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研究生:王騰旭
研究生(外文):Teng-Hsu Wang
論文名稱:番木瓜(Carica papaya)種籽中對N-乙醯半乳糖胺具專一性親醣蛋白之研究
論文名稱(外文):Studies on an N-acetyl Galactosamine Specific Binding Lectin from Papaya (Carica papaya) Seeds
指導教授:蘇南維蘇南維引用關係
指導教授(外文):Nan-Wei Su
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:113
中文關鍵詞:親醣蛋白N-乙醯基半乳糖胺木瓜篩選方法分離純化
外文關鍵詞:LectinsN-acetyl-D-galactosamineCarica papayaScreeningIsolation and Characterization
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植物中廣泛存在的親醣蛋白lectins過去被認為是影響養份吸收的“抗營養物質(anti-nutrient)”,然而近年來的研究卻認為這些可與細胞表面特定醣類結構結合的蛋白質,能夠引起對腫瘤細胞的毒殺作用或抑制其生長,顯示其可能作為對抗惡性腫瘤工具的潛力。本研究利用Lectin可以專一辨識不同醣類結構的特性,建立以聚丙烯醯胺結合不同單醣(包含D-mannose、N-acetyl-D-glucosamine、N-acetyl-D-galactosamine及L-fucose)之共價聚合物作為篩選Lectin之專一性配體(ligand),利用酵素聯結吸附分析法(enzyme-linked adsorbent assay)對植物材料進行篩選,並分別以Con A、WGA、SBA及UEA I等已知Lectin作為正對照組。相較於傳統的血球凝集測試方法,本研究所建立的醣聚合物聯結分析可篩選與辨別樣品中是否具有對特定醣基結構專一性結合能力,且可避免血球來源與表面結合部位無法掌握的缺點。利用此篩選方法自29種植物材料中篩選出數種對特定醣基結構具有專一性結合能力之標的植物材料。其中,木瓜籽中具有對N-acetyl-D-galactosamine (GalNAc)專一性結合活性之Lectin,藉由硫酸銨沉澱劃分、DEAE離子交換管柱層析及重複利用膠體過濾管柱層析等步驟進行純化分離,並將它命名為CPL (C. papaya lectin)。以不同醣類抑制血球凝集反應的結果顯示,CPL對GalNAc與Lactose具有專一性結合能力。進一步利用表面電漿共振技術分析木瓜籽親醣蛋白對GalNAc之親和動力學參數,其平衡解離常數KD達5.5 × 10^-9 M,顯示CPL對GalNAc具有極高的親和力。利用分子篩高效液相層析(Size-exclusion HPLC)分析CPL之分子量得知其原態約為804 kDa,並由38-及40- kDa之次單元所組成。將Jurkat T細胞培養液添加CPL共同培養後,可刺激該細胞大量產生細胞激素IL-2,顯示CPL具有調節免疫機能之潛力。

Lectins are carbohydrate recognizing proteins which prevanlently exist in most, if not all, living organisms. For a long time, plant lectins were commonly perceived as ‘antinutrients’, mainly due to their adverse effects of causing non-pathogenic food-borne poisoning when not properly cooked before consumption. However, recent studies exhibited that several lectins have been found to possess anticancer properties and showed promising potential as anticancer agents.This study provides a new strategy to detect the specific carbohydrate binding capability of lectins. A sugar-polymer based enzyme-linked adsorbent assay was established by applying different monosaccharide-polyacrylamide conjugates, including D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and L-fucose, as capturing agents for screening lectins in biological samples. Four model lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), soybean agglutinin (SBA) and Ulex europaeus agglutinin I (UEA I), were employed as the comparative lectins against each corresponding sugar. The results suggested that this assay is more sensitive than the conventional hemagglutinating methods. On the screening of specific carbohydrate binding capability from 29 plant materials, a specific N-acetyl-D-galactosamine (GalNAc) binding capability was observed in the crude extracts of papaya (Carica papaya) seeds. The GalNAc specific binding protein was subsequently isolated and designated as CPL (C. papaya lectin). Purification of the lectin involved ammonium sulfate fractionation and DEAE anion exchange and repeated gel filtration chromatography. Inhibition of CPL causing hemagglutination on human erythrocytes showed that the lectin shows specificity to GalNAc and lactose. Surface plasmon resonance further revealed that the lectin possesses high specificity toward GalNAc with a dissociation constant of 5.5 × 10^-9 M. The lectin is composed of 38- and 40- kDa subunits with molecular mass of ~804 kDa estimated by size-exclusion high-performance liquid chromatography. Incubation of CPL with Jurkat T cells showed significant induction of IL-2 cytokine, which suggests that CPL has potent immunomodulatory effects on immune cells.

