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

PLoS One. 2020 Jan 8;15(1):e0226928. doi: 10.1371/journal.pone.0226928. eCollection 2020.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Colon / growth & development*
  • Colon / metabolism
  • HEK293 Cells
  • Humans
  • Interleukin-2 Receptor alpha Subunit / metabolism
  • Organ Culture Techniques
  • Organ Specificity
  • Single-Chain Antibodies / metabolism*
  • Thrombospondins / metabolism*
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / immunology
  • Wnt Signaling Pathway*

Substances

  • IL2RA protein, human
  • Interleukin-2 Receptor alpha Subunit
  • Single-Chain Antibodies
  • Thrombospondins
  • RNF43 protein, human
  • Ubiquitin-Protein Ligases
  • ZNRF3 protein, human