Prediction of antigenic determinants of trichosanthin by molecular modeling

The antigenic determinants of trichosanthin were predicted by molecular modeling. First, the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence. Secondly, the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E. Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants: one is composed of two polypeptide segments, Ile201-Glu210 and Ile225-Asp229, which are close to each other in the three-dimensional structure; and the other is the segment Lys173-Thr178. The former region seems to be the more reasonable antigenic determinant than the latter one.

The Structure of Trichosanthin with Space Group C2 at 1. 9 Resolution

The crystal structure of trichosanthin (TCS) in space group C2 hasbeen refined by PROLSQ and XPLOR to an R-factor of 0. 204 for 33, 531 reflectionswith F。≥2-σ(F。) between 6 to 1. 9 A resolution. The root mean square(r. m. s. ) devi-ations of bond lengths and bond angles from the standard values are 0. 015 A and 2.77°, respectively. The overall fold of the molecule of TCS is, in general, similar to oth-er RIPs, but there are some differences in secondary structure and in the active sitecleft. An overlay of the two molecules (shown by Mol. A and Mol. B), which are notrelated by symmetry in an asymmetric unit, results in an r. m. s. deviation of 0. 850Afor the main chain atoms. The backbones of the C-terminus of the two molecules arequite different. The structures of the water in two molecules are not completely analo-gous. In the active site cleft of Mol. B, the bonding sites of three water molecules toprotein are similar to those of N atoms of formycin ring in the structure of pokeweedantiviral protein complexed with formycin 5-monophosphate, and the side chains of twoTyr70 in the two crystalline forms of TCS show a similar orientation, which is differentfrom that of the corresponding residue in ricin A-chain.

The Study of Anti-tumor Activity of Trichosanthin by Cyclic Voltammogram

The anti-tumor activity of Trichosanthin (TCS) has been frequently reported in recent years. In our experiments, electrochemical methods were applied to detect the effects of TCS on human leukemia cells U937. 50 mu g/ml TCS treatment for 40 hours can cause irreversible negative effects on the viability of U937 cells. This effect largely depends on the concentration of TCS and the time period of treatment.

Crystallization and Preliminary Crystallographic Analysis of β-Trichosanthin,an Isoform of Trichosanthin from the Root Tuber of Trichosanthe kirilowii

β-Trichosanthin, a type 1 ribosome-inactivating protein （RIP） isolated from the root tuber of Trichosanthe kirilowii Maxim, is an isoform of trichosanthin. Here we report its crystallization in two crystal forms using the hanging-drop vapor-diffusion method. The form A and form B crystals belong to the orthorhombic space group P212121 and monoclinic space group P21, respectively. X-ray data have been collected to 1.6 and 1.2 A resolution for form A and form B crystals, respectively, using a synchrotron source.

SYNERGIC CYTOTOXICITY TO GASTRIC CANCER CELLS BY COMBINED USE OF TRICHOSANTHIN ANDRECOMBINANT INTERFERON α-2B

Objective To investigate a new approach of the combined use of trichosanthin (TCS) andrecombinant interferon alpha - 2b (rIFN α- 2b) against digestive system cancer cells. Methods Detect separatelythe cytotoxicity of TCS, rIFN α- 2b and their combination against digestive system cancer cell SGC- 7901.Results In the experiment in vitro, TCS, rIFN α- 2b both had direct, dose dependent cytotoxicity againstSGC - 7901. Their combined use demonstrated a toxicity signijicantly higher than that of the two drugs used alone,showing a signilicant synergic effect. This synergic cytotoxicity was confirmed in the animal experiment.Conclusion Combined use of TCS and rIFN α - 2b decreases the therapeutic dose of TCS and its toxic adverseellect, and this synergic effect is favorable to the clinical use of TCS protein against gastric cancer.

EXPERIMENTAL STUDY ON APOPTOSIS OF GASTRIC CANCER CELLS INDUCED BY TRICHOSANTHIN

Objective To investigate whether the apoptosis is involved in the mechanism of cytotoxicity of trichosanthin to gastric cancer cells. Methods Morphologic studies and TL]AIEL stainning were used to ex-plore qualitatively and quantitatively the apoptotic status of gastric cancer cells before and after treatment with trichosanthin. Results In the experiment in vitro , when gastric cancer cells SGC-7901 were treated by trichosanthin (0. 1μg/ml, 36h), some typical apoptotic morphologic changes occured. A significant increase of apoptotic index (AI) was from (3.78±1.11)%, (3.98±1.12)% and (3.85±1.08)%at 36, 42, 48h to (11.30±2.33)%,(10.22±2.00)% and (11.18±1.85)%,respectively(P<0.01). In the experiment in vivo, nude mice with SGC-7901 xenografted tumor were treated by trichosanthin (0. 3mg kg-1 d -1, × 5d, intraperitoneal injection), some typical apoptotic morphologic changes were observed. These apoptotic cells were limited only in xenografted tumor. Al increased significantly from (4.95± 1 .94) % to (8. 75± 1 .38 )%, (P <0.01). Conclusion The apoptosis would be involved in the mechanism of cytotoxicity of trichosanthin to gastric cancer cells.

