臺灣博碩士論文加值系統

我們先前的研究已經證明：由嗜甲醇酵母重組的靈芝蛋白經由蛋白激酶的訊息傳遞路徑來誘發IL-2 表現。接下來，我們將探討rLZ-8與T細胞之間的交互作用，及rLZ-8是透過何種接受體來刺激T 細胞。我們將著重以下三個部分：T細胞接受體(TCR)近端的訊息傳遞之活化，調節IL-2表現的訊息傳遞，還有rLZ-8可能的結合位置(包含CD45)。首先，我們發現rLZ-8強烈地誘發TCR近端訊息傳遞的磷酸化，並隨刺激時間增長有遞增的現象。接下來，我們利用TCR訊息傳遞路徑中不同分子缺陷的細胞株：J.gamma-1(缺乏PLCγ-1)，J.CaM1.6(缺乏細胞內鈣離子反應)，J.RT3-T3.5(缺乏TCR β chain)和J45.01(缺乏CD45)來研究TCR活化中一些分子對IL-2產生的影響。在正常的Jurkat細胞株中，IL-2隨著rLZ-8刺激的時間跟濃度上升而遞增，但是在缺陷的Jurkat細胞株則是有減損的現象，尤其是J45.01。因此，我們更進一步地研究藉由TCR調控IL-2表現的訊息傳遞中，CD45所扮演的角色。首先，利用CD45抑制劑，我們證明抑制CD45的活性可以減少rLZ-8刺激人類T細胞及Jurkat T細胞所產生的IL-2。接下來，用rLZ-8刺激，比較正常Jurkat與CD45缺陷的細胞株之間酪氨酸磷酸化的現象，我們發現正常的Jurkat含有很高量的磷酸化，而J45.01的酪氨酸磷酸化的現象則是有缺失。除此之外，rLZ-8刺激Jurkat很快增加ZAP-70, LAT, PLCγ1 and PKCθ的磷酸化，但是J45.01則否。我們最近的結果發現rLZ-8會結合CD45並藉由TCR的訊息傳遞路徑來調控IL-2的產生，而這個結合可能是藉由醣類的影響。

We previously demonstrated that Pichia pastoris-recombinant Ganoderma lucidum LZ-8 protein (rLZ-8) induces IL-2 expression via protein kinase-dependent signaling pathways. To continue our research, we were interested in the interaction of rLZ-8 and T cell surface membrane. We focused on three parts, i.e. T cell receptor (TCR) the proximal signalings, the signal transduction in the regulation of IL-2 secretion, and the putative rLZ-8 protein binding sites including CD45. Initially, we found that rLZ-8 dramatically induces phosphorylation of TCR-triggered proximal signaling molecules, and the increasing is in a time-dependent fashion. Next, in order to examine the elements involved TCR activation and outcome of IL-2 secretion, we used various TCR signaling-defected cell lines such as J.gamma-1 (PLCγ1-deficient), J.CaM1.6 (intracellular calcium response-deficient), J.RT3-T3.5 (lack αβ heterodimer) and J45.01 (CD45-deficient). In brief, rLZ-8 stimulation of IL-2 is in a time and dose–dependent fashion in wild Jurkat. However, IL-2 secretion is defected in the testing TCR signaling deficient Jurkat cells, especially J45.01. Moreover, we investigated the role of CD45 in the TCR-mediated regulation of IL-2 secretion. First, using a CD45 inhibitor, we demonstrated that CD45 inhibitor reduced IL-2 secretion in both human primary T cells and Jurkat T cells in a dose-dependent manner. Secondary, comparing rLZ-8 stimulated tyrosine phosphorylation profile between Jurkat and J45.01 cells, we found more phosphorylation in wild Jurkat than that in J45.01 cells. In addition, rLZ-8 rapidly increased phosphorylation of ZAP-70, LAT, PLCγ1 and PKCθ in wild Jurkat cells, but not in J45.01 cells. Our current finding provided evidence that rLZ-8 protein interacted with CD45 that allows CD45 to signal through the TCR-mediated signaling to produce IL-2 from human T cells, and free monosaccharides may reduce IL-2 expression due to decrease the interaction.

