Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling

Autoři: Vincent C. Luca aff001;  Yi Miao aff001;  Xingnan Li aff002;  Michael J. Hollander aff003;  Calvin J. Kuo aff002;  K. Christopher Garcia aff001
Působiště autorů: Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Department of Structural Biology, Stanford University School of Medicine, Stanford, California, United States of America aff001;  Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California, United States of America aff002;  Department of Bioengineering, Stanford University, Stanford, California, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of ‘surrogate RSPOs’ that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.

Klíčová slova:

Cell signaling – Colon – Gastrointestinal tract – Organoids – Saccharomyces cerevisiae – Signal processing – Wnt signaling cascade – Yeast


1. Nusse R, Clevers H. Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities. Cell. 2017;169: 985–999. doi: 10.1016/j.cell.2017.05.016 28575679

2. Hao H-X, Xie Y, Zhang Y, Charlat O, Oster E, Avello M, et al. ZNRF3 promotes Wnt receptor turnover in an R-spondin-sensitive manner. Nature. 2012;485: 195–200. doi: 10.1038/nature11019 22575959

3. Janda CY, Dang LT, You C, Chang J, de Lau W, Zhong ZA, et al. Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling. Nature. 2017;545: 234–237. doi: 10.1038/nature22306 28467818

4. Koo B-K, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, et al. Tumour suppressor RNF43 is a stem-cell E3 ligase that induces endocytosis of Wnt receptors. Nature. 2012;488: 665–669. doi: 10.1038/nature11308 22895187

5. de Lau W, Barker N, Low TY, Koo B-K, Li VSW, Teunissen H, et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature. 2011;476: 293–297. doi: 10.1038/nature10337 21727895

6. Xie Y, Zamponi R, Charlat O, Ramones M, Swalley S, Jiang X, et al. Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin. EMBO Rep. 2013;14: 1120–1126. doi: 10.1038/embor.2013.167 24165923

7. Wang D, Huang B, Zhang S, Yu X, Wu W, Wang X. Structural basis for R-spondin recognition by LGR4/5/6 receptors. Genes Dev. 2013;27: 1339–1344. doi: 10.1101/gad.219360.113 23756652

8. Zebisch M, Jones EY. Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3. J Struct Biol. 2015;191: 149–155. doi: 10.1016/j.jsb.2015.05.008 26123262

9. Zebisch M, Xu Y, Krastev C, MacDonald BT, Chen M, Gilbert RJC, et al. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nat Commun. 2013;4: 2787. doi: 10.1038/ncomms3787 24225776

10. Park S, Cui J, Yu WA, Wu L, Carmon K, Liu QJ. Differential activities and mechanisms of the four R-Spondins in potentiating Wnt/β-catenin signaling. J Biol Chem. 2018; jbc.RA118.002743. doi: 10.1074/jbc.RA118.002743 29752411

11. Szenker-Ravi E, Altunoglu U, Leushacke M, Bosso-Lefèvre C, Khatoo M, Thi Tran H, et al. RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6. Nature. 2018;557: 564–569. doi: 10.1038/s41586-018-0118-y 29769720

12. Lebensohn AM, Rohatgi R. R-spondins can potentiate WNT signaling without LGRs. eLife. 2018;7: e33126. doi: 10.7554/eLife.33126 29405118

13. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459: 262–265. doi: 10.1038/nature07935 19329995

14. Ootani A, Li X, Sangiorgi E, Ho QT, Ueno H, Toda S, et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med. 2009;15: 701–706. doi: 10.1038/nm.1951 19398967

15. Zhou W-J, Geng ZH, Spence JR, Geng J-G. Induction of intestinal stem cells by R-spondin 1 and Slit2 augments chemoradioprotection. Nature. 2013;501: 107–111. doi: 10.1038/nature12416 23903657

16. Yan KS, Janda CY, Chang J, Zheng GXY, Larkin KA, Luca VC, et al. Non-equivalence of Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal. Nature. 2017. doi: 10.1038/nature22313 28467820

17. Zhan T, Rindtorff N, Boutros M. Wnt signaling in cancer. Oncogene. 2016;36: onc2016304. doi: 10.1038/onc.2016.304 27617575

18. Hémar A, Subtil A, Lieb M, Morelon E, Hellio R, Dautry-Varsat A. Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains. J Cell Biol. 1995;129: 55–64. doi: 10.1083/jcb.129.1.55 7698995

19. Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC. Structural basis of Wnt recognition by Frizzled. Science. 2012;337: 59–64. doi: 10.1126/science.1222879 22653731

20. Cheng Z, Biechele T, Wei Z, Morrone S, Moon RT, Wang L, et al. Crystal structures of the extracellular domain of LRP6 and its complex with DKK1. Nat Struct Mol Biol. 2011;18: 1204–1210. doi: 10.1038/nsmb.2139 21984209

21. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JMW, Yeung YA, et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol. 2003;21: 163–170. doi: 10.1038/nbt785 12536217

22. Glinka A, Dolde C, Kirsch N, Huang Y-L, Kazanskaya O, Ingelfinger D, et al. LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling. EMBO Rep. 2011;12: 1055–1061. doi: 10.1038/embor.2011.175 21909076

23. Rickert M, Boulanger MJ, Goriatcheva N, Garcia KC. Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its alpha, beta, and gamma(c) receptors. J Mol Biol. 2004;339: 1115–1128. doi: 10.1016/j.jmb.2004.04.038 15178252

24. Spangler JB, Tomala J, Luca VC, Jude KM, Dong S, Ring AM, et al. Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity. 2015;42: 815–825. doi: 10.1016/j.immuni.2015.04.015 25992858

25. Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity. 2013;38: 13–25. doi: 10.1016/j.immuni.2013.01.004 23352221

26. van Loosdregt J, Fleskens V, Tiemessen MM, Mokry M, van Boxtel R, Meerding J, et al. Canonical Wnt signaling negatively modulates regulatory T cell function. Immunity. 2013;39: 298–310. doi: 10.1016/j.immuni.2013.07.019 23954131

27. Malladi S, Macalinao DG, Jin X, He L, Basnet H, Zou Y, et al. Metastatic Latency and Immune Evasion through Autocrine Inhibition of WNT. Cell. 2016;165: 45–60. doi: 10.1016/j.cell.2016.02.025 27015306

28. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006;1: 755–768. doi: 10.1038/nprot.2006.94 17406305

29. Naviaux RK, Costanzi E, Haas M, Verma IM. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J Virol. 1996;70: 5701–5705. 8764092

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