香蕉凝集素分析與其對巨噬細胞之免疫調節作用

Abstract

香蕉 (Musa spp.) 為台灣最重要水果之一，具有豐富營養。由香蕉果肉中發現的香蕉凝集素 (banana lectin, BanLec) 具有增加造血幹細胞庫、抗愛滋病毒及免疫調節特性，並且有良好的高溫及高酸穩定性；上述功效以及加工穩定性使其具有成為保健食品素材甚至蛋白藥物的潛力。但目前尚未有特異性的分析方法可定量香蕉凝集素，使其應用性受限；且香蕉凝集素於抗原呈獻細胞之免疫調節活性亦尚未明瞭。 本研究成功於大腸桿菌中異體表現不具標籤蛋白的香蕉凝集素，亦從香蕉果肉純化取得天然香蕉凝集素。由天然香蕉凝集素或合成之預測抗原表位胜肽片段做為抗原，完成香蕉凝集素專一性多株及單株抗體製備，供後續研究使用。 香蕉凝集素刺激 M1 及 M2 巨噬細胞細胞激素 TNF-α 和IL-10 分泌及活化相關 mRNA 表現，亦提升共刺激分子 CD80 與 CD86 表現。受香蕉凝集素活化之巨噬細胞可進一步刺激 T 細胞分泌 IFN-γ 及 IL-4，香蕉凝集素也能直接刺激T 細胞分泌 IFN-γ 與 B 細胞分泌 IL-6 及 IL-10。香蕉凝集素之活化效應需要其碳水化合物結合區位，因此在甘露糖存在下，其免疫調節活性將受抑制。香蕉凝集素可直接與巨噬細胞、CD3+ T、CD4+ T、CD8+ T 與 CD19+ B 細胞結合；免疫細胞之受體多具醣基化，香蕉凝集素具有四個碳水化合物接合區位，可能透過同時接合免疫細胞受體及共刺激分子活化免疫細胞。 亦利用專一性引子及專一性抗體發展 mRNA 與蛋白層級分析方法，並分析在不同後熟階段之香蕉果肉中凝集素含量，結果顯示後熟過程中香蕉凝集素有先升後降之趨勢，於乙烯催熟後第三天表現量最高。 本研究闡明了香蕉凝集素對於巨噬細胞的活化效應，並且提供香蕉凝集素mRNA 及蛋白之分析平台，未來可進一步發展，為香蕉凝集素用於保健食品工業或釐清香蕉凝集素於植物體內之作用，提供良好之背景研究。

Banana (Musa spp.) is one of the most important fruits in Taiwan and it is full of nutrition. The banana lectin (BanLec) possesses an ability to enhance stem cell pool, anti-HIV, and immune-modulation. It is also stable in high temperature and low pH environments which makes it suitable to be used in food processing. It seems to be a potential ingredient for new functional food or protein medicine. However, there was a lack of suitable method for quantitating BanLec in a specific manner. Besides, the activation of BanLec on antigen presenting cells (APCs) is not clear. This present study successfully expressed recombinant BanLec without tag and enriched rBanLec production. Native BanLec from banana pulp was also purified and further completed the BanLec-specific poly/monoclonal antibody preparation. BanLec activated not only M1 but also M2 type macrophages in both cytokine secretion (TNF-α and IL-10) and mRNA expressions. The co-stimulatory molecules CD80 and CD86 could also be increased by BanLec. BanLec-activated macrophages possessed the function of stimulating T cells secreting IFN-γ and IL-4. On the other hand, BanLec directly stimulated T cells producing TH1 cytokine IFN-γ and B cells producing IL-6 and IL-10, respectively. The activation of BanLec depended on the carbohydrate binding sites that D(+)-mannose eliminated the immune-modulatory effects of BanLec. BanLec could bind to macrophages, CD3+ T, CD4+ T, CD8+ T and CD19+ B cells. Most of the receptors of immune-cells were glycosylated. BanLec possessed four carbohydrate binding sites (CBSs) which might bind to the receptors and co-stimulatory molecules at the same time and activated those cells. The specific relative quantitative methods in mRNA and protein levels of BanLec were developed. It was found that the BanLec in pulp would first raise than dramatically drop during banana ripening. This research clarified the effects of BanLec on macrophage and provided a tool for BanLec analysis in both mRNA and protein level. These results would beneficial to the further research about the usage of BanLec in functional food industry.