中文摘要………………………………………………………………1
Abstract …………………………………………………………… 2
1. Introduction ……………………………………………………4
2. Materials and Methods…………………………………………8
2.1 Cells
2.2 Antibodies and reagents
2.3 IL-2 determinations
2.4 Preparation of membrane protein
2.5 Western Blot Analysis
2.6 Flow Cytometry
3. Results……………………………………………………………13
3.1 Pichia pastoris-recombinant Ganoderma lucidum LZ-8
protein (rLZ-8) induces proximal T cell receptor
(TCR)-mediated signaling pathways
3.2 The TCR downstream signaling molecules participate
in rLZ-8-induced IL-2 expression
3.3 rLZ-8 induces IL-2 expression through CD45
triggering in T cells
3.4 The role of CD45 in the rLZ-8-induced TCR-
mediated signaling pathway
3.5 CD45 is a putative receptor for rLZ-8 on T cells
3.6 Glycosylation-dependent interaction of rLZ-8
with Jurkat T cells
3.7 rLZ-8 induces CD69 expression in T cells
4. Discussion…………………………………………………………21
5. References…………………………………………………………27
6. Figure legends……………………………………………………35

References

1. Sliva, D. 2003. Ganoderma lucidum (Reishi) in cancer treatment. Integr. Cancer Ther. 2:358.
2. Lin, Z. B. 2005. Cellular and molecular mechanisms of immuno-modulation by Ganoderma lucidum. J. Pharmacol. Sci. 99:144.
3. Lin, K. I., Y. Y. Kao, H. K. Kuo, W. B. Yang, A. Chou, H. H. Lin, A. L. Yu, and C. H. Wong. 2006. Reishi polysaccharides induce immunoglobulin production through the TLR4/TLR2-mediated induction of transcription factor Blimp-1. J. Biol. Chem. 281:24111.
4. Song, Y. S., S. H. Kim, J. H. Sa, C. Jin, C. J. Lim, and E. H. Park. 2004. Anti-angiogenic and inhibitory activity on inducible nitric oxide production of the mushroom Ganoderma lucidum. J. Ethnopharmacol. 90:17.
5. Lin, Z. B., and H. N. Zhang. 2004. Anti-tumor and immunoregulatory activities of Ganoderma lucidum and its possible mechanisms. Acta Pharmacol. Sin. 25:1387.
6. Tanaka, S., K. Ko, K. Kino, K. Tsuchiya, A. Yamashita, A. Murasugi, S. Sakuma, and H. Tsunoo. 1989. Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8). An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions.J. Biol. Chem. 264:16372.
7. Murasugi, A., S. Tanaka, N. Komiyama, N. Iwata, K. Kino, H. Tsunoo, and S. Sakuma. 1991. Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidum. J. Biol. Chem. 266:2486.
8. Dayhoff, M. O., W. C. Barker, and L. T. Hunt. 1983. Establishing homologies in protein sequences. Methods Enzymol. 91:524-45.:524.
9. Haak-Frendscho, M., K. Kino, T. Sone, and P. Jardieu. 1993. Ling Zhi-8: a novel T cell mitogen induces cytokine production and upregulation of ICAM-1 expression. Cell Immunol. 150:101.
10. Liu, Y. H., C. F. Tsai, M. C. Kao, Y. L. Lai, and J. J. Tsai. 2003. Effectiveness of Dp2 nasal therapy for Dp2- induced airway inflammation in mice: using oral Ganoderma lucidum as an immunomodulator. J. Microbiol. Immunol. Infect. 36:236.
11. Jhun, B. S., Y. T. Oh, J. Y. Lee, Y. Kong, K. S. Yoon, S. S. Kim, H. H. Baik, J. Ha, and I. Kang. 2005. AICAR suppresses IL-2 expression through inhibition of GSK-3 phosphorylation and NF-AT activation in Jurkat T cells. Biochem. Biophys. Res. Commun. 332:339.
