Acknowledgement:

1. Cloning, over-expression, purification, crystallization, and biochemical study of human NH2-terminal truncated TCTP with IgE-dependent histamine-releasing activity Mi-Sun Kim1, Miyoung Kim, Kyunglim Lee & Dong Hae Shin1 1College of Pharmacy, EwhaWomans University, Seoul 120-750, Republic of Korea;2Institut fürMikrobiologie & Biotechnologie, Universität Bonn, MeckenheimerAllee 168, D-53115 Bonn, Germany II. A trucated TCTP NH2-terminal truncated rat recombinant (Rr) TCTP increases IL-8 and GMCSF secretion from BEAS-2B cells. Since endogenous proteases from neutrophilsand mast cells are present in sites of inflammation, we wondered if TCTP acquires its cytokine-like activity following partial proteolysis by these proteases. To answer this question, we designed five rat TCTP constructs including a rat recombinant TCTP (RrTCTP) construct and four deletion constructs [5]: Del-N11TCTP (residues 11–172), Del-N35TCTP (residues 35–172), Del-C112TCTP (residues 1–112), and Del-N39C110TCTP (residues 39–110) (Figure 1A). RrTCTP and the RrTCTP deletion derivatives were tested for their ability to stimulate the secretion of IL-8 from BEAS-2B bronchial epithelial cells. When BEAS-2B cells were treated with RrTCTP, IL-8 release was stimulated in a dose- and time-dependent fashion (Figure 1, A and B). Under the same experimental condition, Del-N11TCTP and Del-N35TCTP stimulated IL-8 release about 4–9 fold better than RrTCTP; Del-C112TCTP stimulated IL-release at about the same level as RrTCTP; Del-N39C110TCTP did not show any stimulatory activity, and this may be due to an alteration in its tertiary structure by excessive deletion. The level of GM-CSF secretion was somewhat lower than that of IL-8 secretion in BEAS-2B cells. We could measure GM-CSF secretion by Del-N11TCTP and Del-N35TCTP derivatives, but not by RrTCTP (Figure 1C). These results suggest that NH2-terminal truncation increases the cytokine-like activity of RrTCTP. Translationally controlled tumor protein (TCTP) is a multifunctional protein with microtubule-stabilizing activity, guanine nucleotide dissociation inhibitory activity, Na,K-ATPase suppressing activity and histamine-releasing activity. It was recently reported that NH2-terminal truncated TCTP shows cytokine releasing activity compared to full-length TCTP. For this reason, TCTP is also known as IgE-dependent histamine-releasing factor (HRF). Interestingly, NH2-terminal truncated TCTP forms a dimer through an intermolecular disulfide bond. Therefore, the three-dimensional structure of the dimeric form with IgE-dependent histamine-releasing activity may be quite different from monomeric full-length TCTP. In order to develop a therapeutic peptide antiallergic drug, the three-dimensional structure of NH2-terminal truncated TCTP is essential. Therefore, we have cloned, over-expressed, and purified NH2-terminal truncated TCTP. Since it forms a dimer through a disulfide bond, several oxidizing reagents I. Various functions of human TCTP Translationally controlled tumor protein (TCTP), also variously known as IgE-dependent histamine-releasing factor (HRF), p23/p21, and fortilin, is a multifunctional protein distributed in all normal cell types [1, 2]. It exhibits a high degree of homology among various species, suggesting that TCTP may play an essential role in cellular processes. Apart from its various intracelluar functions, TCTP was reported to be involved, extracellularly [3], in human allergic response as an HRF which appears outside of macrophages and activates mononuclear cells, in bronchoalveolarlavage fluids (BALF) from patients with eosinophilic pneumonia and asthma, in nasal washings, and in skin blister fluids during the late allergic reaction. Human recombinant HRF (HrHRF) directly stimulates the secretion of histamine, IL-4, and IL-13 from a subpopulation of basophils, and also enhances their secretion from all IgE-bearing basophils activated by anti-IgE Ab. HrHRF stimulates the secretions of IL-8 and GM-CSF in primary cultures of human bronchial epithelial cells and human bronchial epithelial cell line, BEAS-2B. HrHRF has been recently reported to be much less effective than HRF produced from activated mononuclear cells (HRFmn). HRF exhibiting cytokine-like activity in vivo might be a modified form of intracellular