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

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