neo Trichosanthin及其突变体Y70A neo Trichosanthin的晶体结构研究

天花粉蛋白（Ｔｒｉｃｈｏｓａｎｔｈｉｎ，ＴＣＳ）的一个亚型，ｎｅｏＴｒｉｃｈｏｓａｎｔｈｉｎ（ｎ－ＴＣＳ），及其突变体Ｙ７０Ａｎ－ＴＣＳ被克隆和表达为重组蛋白。用悬滴汽相扩散法得到ｎ－ＴＣＳ和Ｙ７０Ａｎ－ＴＣＳ的晶体。在ＭａｒＲｅｓｅａｒｃｈ面探测器系统上分别收集了０．２０ｎｍ和０．２０５ｎｍ分辨率的Ｘ射线衍射数据。用同晶差值傅立叶法解析了结构。最后晶体学Ｒ因子分别为０．１８３和０．１８４。键长的ｒ．ｍ．ｓ．偏差分别为０．００１６ｎｍ和０．００１４ｎｍ，键角的ｒ．ｍ．ｓ．偏差分别为２．９３０°和２．７３２°。ｎ－ＴＣＳ虽然与ＴＣＳ有１１个氨基酸的差别，但它们都不是活性口袋区的关键残基。并且这些氨基酸的替换绝大多数属于保守替换。和ＴＣＳ相比，这１１个残基及其附近残基的主链间形成的保守氢键在ｎ－ＴＣＳ中没被破坏，它们所处的二级结构也保持不变。所以ｎ－ＴＣＳ的整体结构与ＴＣＳ十分相近。Ｙ７０Ａｎ－ＴＣＳ的晶体在收集数据前曾用含５＇ＡＭＰ的缓冲液浸泡过，活性口袋区没发现可与Ａｄｅｎｉｎｅ相匹配的密度。用ＨＰＬＣ检测Ｙ７０Ａｎ－ＴＣＳ与５＇ＡＭＰ作用后产物，层析图谱上相应位置也没有出现Ａｄｅｎｉｎｅ。上述结果表明，７?

The influence of Trichosanthin on the induction of IgE responses to ovalbumin under adjuvant-free condition

Trichosanthin(TCS) is a potent allergen in mice. It can reproducibly induce specific IgE responses in C57BL/6J mice without the help of adjuvant alum. TCS can bring out the IgE responses to ovabumin(OVA), while OVA itself could not induce IgE responses to it. How- ever, TCS only works when QVA immunization is given one day after TCS immunization. Either time lag in OVA immunization, or immunization of both antigens at the same time, or OVA immunization given first, all has no effect on the induction of IgE responses to OVA. Through analysis of the antibody specificity of hybridoma clones, it indicated that specific antibodies to TCS or OVA were secreted by independent B cell clones. The IgE antibodies showed no polyreactivity to different antigens.

Human immune suppression is inducible by trichosanthin via CD8 cell-mediated pathway

Thichosanthin(Tk), a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb, showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication. Owing to sequence homology of the peptide with a ribosomeinactivating protein, the downward activity of Tk was suggested to be related to its cytotoxic property. We report here, however, that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic, mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5 μg/ml. The lowresponsiveness caused by Tk was not due to toxic cytolysis. Rather, evidences suggested that, in the dose range adopted, the Tk-induced inhibition was attributable, at least in part, to immune suppression, in view of (1) Tk was more effective in the early stage of alloreactivity; (2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured; (3) Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture; (4) Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells; (5) Tkinduced immune suppression was diminished by depletion of CD8+ cells from the culture, and, finally; (6) Adding CD8+ cells back to the culture could restore the suppres Trichosanthin-induced humall immune suppression sion. Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.

