小孢子靈芝免疫調節蛋白質GMI之功能與結構分析

Functional and structural analysis of GMI, a fungal immunomodulatory protein from Ganoderma microsporum

翁瑞芸(Jui-Yun Weng)

指導教授 ： 黃慶璨

國立臺灣大學/生命科學院/生化科技學系/碩士(2011年)

https://doi.org/10.6342/NTU.2011.02191

摘要

靈芝屬真菌具有許多對人體的藥理活性成份，其中小分子的真菌免疫調節蛋白質為其中的功能成份之一，在比對不同種來源的免疫調節蛋白質結構，發現其C端的第一個loop是構形變異大的區域，在此立體結構上的差異是否影響不同種免疫調節蛋白質的活性差異是本研究欲探討的主題，因此本研究使用自小孢子靈芝(Ganderma microsporum)選殖出的免疫調節蛋白GMI，應用前人將第6個胺基酸leucine突變為cysteine (GMI-L6C)後，能使免疫調節蛋白質因雙硫鍵鍵結而增加同源雙體的單體間穩定結合的比例的基礎，使用GMI-L6C作為研究免疫調節蛋白質末端loop區域的目標蛋白質，對此區域進行一系列的序列突變，以排除雙體不穩定所造的實驗變因。本研究將在此loop上胺基酸序列進行不同電性或大小之殘基突變，或蛋白質序列的刪減：如將C端第一條b-sheet刪除使此loop結構變為較自由的形式，或刪除部份loop序列。為測試突變後的重組蛋白質的免疫活性是否改變，選用人類淋巴癌Jurkat T細胞作為本研究的免疫分析工具，分析經蛋白質免疫刺激後細胞激素IL-2的分泌量，探討此loop結構於不同細胞激素刺激表現上扮演之角色。另外為更深入探討雙體結構的重要性，本研究對野生型GMI進行第六個胺基酸的點突變，以獲得不穩定雙體結構的突變型免疫調節蛋白質GMI-L6K及GMI-L6F。
在GMI及GMI-L6C以E. coli表現系統表現時，重組的目標蛋白質不具有刺激Jurkat細胞的免疫活性。以嗜甲醇酵母菌Pichia pastoris表現的蛋白質在5 ug/ml的刺激濃度下，GMI-L6C可刺激IL-2分泌量達709.7±199.1 pg/ml，GMI-L6K與GMI-L6F則低於10 pg/ml，第98個胺基酸的點突變GMI-L6C&D98N及GMI-L6C&D98A皆低於50 pg/ml。顯示破壞雙體的穩定性可能導致免疫活性的喪失，以及第98個胺基酸在GMI蛋白質分子免疫功能上的重要性。

關鍵字

真菌免疫調節蛋白質(FIPs) ； 小孢子靈芝 ； GMI ； GMI-L6C ； 細胞激素表現

並列摘要

Fungal immunomodulatory proteins (FIPs) were immune functional proteins existed in the medical fungus. Based on the protein structural alignment of different species, the first loop from C terminus showed great difference in the conformation. Therefore, in this study, we tried to investigate that whether the stereo conformational difference in this loop area contributes to the immune activity.
Here we used the mutated recombinant FIP, GMI-L6C, as the topic to study the relationship between immune activity and function. GMI-L6C was a mutated protein from GMI, which was the FIP from Ganoderma microsporum. The sixth amino acid was mutated from leucine to cysteine. The disulfide bond stabilized the homo dimer structure of GMI-L6C. Using GMI-L6C as the base to conduct the site-specific mutagenesis in the final loop amino acid sequence, the effect was reduced by its shift between dimer and momer form while changing the resudues. In addition, the mutations were carried out in 2 sets of single amino acid mutation based on either residue charge or size and sequence deletion. For deletion, the b-sheet sequence downstream the final loop was deleted to let the loop structure as a free form tail, or delete the amino acid on the loop region to shorten the loop size. Human T cell lymphoblast-like cell line (Jurkat cell line) was used as the assay cells to test the immune activity mutated GMI-L6C protein. IL-2 secretion by Jurkat T cell after protein stimulation indicated the mutagenesis effect on the loop structure and its immune functional change.
On the other side, to further investigate the importance of FIP dimer structure, the sixth amino acid mutagenesis protein GMI-L6K and GMI-L6F were used to express the proteins, and the unstable dimer FIP were expected to be obtained.
Taken together the recombinant protein GMI and GMI-L6C were not able to stimulation Jurkat T cell to secrete IL-2 in E. coli expression system. In methyltrophic yeast Pichia pastoris system, the IL-2 secretion was up to 709.7±199.1 pg/ml in the recombinant protein GMI-L6C. The result of GMI-L6K and GMI-L6F were lower than 10 pg/ml and the 98th amino acid mutated protein GMI-L6C&D98N and GMI-L6C&D98A were lower than 50 pg/ml in the secretion of IL-2. The results suggested that the unstable dimer form of GMI decreased the immune activity and showed the importance of the 98th amino acid in modulating immune function.

並列關鍵字

Fungal immunomodulatory proteins (FIPs) ； Ganoderma microsporum ； GMI ； GMI-L6C ； cytokine expression

參考文獻

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