Abstract
Translationally controlled tumor protein (TCTP), also called histamine releasing factor, is an evolutionarily conserved multifunctional protein in eukaryotes. We previously reported that extracellular TCTP acquires its cytokine-like function following dimerization. This study aims to identify the functional domain involved in the cytokine-like function of dimerized TCTP (dTCTP). We performed X-ray crystallographic studies and a deletion mutant of dTCTP which lacks the flexible loop domain. Synthetic peptides corresponding to TCTP domains and antibodies developed against them were examined for the anti-allergic effect. In an OVA-induced airway inflammation mouse model, inhibitory effect of synthetic peptides was evaluated. dTCTP was mediated by dimers between Cys172s of TCTP monomers. Synthetic peptides corresponding to the flexible loop and helix 2 domain of TCTP, and antibodies against them inhibited dTCTP-induced IL-8 release. In particular, the TCTP mutant lacking the flexible loop domain decreased the inflammatory cytokine activity of dTCTP. We conclude that the flexible loop and helix 2 domain of TCTP are the functional domains of dTCTP. They may have the potential to be therapeutic targets in the suppression of allergic reactions induced by dTCTP.
Highlights
Histamine-releasing factor (HRF) was identified in the sera of allergic patients and it showed histamine releasing activity in human basophils, which in turn provoked allergic reactions[1,2]
In order to understand the role of flexible loop (FL) domain in dimeric Translationally controlled tumor protein (TCTP), we constructed full length human TCTP (f-TCTP) and the FL domain deleted mutant TCTP (∆-TCTP) expression vectors and produced recombinant full length TCTP (f-TCTP) and Δ-TCTP proteins
We found that the molecular weights of most of the expressed proteins corresponded to the monomers (25 kDa for f-TCTP and 17 kDa for Δ-TCTP), but two weak bands were observed (Fig. 1A,B)
Summary
Histamine-releasing factor (HRF) was identified in the sera of allergic patients and it showed histamine releasing activity in human basophils, which in turn provoked allergic reactions[1,2]. In a study of the possible function of the FL of TCTP, Yarm[8] reported that a polo-like kinase reduces microtubule stabilizing activity of TCTP by phosphorylating Ser 46 and Ser 64 residues in the loop region of TCTP. Suggestions for NMR and crystal structures of TCTP have been deposited in the Protein Data Bank These include different structures of monomeric TCTPs from several species and just one of human dimeric TCTP (dTCTP). They all share a high topological similarity. In both forms, a long middle loop located outside of the core domain of TCTP shows high flexibility. Based on the structures of human dTCTP and this mutant, we predicted the mechanism of action of each functional domain
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