Trichosanthin inhibits T cell activation by interfering with the recruitment of ZAP-70 to CD3 ζchain

Plant protein Trichosanthin (Tk) has been shown in our previous experiments to suppress antigenic response of T cells. Here we explored its inhibitory mechanisms on the proliferation of human Jurkat leukemia T cell triggered by anti-CD3 McAb. By examination of tyrosine phosphorylation of cell lysate, we were able to show that Tk could interfere with the PTK-related activity in the TCR/CD3initiated signal transduction in addition to blocking the phosphorylation of PKC. As shown in our experiment,the expression intensity of ZAP-70, a kind of protein tyrosine kinase, was not changed but its phosphorylation could be inhibited. When physical link between CD3(chain and ZAP-70 was further examined by using coimmunoprecipitation after pluse-treatment of the cell line with Tk, the anti-CD3 McAb-induced recruitment of ZAP70 to CD3 ζ chain was observed to be blocked in some extent. This may account for, at least in part, how Trichosanthin was able to inhibit the TCR-triggered T cell proliferation.

APOPTOSIS IN GASTRIC CANCER INDUCED BY TRICHOSANTHIN AND RELATIONSHIP OF THIS APOPTOSIS WITH EXPRESSION OF C-MYC

Objective To investigate the apoptosis in gastric cancer induced by trichosanthin (TCS) and the relationship between this apoptosis and expression of c-myc. Methods In in vitro experiment, morphologic test and TUNEL staining method were used to detect quantitatively and qualitatively the apoptosis status of gastric adenocarcinoma cell line SGC-7901 before and after the treatment of 0.1μg/ml TCS. Immunohistochemical staining method and Northern Blot hybridization were used to detect the expression status of apoptosis-related genes c-myc before and after TCS treatment. Results Some typical apoptotic morphologic changes appeared after 36h treated by TCS. The apoptotic indexes increased significantly in the TCS treated group than those of the untreated group after 36h,42h and 48h (P<0.01). After 24h treated by TCS, an increased expression of c-myc protein product occurred, the staining density was increased from + or ++ to +++ (P< 0.01), and the expression of c-myc RNA increased significantly (P<0.05). Conclusion TCS is able to induce the apoptosis in gastric cancer. This apoptosis may be mediated by up-expression of apoptosis-regulated gene c-myc.

Using Xe as a heavy atom for phase determination of protein trichosanthin structure

Under gas pressures of 1–3 MPa, xenon can be bound to discrete sites in hydrophobic cavities of protein,so as to use Xe as a heavy atom for determining phases in protein crystallography. Using single anomalous scattering diffraction method, we demonstrate that an interpretable electron density map can be obtained for protein trichosanthin from a single xenon derivative. We found, for the first time, a pre-existing hydrophobic cavity just under the protein surface of trichosanthin.

Construction of normal human IgE phage antibody library and the screening of the anti-trichosanthin IgE

Allergen specific IgE response is the major cause of immediate hypersensitivity. However the number of IgEproducing B cells and the amount of IgE, especially the specific IgE, are so low, it greatly impedes the study of the allergic-specifc antibody responses. Here we report the construction of a normal human IgE combinatorial library The repertoire of IgE VH genes and of K genes were separately amplified from normal human peripheral blood lymphocytes through RT-PCR, and were then constructed to form the phage surface display human Fab(IgEVH) library. A plant protein allergen, trichosanthin(TCS), was used to affinity-enrich and to screen the anti-TCS phage HuFab clones from the library. Human IgE(Fab) to TCS were detected.

Structural analysis and molecular modeling of twoantitrichosanthin IgE clones from phage antibody library

Recently we constructed a murine IgE phage surfacedisplay library and screened out two IgE (Fab) cloneswith specific binding activity to Trichosanthin (TCS). Inthis work, the Vε and Vκ genes of the two clones weresequenced and their putative germline gene usages werestudied. On the basis of the known 3D structure of Trichosanthin and antibody, molecular modeling was carriedout to study the antigen-antibody interaction. The possible antigenic determinant sites on the surface of TCSrecognized by both the clones were analyzed, and the reaction forces between TCS and two Fab fragments werealso analyzed respectively.

