Dimerization of Translationally Controlled Tumor Protein Is Essential For Its Cytokine-Like Activity

BACKGROUND:Translationally Controlled Tumor Protein (TCTP) found in nasal lavage fluids of allergic patients was named IgE-dependent histamine-releasing factor (HRF). Human recombinant HRF (HrHRF) has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn). METHODS AND FINDINGS:We found that only NH(2)-terminal truncated, but not C-terminal truncated, TCTP shows cytokine releasing activity compared to full-length TCTP. Interestingly, only NH(2)-terminal truncated TCTP, unlike full-length TCTP, forms dimers through intermolecular disulfide bonds. We tested the activity of dimerized full-length TCTP generated by fusing it to rabbit Fc region. The untruncated-full length protein (Fc-HrTCTP) was more active than HrTCTP in BEAS-2B cells, suggesting that dimerization of TCTP, rather than truncation, is essential for the activation of TCTP in allergic responses. We used confocal microscopy to evaluate the affinity of TCTPs to its putative receptor. We detected stronger fluorescence in the plasma membrane of BEAS-2B cells incubated with Del-N11TCTP than those incubated with rat recombinant TCTP (RrTCTP). Allergenic activity of Del-N11TCTP prompted us to see whether the NH(2)-terminal truncated TCTP can induce allergic airway inflammation in vivo. While RrTCTP had no influence on airway inflammation, Del-N11TCTP increased goblet cell hyperplasia in both lung and rhinal cavity. The dimerized protein was found in sera from allergic patients, and bronchoalveolar lavage fluids from airway inflamed mice. CONCLUSIONS:Dimerization of TCTP seems to be essential for its cytokine-like activity. Our study has potential to enhance the understanding of pathogenesis of allergic disease and provide a target for allergic drug development

Catabolism of the murine IgGl molecule: Evidence that both CH2‐CH3 domain interfaces are required for persistence of IgGl in the circulation of mice

Site‐directed mutagenesis of a recombinant Fc‐hinge fragment has previously been used to identify a region of the murine IgG 1 molecule that controls catabolism, and this site encompasses amino acid residues at the interface of the CH2 and CH3 domains. In the current study the nature of this ‘catabolic site’ has been further analysed using recombinant techniques. Fc‐hinge, CH2‐hinge, CH2 and CH3 fragments have been expressed in Escherichia coli, purified and analysed in pharmacokinetic studies in mice. The CH2‐hinge has been analysed as both a monomer and dimer, and the dimer has a longer β phase half‐life (61.6 h) than the monomer (29.1 h). This suggests that two catabolic sites per Fc fragment are required for serum persistence. The need for two functional sites per molecule has been confirmed by the analysis of a hybrid Fc‐hinge fragment comprising a heterodimer of one Fc‐hinge with the wild type (WT) IgGl sequence and a mutant Fc‐hinge with a defective catabolic site (mutated at His310, Gln311, His433 and Asn434). This hybrid is cleared with a β phase half‐life of 37.9 h and this is significantly shorter than that of the WT Fc‐hinge fragment (82.9 h). In contrast to the CH2‐hinge dimer, the CH3 domain is cleared rapidly (β phase half‐life of 21.3 h) indicating that the region of this domain (His433 and Asn434) previously identified as being involved in the control of catabolism is not sufficient in the absence of the CH2 domain for the serum persistence of an IgG fragment. The data extend our earlier observations concerning a region of the murine IgGl molecule that is involved in the control of catabolism and have implications for the design of engineered antibodies for therapy.</p