12. Nel, A. E. 2002. T-cell activation through the antigen receptor. Part 1: signaling components, signaling pathways, and signal integration at the T-cell antigen receptor synapse. J. Allergy Clin. Immunol. 109:758.
13. kolitz, J. E., K. Welte, K. W. Sykora, and R. Mertelsmann. 1985. Interleukin 2: a review. Arzneimittelforschung. 35:1607.
14. Gaffen, S. L., and K. D. Liu. 2004. Overview of interleukin-2 function, production and clinical applications. Cytokine. 28:109.
15. Tchilian, E. Z., and P. C. Beverley. 2006. Altered CD45 expression and disease. Trends Immunol. 27:146.
16. Koretzky, G. A., J. Picus, T. Schultz, and A. Weiss. 1991. Tyrosine phosphatase CD45 is required for T-cell antigen receptor and CD2-mediated activation of a protein tyrosine kinase and interleukin 2 production. Proc. Natl. Acad. Sci. U. S. A. 88:2037.
17. Hermiston, M. L., Z. Xu, and A. Weiss. 2003. CD45: a critical regulator of signaling thresholds in immune cells. Annu. Rev. Immunol. 21:107-37. Epub;2001 Dec;19:107.
18. Paterson, R. R. 2006. Ganoderma - a therapeutic fungal biofactory. Phytochemistry. 67:1985.
19. Yamamori, T., O. Inanami, H. Sumimoto, T. Akasaki, H. Nagahata, and M. Kuwabara. 2002. Relationship between p38 mitogen-activated protein kinase and small GTPase Rac for the activation of NADPH oxidase in bovine neutrophils. Biochem. Biophys. Res. Commun. 293:1571.
20. Asehnoune, K., D. Strassheim, S. Mitra, J. Y. Kim, and E. Abraham. 2004. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J. Immunol. 172:2522.
21. Hsu, H. Y., and M. H. Wen. 2002. Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression. J. Biol. Chem. 277:22131.
22. Baba, M., M. B. Yong, M. Nonaka, Y. Matsuishi, M. Hirano, N. Nakamura, N. Kawasaki, N. Kawasaki, and T. Kawasaki. 2007. Glycosylation-dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion. J. Leukoc. Biol. 81:1002.
23. Musci, M. A., S. L. Beaves, S. E. Ross, T. Yi, and G. A. Koretzky. 1997. Surface expression of hemopoietic cell phosphatase fails to complement CD45 deficiency and inhibits TCR-mediated signal transduction in a Jurkat T cell clone. J. Immunol. 158:1565.
24. Motto, D. G., M. A. Musci, and G. A. Koretzky. 1994. Surface expression of a heterologous phosphatase complements CD45 deficiency in a T cell clone. J. Exp. Med. 180:1359.
25. Hsieh, K. Y., C. I. Hsu, J. Y. Lin, C. C. Tsai, and R. H. Lin. 2003. Oral administration of an edible-mushroom-derived protein inhibits the development of food-allergic reactions in mice. Clin. Exp. Allergy. 33:1595.
26. Lin, W. H., C. H. Hung, C. I. Hsu, and J. Y. Lin. 1997. Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis. J. Biol. Chem. 272:20044.
27. Miyasaka, N., H. Inoue, T. Totsuka, R. Koike, K. Kino, and H. Tsunoo. 1992. An immunomodulatory protein, Ling Zhi-8, facilitates cellular interaction through modulation of adhesion molecules. Biochem. Biophys. Res. Commun. 186:385.
28. Ko, J. L., C. I. Hsu, R. H. Lin, C. L. Kao, and J. Y. Lin. 1995. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence. Eur. J. Biochem. 228:244.
