Molecular Engineering of the Helminth <scp>TGF</scp> ‐β Mimetics, <scp>TGM1</scp> and <scp>TGM4,</scp> Reveals a Novel Antagonist of <scp>TGF</scp> ‐β Signaling in Fibroblasts

The transforming growth factor-β (TGF-β) mimics TGM1 and TGM4 are modular proteins expressed by the parasite Heligmosomoides polygyrus. Each comprises 5 complement control protein (CCP) domains binding the TGF-β receptors TGFBR1 (Domains 1 and 2) and TGFBR2 (Domain 3), whereas Domains 4 and 5 bind cell surface co-receptors including CD44. Despite sharing 86% amino acid identity, TGM1 and TGM4 differ functionally in key respects of affinity for mammalian TGFBRs and interactions with cell surface co-receptors. Although both induce T cell expression of the transcription factor Foxp3, TGM1 activates intracellular SMAD signaling in fibroblasts, whereas conversely, TGM4 blocks SMAD responses to TGF-β or TGM1 in fibroblasts. By recombining and/or deleting domains, we identified that the TGFBR1-binding Domains 1 and 2 of TGM4 confer antagonist activity, which is dependent on the CD44 co-receptor interacting domains (Domains 4 and 5); antagonism is enhanced but not fully dependent on binding to TGFBR2. TGMs lack interchain disulfide bonds, in distinction to TGF-β, which is a covalently linked dimeric cytokine. Dimerization of TGM4 as an Fc fusion protein converted it from an inhibitor to an activator of SMAD signaling in fibroblasts and enhanced Foxp3 induction, indicating that high avidity interactions (either monomer/dimer on T cells or only dimer on fibroblasts) drive TGF-β signaling. However, when TGM4 is below an avidity threshold, it binds receptors in an antagonistic mode. These results advance our understanding of the mechanism of action of these parasite-encoded products and uncover new tools modulating TGF-β functions in health and disease.