Structure-based mapping of the TβRI and TβRII receptor binding sites of the parasitic TGF-β mimic, Hp-TGM

Abstract

TGF-β is a secreted signaling protein involved in many physiological processes: organ development, production and maintenance of the extracellular matrix, as well as regulation of the adaptive immune system. As a cytokine, TGF-β stimulates the differentiation of CD4+ T-cells into regulatory T-cells (Tregs) that act to promote peripheral immune tolerance. The murine parasite Heligmosomoides polygyrus takes advantage of this pathway to induce inducing Foxp3+ Tregs in a similar manner using a TGF-β mimic (TGM), comprised of five tandem complement control protein (CCP) domains, designated D1-D5. Despite having no structural homology to TGF-β or to TGF-β family proteins, TGM binds directly to the TGF-β type I and type II receptors, TβRI and TβRII. To further investigate, NMR titration, and SPR and ITC binding experiments were performed, showing that TGM-D2, with the aid of D1, binds TβRI and TGM-D3 binds TβRII. Competition ITC experiments showed that TGM-D3 competes with TGF-β for binding to TβRII, consistent with TGM-D3-induced NMR chemical shift perturbations of TβRII which aligned with the solvent inaccessible areas of TβRII upon binding TGF-β. Thus, TGM-D3 binds to the same edged β-strand of TβRII that is used to bind TGF-β. Competition ITC experiments demonstrated that TGM-D1D2 and TGF-β3:TβRII compete for binding to TβRI, while TGM-D2-induced NMR chemical shift perturbation of TβRI showed that TGM-D2 binds to the same pre-helix extension of TβRI as does the TGF-β/TβRII binary complex. The solution structure of TGM-D3 revealed that while it has the overall structure of a CCP domain, TGM-D3 has an insertion in the hypervariable loop uncommon to CCP domains. These findings suggest that parasitic TGM, despite its lack of structural similarity to TGF-β, evolved to take advantage of the binding regions of the mammalian TGF-β type I and type II receptors. The structure of this TGM domain, along with the predicted structure of other H. polygyrus secreted proteins reported in the literature, suggest that TGM is part of a larger family of evolutionarily-adapted immunomodulatory CCP-containing proteins.

Induction of stable human FOXP3+ Tregs by a parasite‐derived TGF‐β mimic

“… 16 A recent study of Hp‐ TGM binding to human receptors identified that domain 3 competes for the same binding sites as TGF‐β on TBRII, while domains 1 and 2 are required for optimal binding to TBRI, with the role of domains 4 and 5 in full‐length Hp‐ TGM remaining unclear. 31 It is important for future studies to understand which domains are required for Hp ‐TGM signaling in human cells, to solve the crystal structure of Hp ‐TGM and TβRI/II, and determine if Hp ‐TGM is able to bind other receptors. These data will provide clues as to how TGF‐β signaling pathways could be targeted therapeutically.…”

The Immune Response to Nematode Infection

“…The expression of immunity is inhibited by galectin in some parasites [79] and our pilot trials show that this is also true for H. polygyrus (unpublished observations). Two molecules influence the production of regulatory T cells: TGM which is a TGFβ mimic [108], and a large immunosuppressive molecule (MW 226,476) recently discovered by us (EGF-M) that contains 24 Epidermal Growth Factor-like (EGF-like) domains, 2 SEA domains and one von Willebrand factor A domain. Our pilot trials suggest that this molecule or its cleavage products bind epidermal growth factor receptor (EGFR), disrupt normal signalling and modulate the immune response to nematode infection.…”