小孢子靈芝免疫調節蛋白質 GMI 部分片段之生理活性分析

Abstract

自小孢子靈芝 (Ganoderma microsporum) 分離出之真菌免疫調節蛋白質 (Fungal immunomodulatory protein) GMI 已證實能藉由與細胞膜表面受器作用，活化下游訊息傳遞路徑，進而具有調節細胞免疫反應 (Cell immune) 或抗腫瘤 (Anti-tumor) 等功效。本研究探討位於 GMI 部分片段 (Partial fragments) 之生理活性。藉由二級結構的比對及生物資訊軟體的分析，篩選出一個 α-helix 片段及三個 β-sheet 短胜肽 (Peptides) 片段進行化學合成，生產高純度及高產量之短胜肽。據細胞免疫活性測試結果顯示野生型蛋白質 GMI 及突變型蛋白質 GMI-L6C 皆能有效促進人類淋巴癌 T 細胞株 Jurkat 分泌 IL-2，而 α-helix 短胜肽 (GMIαL6C) 及三個 β-sheet 短胜肽 (GMIβ1531、GMIβ4567 及 GMIβ85103) 則無法促使 Jurkat 分泌 IL-2。在抗腫瘤活性測試中，GMI 及 GMI-L6C 皆能降低人類肺癌細胞株 A549 的存活率及移動能力，雙體的 α-helix 短胜肽 (GMIαL6C) 具抑制 A549 存活率及移動效果，而三個 β-sheet 短胜肽 (GMIβ1531、GMIβ4567 及 GMIβ85103) 則不具影響 A549 存活率及移動的能力。藉由細胞免疫及抗腫瘤試驗結果，GMI-L6C 相較於 GMI 可提升蛋白質免疫調節及抗腫瘤能力，證實穩定雙體構形與其生理活性的必要性；α-helix 短胜肽具抗腫瘤效果，顯示 α-helix 於 GMI 抗腫瘤活性的重要性；而三個 β-sheet 短胜肽則不具生理活性。

GMI, a fungal immunomodulatory protein (FIP) isolated from Ganoderma microsporum had been reported that it could interact with cell surface receptors and trigger downstream signal transduction. According to these evidences, FIPs exhibit potent immunomodulation and anti-tumor effect. In this study, we investigated the partial fragments within GMI and analyzed their bioactivities. By analyzing the secondary structure alignment and bioinformatics data, selected one α-helix and three β-sheet amino acid sequences. In order to get high quality and output target peptides, these peptides were produced by chemical synthesis. In terms of cell immune assay, wild type protein GMI and mutative protein GMI-L6C could enhance Jurkat T cells to secret IL-2 but α-helix-peptide and three β-sheet-peptides could not. By analyzing the results of anti-tumor tests, GMI and GMI-L6C could inhibit A549 viability and migration. Interestingly, α-helix-peptide could affect A549 viability and migration. However, three β-sheet-peptides could not inhibit A549 neither viability nor migration. Based on cell immune and anti-tumor assays, GMI-L6C exhibited better immunomodulatory and anti-tumor ability. It’s indicated dimerization is important to its bioactivity. By α-helix-peptide exhibiting anti-tumor activity, it suggested the importance of α-helix in anti-tumor activity of GMI. On the contrary, β-sheet-peptides didn’t have immunomodulatory and anti-tumor activity.