A Novel Splice Variant of Human TGF-β Type II Receptor Encodes a Soluble Protein and Its Fc-Tagged Version Prevents Liver Fibrosis in vivo

Marcela Soledad Bertolio,Alejandra Carrea,Anabela La Colla,Ricardo Alfredo Dewey,Ana Romo,Andrea Nancy Chisari,Stella Maris Echarte,Tania Melina Rodríguez,Gabriela Canziani,German Patricio Barletta,Sabrina Campisano,Alexander Miguel Monzon

doi:10.3389/fcell.2021.690397

Abstract

We describe, for the first time, a new splice variant of the human TGF-β type II receptor (TβRII). The new transcript lacks 149 nucleotides, resulting in a frameshift and the emergence of an early stop codon, rendering a truncated mature protein of 57 amino acids. The predicted protein, lacking the transmembrane domain and with a distinctive 13-amino-acid stretch at its C-terminus, was named TβRII-Soluble Endogenous (TβRII-SE). Binding predictions indicate that the novel 13-amino-acid stretch interacts with all three TGF-β cognate ligands and generates a more extensive protein–protein interface than TβRII. TβRII-SE and human IgG1 Fc domain were fused in frame in a lentiviral vector (Lv) for further characterization. With this vector, we transduced 293T cells and purified TβRII-SE/Fc by A/G protein chromatography from conditioned medium. Immunoblotting revealed homogeneous bands of approximately 37 kDa (reduced) and 75 kDa (non-reduced), indicating that TβRII-SE/Fc is secreted as a disulfide-linked homodimer. Moreover, high-affinity binding of TβRII-SE to the three TGF-β isoforms was confirmed by surface plasmon resonance (SPR) analysis. Also, intrahepatic delivery of Lv.TβRII-SE/Fc in a carbon tetrachloride-induced liver fibrosis model revealed amelioration of liver injury and fibrosis. Our results indicate that TβRII-SE is a novel member of the TGF-β signaling pathway with distinctive characteristics. This novel protein offers an alternative for the prevention and treatment of pathologies caused by the overproduction of TGF-β ligands.

Highlights

Transforming growth factor-β (TGF-β) is a multifunctional cytokine involved in critical processes, including immune regulation and wound healing together with cell proliferation, maturation, and differentiation (Derynck and Budi, 2019)
We found the presence of the 433 bp band in CD3+, CD19+, and CD14+ cells isolated by immunomagnetic separation, and in granulocytes obtained from Ficoll density gradient (Figure 1C)
The new TβRII splice variant has the capacity to encode a protein of 80 amino acid (AA) residues, which includes an ER signal sequence of 23 AA, and a mature protein of 57 AA, lacking the transmembrane domain (TMD) (Figure 1E)

Summary

Introduction

Transforming growth factor-β (TGF-β) is a multifunctional cytokine involved in critical processes, including immune regulation and wound healing together with cell proliferation, maturation, and differentiation (Derynck and Budi, 2019). Three TGF-β isoforms have been identified in mammals: TGF-β1, TGF-β2, and TGF-β3 They are encoded by distinct genes sharing 64–82% sequence identity. These genes are regulated developmentally and in a tissue-specific manner (Moses et al, 2016). Canonical signaling starts when mature, dimeric TGF-β isoforms bind cell surface receptor complexes comprising “type II” (TβRII) and “type I” (TβRI) receptors (Heldin and Moustakas, 2016). It is known that the betaglycan coreceptor (former TβRIII), is necessary for efficient binding of TGFβ2 and subsequent signaling (López-Casillas et al, 1993) This coreceptor binds all three TGF-βs showing a comparable affinity for TGF-β2 (Dong et al, 2007). TGF-β2 binds to betaglycan and recruits TβRII and TβRI to phosphorylate TβRI eliciting downstream signaling (López-Casillas et al, 1993)

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