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.
Highlights
The Wnt/β-catenin pathway controls cell fate determination and tissue homeostasis in all metazoans[1]
Our surrogate RSPO design concept involves the fusion of two components: (i) a RNF43- or ZNRF3-binding protein and (ii) a tissue-specific targeting protein that binds to a surface receptor
After several rounds of selection, we isolated a panel of different RNF43- or ZNRF3-specific scFvs (S1 Fig), and the clones “R5” and “Z6” were chosen for incorporation into surrogate RSPOs based on their ability to robustly bind to RNF43 and ZNRF3, respectively, in a fluorescence-based assay (Fig 2B)
Summary
The Wnt/β-catenin pathway controls cell fate determination and tissue homeostasis in all metazoans[1]. Pleiotropic Wnt signaling exhibits differential effects on a wide array of cell types and is tightly regulated by several host-encoded enhancers and inhibitors. Rspondin proteins (RSPO1-4 in mammals) potentiate Wnt signaling by antagonizing negative. Surrogate R-spondin proteins decision to publish, or preparation of the manuscript
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