Resistance to rice blast (Pyricularia oryzae) caused by the expression of trichosanthin gene in transgenic rice plants transferred through agrobacterium method

The gene of trichosanthin has been transferred into rice plants through agrobacterium method. The single copy insertion and the expression of foreign gene have been proved in regenerated plants. In antifungal assay the degrees of rice blast (Pyricularia oryzae) infection of the transgenic plants expressing trichosanthin and expressing GUS gene as control have been evaluated. The differences such as the time of disease symptom observed, the number of infected plants and damaged leaves, the growth of infected plants of the two transgenic plants after being inoculated by rice blast (Pyricularia oryzae) are significant. The transgenic plants with trichosanthin gene grew faster than the plants with GUS gene, even when humidity environment was removed. The results show that the transgenic plants that expressed trichosanthin are able to delay the infection of rice blast compared with the plants as control. In addition, no damage caused by the expression of trichosanthin gene in transgenic plants has been

Trichosanthin enhances anti-tumor immune response in a murine Lewis lung cancer model by boosting the interaction between TSLC1 and CRTAM

Trichosanthin(TCS),extracted from the Chinese medicinal herb Trichosanthes kirilowi,has shown promise for the inhibition of tumor growth.However,its immunomodulatory effect on tumor–host interaction remains unknown.In this study,we focused on the effect of TCS on murine anti-tumor immune response in the 3LL Lewis lung carcinoma tumor model and explored the possible molecular pathways involved.In addition to inhibiting cell proliferation and inducing apoptosis in the 3LL tumor,TCS retarded tumor growth and prolonged mouse survival more significantly in C57BL/6 immunocompetent mice than in nude mice.This reflected the fact that the host immune system was involved in tumor eradication.Using FACS analysis,we found that TCS increased the percentage of effector T cells,particularly Interferon-gamma(IFN-c)producing CD41 and CD81 T cells from tumor-bearing mice.TCS also promoted the vigorous proliferation of antigen-specific effector T cells,markedly increased Th1 cytokine secretion and elicited more memory T cells in tumor-bearing mice,consequently enhancing the anti-tumor response and inducing immune protection.Furthermore,we found that TCS upregulated the expression of tumor suppressor in lung cancer 1(TSLC1)in 3LL tumor cells and the expression of its ligand,class I-restricted T cell-associated molecule(CRTAM),in effector T cells.Blocking TSLC1 expression with small interfering RNA(siRNA)significantly eliminated the effects of TCS on the proliferation and cytokine secretion of effector T cells,suggesting that TCS enhances anti-tumor immune response at least partially by boosting the interaction between TSLC1 and CRTAM.Collectively,our data demonstrate that TCS not only affects tumor cells directly,but also enhances anti-tumor immunity via the interaction between TSLC1 and CRTAM.These findings may lead to the development of a novel approach for tumor regression.

Trichosanthin Induced Th2 Polarization Status

Trichosanthin is extracted from the root tuber of Chinese medicinal herb Trichosanthes kirilowii maximowicz （Tian Hua Fen）. TCS has abortifacient, anti-tumor, anti-HIV and immunoregulatory functions. It has been proved that it could inhibit immune response and arouse a T helper 2 response in the draining lymph node. In the current study the effect of TCS on mouse splenocytes was investigated. We stimulated C57BL/6 mice with TCS both in vivo and in vitro and analyzed the change of type 1 and type 2 cytokines in mouse splenocytes. The results showed that TCS could induce the expression of IL-4, one of the major T helper 2 （Th2） cytokines, and inhibit the expression of IFN-γ, an important Thl cytokine in spleen lymphocytes both in vivo and in vitro. It is also shown the kinetics of Thl-to-Th2 transition after TCS stimulation in vivo in C57BL/6 mice. We found that type 2 cytokines, such as IL-10, TGF-β and IL-4 were increased regularly but IFN-γ, was decreased at day 3 and then increased. However the mechanism for cytokine change is not clear.

N-glycosidase mechanism of Trichosanthin

The interactions between TCS and AMP, ADO, TUB., CMP were studied by crystallographic method, which confirmed that TCS specially recognizes ADE, but does not recognize cytosine. When TCS interacts with mononucletide, the ligand with or without phosphate is not important. The proton H + of guanidinum group of Arg163 is shared by N 3 of ADE, but is not received by N 7. This enriches the model of N glycosidase mechanism about TCS and AMP. This model should suit to the interaction between RIPs and AMP, and reflect the main interaction between RIPs and rRNA. However, the interaction between RIPs and rRNA is more complicated. To comprehensively understand recognition and catalysis of RIPs against rRNA, at least the RIP/RNA segment complex crystal structure should be determined. This RNA segment should contain stem and loop with GAGA sequence.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

Trichosanthin can spontaneously penetrate phospholipid monolayer under acid condition

Tianhuafen is a kind of traditional Chinese medicine for abortion, which has long been used in China. The basic protein trichosanthin (TCS) is responsible for such activity. As a ribosome inactivating protein (RIP), trichosanthin removes A4304 in the 28s rRNA via the N-glycosidase activity and inactivates the ribosome. However, it remains unclear how TCS