Effect of the dimerization of the Ganoderma microsporum protein GMI on its immune-modulatory function

靈芝是中國傳統醫學的珍貴藥材，具有滋補強身之效。近代科學研究也證實靈芝具有抗腫瘤與增強免疫力之功效，而靈芝的藥性成分可分為三萜類、多醣體、真菌免疫調節蛋白質三類。本研究探討選殖自小孢子靈芝Ganoderma microsporum的免疫調節蛋白質GMI，由嗜甲醇酵母菌Pichia pastoris以甲醇誘導方式進行胞外表現重組蛋白質GMI，並利用醱酵槽大規模生產。蛋白質純化後進行免疫活性測試結果顯示GMI能促進T細胞株Jurkat分泌IL-2、活化小鼠骨髓樹突細胞表面蛋白質作抗原呈獻且刺激其分泌IL-12、降低巨噬細胞株Raw264.7因添加LPS造成的促發炎細胞激素分泌量。 GMI由111個胺基酸組成，分子量為12.6 kDa，具高度水溶性。蛋白質於水溶液中的單體間會因N端α-helix形成分子間疏水性作用力及氫鍵，使兩單體結合為同源雙體結構。本篇研究利用點突變方式，將GMI蛋白質N端α-helix的第六個胺基酸，由原本的leucine (L)突變為cycteine (C)、lysine (K)或phenylalanine (F)，藉此探討不同程度雙體化的GMI是否會對免疫活性造成差異，並以膠體電泳和奈米微粒分析儀探討突變型蛋白質的雙體化程度，佐以免疫細胞實驗。結果顯示雙體化比率最高的突變型GMI-L6C，和野生型GMI相比，免疫調節活性顯著提升；而無法形成雙體的突變型組別(GMI-L6K、GMI-L6F)則免疫調節活性下降。由此確認真菌類免疫調節蛋白質雙體化所扮演的角色，提供未來真菌免疫調節蛋白質藥理活性提升的參考。Lingzhi, a popular medicinal edible mushroom, has been used in Asia for the past 2000 years. The antitumoral and immune-stimulating effects of Lingzhi are attributed to polysaccharides, triterpenoids and fungal immunomodulatory proteins (FIPs). This study investigated GMI, a FIP cloned from Ganoderma microsporum and expressed in a methytrophic yeast, Pichia pastoris. The native form GMI is a noncovalently linked homodimer, and the monomer consists of 111 amino acids, with molecular weight of 12.4 kDa. In this study, three different point mutation GMIs here made at the sixth residue, from leucine (L) to cysteine (C), lysine (K) and phenylalanine (F) respectively, resulting of the mutant GMIs, GMI-L6C, GMI-L6K and GMI-L6F. These mutants and wild type GMI here used to study the role of dimerization in immune modulatory functions and protein structure. GMI-L6C is able to form a stable homodimer via the formation of a disulfide bond. GMI-L6K and GMI-L6F tend to be pure monomer due to the positive charge or steroic conformation. Based on the results of the immune cell assays and nanoparticle analyzer, homodimer form GMI-L6C is able to increase immune cells function, while the monomer form GMI-L6K and GMI-L6F are unable to enhance immune activity