Zhu-Salzman K, Shade R E, Koiwa H, Salzman R A, Narasimhan M, Bressan R A, Hasegawa P M, Murdock L L
Department of Entomology, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 1998 Dec 8;95(25):15123-8. doi: 10.1073/pnas.95.25.15123.
Griffonia simplicifolia leaf lectin II (GSII), a plant defense protein against certain insects, consists of an N-acetylglucosamine (GlcNAc)-binding large subunit with a small subunit having sequence homology to class III chitinases. Much of the insecticidal activity of GSII is attributable to the large lectin subunit, because bacterially expressed recombinant large subunit (rGSII) inhibited growth and development of the cowpea bruchid, Callosobruchus maculatus (F). Site-specific mutations were introduced into rGSII to generate proteins with altered GlcNAc binding, and the different rGSII proteins were evaluated for insecticidal activity when added to the diet of the cowpea bruchid. At pH 5.5, close to the physiological pH of the cowpea bruchid midgut lumen, rGSII recombinant proteins were categorized as having high (rGSII, rGSII-Y134F, and rGSII-N196D mutant proteins), low (rGSII-N136D), or no (rGSII-D88N, rGSII-Y134G, rGSII-Y134D, and rGSII-N136Q) GlcNAc-binding activity. Insecticidal activity of the recombinant proteins correlated with their GlcNAc-binding activity. Furthermore, insecticidal activity correlated with the resistance to proteolytic degradation by cowpea bruchid midgut extracts and with GlcNAc-specific binding to the insect digestive tract. Together, these results establish that insecticidal activity of GSII is functionally linked to carbohydrate binding, presumably to the midgut epithelium or the peritrophic matrix, and to biochemical stability of the protein to digestive proteolysis.
西非吊灯树叶片凝集素II（GSII）是一种抵御某些昆虫的植物防御蛋白，由一个结合N - 乙酰葡糖胺（GlcNAc）的大亚基和一个与III类几丁质酶具有序列同源性的小亚基组成。GSII的大部分杀虫活性归因于凝集素大亚基，因为细菌表达的重组大亚基（rGSII）抑制了豇豆象鼻虫Callosobruchus maculatus（F）的生长和发育。将位点特异性突变引入rGSII以产生具有改变的GlcNAc结合能力的蛋白质，并在将不同的rGSII蛋白质添加到豇豆象鼻虫的食物中时评估其杀虫活性。在接近豇豆象鼻虫中肠腔生理pH值的pH 5.5条件下，rGSII重组蛋白被分类为具有高（rGSII、rGSII - Y134F和rGSII - N196D突变蛋白）、低（rGSII - N136D）或无（rGSII - D88N、rGSII - Y134G、rGSII - Y134D和rGSII - N136Q）GlcNAc结合活性。重组蛋白的杀虫活性与其GlcNAc结合活性相关。此外，杀虫活性与对豇豆象鼻虫中肠提取物蛋白水解降解的抗性以及与昆虫消化道的GlcNAc特异性结合相关。总之，这些结果表明，GSII的杀虫活性在功能上与碳水化合物结合相关，可能与中肠上皮或围食膜有关，并且与蛋白质对消化性蛋白水解的生化稳定性有关。
