金針菇免疫調節蛋白FIP-fve的醣類結合模組與誘發干擾素-γ活性之研究

FIP-fve為一種從金針菇 (Flammulina velutipes) 純化出來的免疫調節蛋白，具有刺激人類週邊血單核球細胞產生干擾素-γ的活性，經由蛋白質3D結構預測，發現FIP-fve的結構與澱粉酶之澱粉結合區的醣類結合模組相類似，這暗示著澱粉具有破壞FIP-fve刺激人類週邊血單核球細胞產生干擾素-γ的效果。本實驗藉由ELISA實驗觀察人類週邊血單核球細胞分泌干擾素-γ的情形。我們的研究證明starch、dextrin, cyclodextrin, maltotriose, maltose與neuraminic acid具有抑制由FIP-fve刺激而分泌的干擾素-γ。雖然這些醣類可以抑制干擾素-γ的分泌，但由FIP-fve刺激造成人類週邊血單核球細胞聚集的狀況卻不受影響。利用點突變的方式產生突變的FIP-fve (W26G, D36G, T92A, I93A, W113G)，破壞FIP-fve結構中預測與醣類之結合位，並以ELISA與凝血測試分析這些突變FIP-fve的蛋白活性，FIP-fve的醣類結合位經突變後，造成其免疫調節活性作用降低。突變的FIP-fve T30N，並沒有改善其刺激人類週邊血單核球細胞產生干擾素-γ的活性。此外，對人類週邊血單核球細胞處理tunicamycin與去醣化酵素也會減少干擾素-γ分泌量。綜合以上實驗結果，干擾素-γ的產生與FIP-fve的醣類結合模組和人類週邊血單核球的醣化受器息息相關。

關鍵字

真菌免疫調節蛋白； FIP-fve ；干擾素-γ ；人類週邊血單核球細胞

並列摘要

FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes and it has the function to stimulate IFN-γ production in human peripheral mononuclear cells (hPBMC). Prediction of three-dimensional structure, we found that the structure of FIP-fve is similar with the starch-binding carbohydrate binding module of amylase. It suggested that starch may abolish the function of FIP-fve to stimulate IFN-γ production in hPBMC. In this study, we investigated the level of IFN-γ secreted from hPBMC with ELISA. Our study had demonstrated that starch, dextrin, cyclodextrin, maltotriose, maltose, and neuraminic acid suppressed the secretion of IFN-γ stimulated by FIP-fve. Though the above carbohydrates inhibited the secretion of IFN-γ but not affected on aggregation of the hPBMCs stimulated by FIP-fve. To truncate the putative carbohydrate binding site in the FIP-fve, we constructed numbers of FIP-fve mutants by site-direct mutagenesis (W26G, D36G, T92A, I93A, W113G), the activity of mutant FIP-fve was investigated by ELISA and human red blood hemagglutination. The immunomodulatory activity of FIP-fve proteins was disrupted after truncating the carbohydrate binding site. The mutant FIP-fve T30N this mutant FIP-fve did not improve the immunomodulatory activity for IFN-γ stimulation than wild type FIP-fve in hPBMCs. Furthermore, hPBMCs were treated with tunicamycin and deglycosylation enzymes that decreased IFN-γ production. In conclusion, IFN-γ production is correlated with carbohydrate binding module of FIP-fve and glycosylation receptors of hPBMCs.

並列關鍵字

fungal immunomodulatory protein ； FIP-fve ； interferon-γ ； human peripheral blood mononuclear cells

參考文獻

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金針菇免疫調節蛋白FIP-fve的醣類結合模組與誘發干擾素-γ活性之研究

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