摘要..................................................ii
Abstract..............................................iii
目錄..................................................iv
圖目錄................................................vii
表目錄................................................viii
縮寫表................................................ix
第一章、研究動機......................................1
第二章、研究背景與文獻整理............................2
第2-1節、醣生物學(Glycobiology)簡介...................2
第2-2節、Lectin簡介...................................6
一、定義範圍..........................................6
二、來源與分類........................................9
三、生理活性與機能性相關研究..........................22
第2-3節、Lectin之檢測方法.............................27
第2-4節、實驗設計大綱.................................28
第三章、Lectin新檢測方法之建立........................29
第3-1節、前言.........................................30
第3-2節、材料與方法...................................31
一、 試驗材料與試劑...................................31
二、 表面結合特定醣聚合物之酵素聯結吸附分析法基本操作流程..............................31
三、 特定醣類聚丙烯胺聚合物(Sugar-PAAs)之表面吸附程度探討................................32
四、 建立對照標準Lectin分析檢量線.....................34
五、 對照Lectin之非特異性醣結合交互分析...............34
六、 Lectin與醣聚合物交互作用之游離醣類競爭抑制.......34
七、 血液凝集活性分析方法.............................35
第3-3節、結果.........................................36
一、 Sugar-PAAs之表面吸附程度探討.....................36
二、 建立標準曲線及可分析濃度範圍.....................38
三、 對照Lectin之非特異性醣結合交互分析...............40
四、 Lectin與醣聚合物交互作用之游離醣競爭抑制.........42
五、 檢測極限與靈敏度之探討...........................45
第3-4節、討論.........................................47
第四章、生物材料中新穎Lectin之篩選....................49
第4-1節、前言與研究目的...............................50
第4-2節、材料與方法...................................51
一、 試驗材料.........................................51
二、 樣品前處理與分析.................................51
三、 篩選試驗:以最適化之酵素聯結吸附分析法檢測可與特定醣基結合之食材萃取液......................................51
第4-3節、結果與討論...................................53
一、 以酵素聯結吸附分析篩選萃取液中具特定單醣結合之能力 .............................................53
二、 篩選結果之綜合比較與探討.........................57
第五章、木瓜種籽中對N乙醯半乳糖胺專一性結合Lectin之分離純化與生化性質研究........................................61
第5-1節、前言.........................................62
第5-2節、材料與方法...................................64
一、 試驗材料與試劑...................................64
二、 木瓜種籽前處理與蛋白質萃取.......................64
三、 木瓜種籽萃取物劃分與親醣蛋白之分離純化...........64
四、 純化木瓜籽Lectin之原態分子量評估:分子篩高效液相層析法 .............................................66
五、 純化木瓜籽Lectin之醣類專一性血球凝集抑制試驗.....66
六、 表面電漿共振系統測定木瓜籽Lectin對N-乙醯基半乳糖胺之親和動力學..............................................66
七、 木瓜籽Lectin對人類Jurkat T細胞與THP-1單核球細胞之細胞毒性與誘導細胞激素分泌之影響............................67
第5-3節、試驗結果.....................................69
一、 木瓜籽初步萃取與前處理...........................69
二、 陰離子交換管柱層析...............................69
三、 膠體過濾管柱層析.................................71
四、 純化之木瓜籽Lectin之醣類專一性血球凝集抑制.......76
五、 表面電漿共振方法探討木瓜籽Lectin對N-乙醯基半乳糖胺之親和動力學..............................................78
六、 木瓜籽Lectin對人類Jurkat T細胞與THP-1單核球細胞之細胞毒性與誘導細胞激素分泌之影響............................80
第5-4節、討論.........................................83
一、 分離方法探討.....................................83
二、 分子量與結構探討.................................84
三、 醣類專一性與親和動力學探討.......................85
四、 生理活性與免疫調節機能探討.......................87
第六章、結論..........................................88
參考文獻 .............................................89
附錄..................................................100
一、 本研究所使用之儀器設備...........................100
二、 各項緩衝液或溶液調配方法.........................102
三、 SDS-PAGE分析.....................................102
四、 相關著作.........................................106

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