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

Abstract

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.