利用親和性層析法分離木瓜籽凝集素

凝集素(Lectin)為一類具有選擇性辨識特定醣類分子的蛋白質或醣蛋白，具有多種生理功能，如調控細胞分化、細胞附著、免疫反應等。近年來，廣泛作為研究醣生物學的分析工具，且發現凝集素有抗癌與免疫調節等生理機能。本實驗室先前的研究結果顯示，由番木瓜(Carica papaya LINN.)種籽中具有對於N-acetyl-D-galactosamine (GalNAc) 有良好親和性的凝集素蛋白─ C. papaya lectin (CPL)，並進行部分生化特性分析，顯示CPL 可以刺激Jurkat 細胞分泌IL-2，具有調節免疫細胞活性的潛力。但是，CPL分離純化步驟繁多且回收量率低，故本研究使用自行衍生製備的膠體lactose-Sepharose 6B (LS6B)及 GalNAc-Sepharose 6B (GS6B)，經由改變管柱順序與流洗條件，並搭配凝血活性追蹤，建立一套高效率的純化方法。研究顯示，此兩階段的親和性管柱層析：先通入LS6B，再通入GS6B管柱，接續分別以0.5 M 乳糖溶液、0.1 M GalNAc溶液流洗。其中，GalNAc溶液可流洗出滯留於GS6B管柱上的具凝血活性之蛋白，膠體過濾管柱分析結果顯示該蛋白即為CPL。與先前本研究室文獻報告的數據比較，以兩階段親和性管柱層析確實能有效純化CPL的比活性及提高其回收率。另一方面，以乳糖溶液洗出的區分液蛋白(簡稱LpGLac)，無論在血球凝集試驗或ELISA的結果都不具有醣類結合能力，可見其並非一類新的凝集素，目前推測LpGLac是藉由蛋白上所鍵結的醣基片段能為CPL所辨識、結合，而被滯留於親和性管柱上。此外，本研究亦將純化所得之具凝血活性的區分液及LpGLac處理小鼠巨噬細胞，結果顯示兩者皆能夠誘發巨噬細胞分泌促發炎細胞激素(pro-inflammatory cytokines)，具有免疫調節活性。

關鍵字

番木瓜；植物凝集素；免疫調節活性；促發炎細胞激素

並列摘要

Lectins are defined as sugar-binding and cell-agglutinating proteins of non-immune origin. They are ubiquitous in nature, being found in all kinds of organisms, from viruses to humans. Recently, our group isolated an N-Acetyl-D-galactosamine (GalNAc)-specific lectin from the seeds of papaya (Carica papaya), named CPL. In this study, an effective affinity chromatographic method for CPL isolation and purification was established, based on the specificity of CPL. By using two steps affinity chromatography through lactose-Sepharose 6B and GalNAc-Sepharose 6B resins, with sequential elution by lactose and GalNAc solution. The hemagglutination activity of GalNAc-elution fractions (LpGLac-GalNAc) was 1461 fold higher than original papaya seed crude extracts. Through the gel filtration chromatography, the results showed that the purified CPL was appeared in LpGLac-GalNAc; also, this purification protocol obtained higher purity and higher protein yield of CPL than previous research (Wang et al., 2011). At the same time, we discovered a lactose desorbed protein, designed as LpGLac, in the process of affinity purification. The results of SDS-PAGE, hemagglutination assay and ELISA assay indicated that LpGLac was recognized by CPL and might compete with lactose for carbohydrate binding domain on CPL, which resulted in desorbing from affinity column by lactose solution. In addition, we investigated the immunomodulatory effects of purified protein on murine macrophages RAW 264.7 cell line in vitro. Incubation of LpGLac and LpGLac-GalNAc with RAW cells showed significant induction of pro-inflammatory cytokines, IL-6 and TNF-α, as well as nitric oxide, which suggests that both of purified proteins have potent immunomodulatory effects on immune cells.

並列關鍵字

papaya ； lectin ； immunomodulatory effect ； affinity purification ； pro-inflammation cytokines

參考文獻

王騰旭、陳似蘭、李敏雄、蘇南維 (2008)。數種植物材料之親醣蛋白活性檢測。台灣農業化學與食品科學 46, 14-21。

龔彥綸 (2010)。番木瓜種子凝集素之純化與生化特性探討。台大農業化學系碩

王騰旭 (2010)。番木瓜(Carica papaya)種籽中對N-乙醯半乳糖胺具專一性親

Baxevanis, C. N., Voutsas, I. F., Soler, M. H., Gritzapis, A. D., Tsitsilonis, O. E., Stoeva, S., Voelter, W., Arsenis, P. and Papamichail, M. (1998). "Mistletoe lectin I-induced effects on human cytotoxic lymphocytes. I. Synergism with IL-2 in the induction of enhanced LAK cytotoxicity." Immunopharmacol Immunotoxicol 20(3): 355-372.

Bendtzen, K. (1994). "Cytokines and natural regulators of cytokines." Immunol Lett 43(1-2): 111-123.

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利用親和性層析法分離木瓜籽凝集素

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