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

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.