Zhan-Yun Guo (1)
  
  1. Research Center for Translational Medicine at East Hospital, College of Life Sciences and Technology, Tongji University, 1239 Siping Road, Shanghai, 200092, China
  
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Biochemistry
  Analytical Chemistry
  Biochemical Engineering
  Life Sciences
  Proteomics
  Neurobiology
  
• 出版者：Springer Wien
• ISSN：1438-2199

文摘

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. Keywords Peptide hormone Receptor-binding Ghrelin Bioluminescence NanoLuc