29. Palacios, E. H., and A. Weiss. 2004. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene. 23:7990.
30. Harder, T. 2004. Lipid raft domains and protein networks in T-cell receptor signal transduction. Curr. Opin. Immunol. 16:353.
31. Appleman, L. J., and V. A. Boussiotis. 2003. T cell anergy and costimulation. Immunol. Rev. 192:161-80.:161.
32. Call, M. E., and K. W. Wucherpfennig. 2005. The T cell receptor: critical role of the membrane environment in receptor assembly and function. Annu. Rev. Immunol. 23:101-25.:101.
33. Razzaq, T. M., P. Ozegbe, E. C. Jury, P. Sembi, N. M. Blackwell, and P. S. Kabouridis. 2004. Regulation of T-cell receptor signalling by membrane microdomains. Immunology. 113:413.
34. Houtman, J. C., R. A. Houghtling, M. Barda-Saad, Y. Toda, and L. E. Samelson. 2005. Early phosphorylation kinetics of proteins involved in proximal TCR-mediated signaling pathways. J. Immunol. 175:2449.
35. Irvin, B. J., B. L. Williams, A. E. Nilson, H. O. Maynor, and R. T. Abraham. 2000. Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line. Mol. Cell Biol. 20:9149.
36. Marhaba, R., M. J. Dumaurier, C. Pelassy, M. Batoz, J. F. Peyron, J. P. Breittmayer, and C. Aussel. 1997. The protein tyrosine kinase p56(lck) regulates the serine-base exchange activity in Jurkat T cells. FEBS Lett. 405:163.
37. Straus, D. B., and A. Weiss. 1992. Genetic evidence for the involvement of the lck tyrosine kinase in signal transduction through the T cell antigen receptor. Cell. 70:585.
38. Goldsmith, M. A., and A. Weiss. 1987. Isolation and characterization of a T-lymphocyte somatic mutant with altered signal transduction by the antigen receptor. Proc. Natl. Acad. Sci. U. S. A. 84:6879.
39. Roth, M. S., F. S. Collins, and D. Ginsburg. 1988. Sizing of the human T cell receptor alpha locus and detection of a large deletion in the Molt-4 cell line. Blood. 71:1744.
40. Huntington, N. D., and D. M. Tarlinton. 2004. CD45: direct and indirect government of immune regulation. Immunol. Lett. 94:167.
41. Stone, J. D., L. A. Conroy, K. F. Byth, R. A. Hederer, S. Howlett, Y. Takemoto, N. Holmes, and D. R. Alexander. 1997. Aberrant TCR-mediated signaling in CD45-null thymocytes involves dysfunctional regulation of Lck, Fyn, TCR-zeta, and ZAP-70. J. Immunol. 158:5773.
42. Wang, Y., and P. Johnson. 2005. Expressin of CD45 lacking the catalytic protein tyrosine phosphatase domain modulates Lck phosphorylation and T cell activation. J. Biol. Chem. 280:14318.
43. Zhang, M., M. Moran, J. Round, T. A. Low, V. P. Patel, T. Tomassian, J. D. Hernandez, and M. C. Miceli. 2005. CD45 signals outside of lipid rafts to promote ERK activation, synaptic raft clustering, and IL-2 production. J. Immunol. 174:1479.
44. Sato, T., K. Furukawa, M. Autero, C. G. Gahmberg, and A. Kobata. 1993. Structural study of the sugar chains of human leukocyte common antigen CD45. Biochemistry. 32:12694.
45. Liu, Y. H., C. F. Tsai, M. C. Kao, Y. L. Lai, and J. J. Tsai. 2003. Effectiveness of Dp2 nasal therapy for Dp2- induced airway inflammation in mice: using oral Ganoderma lucidum as an immunomodulator. J. Microbiol. Immunol. Infect. 36:236.
46. van Vliet, S. J., S. I. Gringhuis, T. B. Geijtenbeek, and K. Y. van. 2006. Regulation of effector T cells by antigen-presenting cells via interaction of the C-type lectin MGL with CD45. Nat. Immunol. 7:1200.