TCTP. The observed differences in the activity of HRFmn and HrHRF might arise from modifications occurring in TCTP in the allergic environment. III. A dimeric TCTP: There are several reports that TCTP homologues form dimeric structures [4]. To elucidate the oligomeric structure of TCTP, the apparent molecular weights of Del-N35TCTP and Del-N35TCTP-C172S were determined using size exclusion column chromatography (Figure 3B) [5]. Del-N35TCTP showed two peaks, a major peak of approximately 45 kDa corresponding to the dimeric form and a minor peak of about 29 kDa corresponding to the monomericform (Figure 2A-i). Del-N35TCTP-C172S also showed two peaks, but the major peak corresponded to the monomericform (Figure 3A-ii) We also tested whether the NH2-terminal truncated RrTCTP can be dimerized by oxygen species such as H2O2. RrTCTPand Del-N35TCTP were completely reduced with 20 mM DTT, and then oxidized by increasing concentrations of H2O2 (Figure 2B). Whereas RrTCTP showed only minimal fraction of dimers, all the Del-N35TCTP migrated in the dimer position in non-reducing SDS-polyacrylamidegels in the presence of H2O2. This implies that only the NH2-terminal truncated TCTP can be modified by reactive oxygen species at sites of inflammation. Figure 1. NH2-terminal truncated RrTCTPs show increased activity in BEAS-2B cells. (A) BEAS-2B cells were treated with 1 or 10 mm/ml of RrTCTPor various RrTCTP derivatives for 48 h. The resulting IL-8 in supernatant was analyzed by ELISA (*** p,0.001, significantly different from the NC value, n = 3). (B) 10 mm/ml of RrTCTP or RrTCTP derivatives were added to BEAS-2B cells and incubated for 1, 6, or 24 h (** p,0.01, *** p,0.001, significantly different from the NC value, n = 3). (C) 10 mm/ml of RrTCTP or RrTCTP derivatives were added to BEAS-2B cells and incubated for 48 h.Theresulting GM-CSF in supernatant was analyzed by ELISA (*** p,0.001, significantly different from the NC value, n = 3). IV. Purification and crystallization of human HRF Ligation independent cloning (LIC) was developed for the directional cloning of PCR products without restriction enzyme digestion or ligation reactions [6]. The N-terminal 34 amino acid truncated human TCTP gene was amplified by PCR and was annealed into the pB2 vector that expresses the cloned gene fused to a non-cleavable N-terminal His6-tag. HRFwas expressed using Escherichia coli BL21 (DE3) grown in LB media at 37°C with 1 mM IPTG for 3 hours. was obtained after Ni-affinity chromatography and anionic exchange chromatography. KDa 70 50 35 2520 15 (A) (B) Figure 2. (A) Del-N35TCTP (i) and Del-N35TCTP-C172S (ii) were loaded onto Superdex 200 HR 10/30 gel filtration column (Amersham Pharmacia Biotech). The column was equilibrated and eluted with 50 mM sodium phosphate, pH 7.0 containing 150 mMNaCl. The eluting positions were calibrated with the molecular size marker (Sigma) and the peak positions of molecular mass standards are indicated by vertical arrow. Figure 3. SDS-PAGE of purified HRF after anion chromatography. Screening for crystallization conditions was performed using the sparse-matrix method with several screens from Hampton Research (Hampton Research, Laguna Niquel, CA). The crystallization robot “Hydra Plus-One” (Matrix Technologies, Hudson, NH) was used to set the screens using the sitting drop vapor diffusion method at room temperature. The 96 well Intelli-plate (Art Robbins Instrument, Salt Lake City, UT) was used and a sitting drop was made by mixing 0.2 ㎕ of protein solution (~10 mg/ml) and 0.2 ㎕ of reservoir solution equilibrated over 90 ㎕ of reservoir solution. (B) Del-N11HRF, Del-N35HRF, and their mutants were analysed by reducing or non-reducing 15% SDSPAGE Acknowledgement: This work was supported partly by grant No. R15-2006-020 from NCRC program of MOST and KOSEF and by the Seoul R&BD program (ST0900801). Kim, M. issupported by a Brain Korea 21 grant from the Ministry of Education and Human Resources Development. References: [1] McDonald S.M., et al.(1995), Science269: 688-690 [2] Sanchez, J. C.(1997)Electrophoresis18: 150-155 [3] Pasmans, S. G. et al.(1994) Int. Arch. Allergy Immunol.103: 44-52 [4] Yoon, T. et al. (2000) Arch. Biochem. Biophys. 384: 379-382 [5] Kim,M.et al. (2009) Pros One4: e6464 [6] Kim S. H. et al. (2005) J. Struct. Funct. Genomics 6:63-70