伞枝犁头霉分泌的糖蛋白与人血管内皮细胞凋亡相关

目的 部分纯化伞枝犁头霉分泌的毒力因子,并研究其诱导人血管内皮细胞凋亡的机制.方法 用Con A Lectin亲和层析法纯化伞枝犁头霉分泌的糖蛋白,并在流式细胞仪下检测不同蛋白成分对人血管内皮细胞凋亡的影响.用变性和非变性法去除伞枝犁头霉糖蛋白的糖基,并分别检测其蛋白和寡糖成分诱导凋亡的活性.Western blot法分析伞枝犁头霉糖蛋白诱导人血管内皮细胞caspase激酶活化情况.用caspase抑制剂检测caspase-8和-9在凋亡反应中的作用.XTT法检测caspase抑制剂能否终止细胞存活率的抑制.结果 流式细胞仪分析显示伞枝犁头霉分泌的总蛋白和糖蛋白能以剂量依赖的方式诱导人血管内皮细胞凋亡,而非糖蛋白则无此活性.纯化的去糖基化蛋白和寡糖成分都不能独立诱导人血管内皮细胞凋亡.在凋亡信号传导途径中,caspase-9、-3和细胞色素C显著活化,而caspase-8未见活化.Caspase-9抑制剂能够终止伞枝犁头霉糖蛋白诱导的凋亡反应,而caspase-8抑制剂则无此效应.Caspase-9和-3抑制剂终止了人血管内皮细胞活性下降.结论 伞枝犁头霉分泌的糖蛋白具有诱导人血管内皮细胞凋亡的活性.糖蛋白结构的完整性对其诱导凋亡的活性是必需的.内源性凋亡信号传导途径介导了伞枝犁头霉糖蛋白诱导的凋亡反应.

Abstract：

Objective To partially purify the toxic factor secreted by A. corymbifera and to analyze the mechanism of A. corymbifera-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Methods Glycoprotein secreted by A. corymbifera was purified by Con A Lectin chromatography. The influence of different protein fractions on HUVEC apoptosis was determined by flow eytometer. Both denaturing and nondenaturing deglycosylation of purified glycoprotein was performed and the ability of the protein moiety and carbohydrate moiety to induce HUVEC apoptosis was evaluated respectively. Activation of related caspases during A. corymbifera-induced apoptosis was analyzed by Western blot. The role of caspase-8 and -9 in HUVEC apoptosis was investigated using caspase inhibitors. Caspase inhibitors were used to stop the suppression of HUVEC viability by XTT assay. Results Flow cytometric analysis shows the total protein as well as the glycoprotein fraction of A. corymbifera may induce HUVEC apoptosis in a dose dependent manner. In contrast, similar activity was not observed in the non-glycoprotein fraction. Neither deglycosylated protein nor carbohydrate moiety is able to induce HUVEC apoptosis alone. In the apoptotic signaling pathway, caspase9, caspase-3 and cytochrome C were activated significantly, except caspase-8. Moreover, caspase-9 inhibitor, instead of caspase-8 inhibitor, completely abrogates A. corymbifera-induced HUVEC apoptosis. Caspase9 and caspase-3 inhibitors completely waived the suppression of HUVEC viability by A. corymbifera. Conclusion Glycoprotein secreted by A. corymbifera is associated with HUVEC apoptosis. Intact glycoprotein is essential for the apoptotic progress. Intrinsic apoptotic signaling pathway mediates A. corymbifera-induced HUVEC apoptosis.

Glycoprotein secreted by Absidia corymbifera is associated with apoptosis of human umbilical vein endothelial cells

Objective To partially purify the toxic factor secreted by A. corymbifera and to analyze the mechanism of A. corymbifera-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Methods Glycoprotein secreted by A. corymbifera was purified by Con A Lectin chromatography. The influence of different protein fractions on HUVEC apoptosis was determined by flow eytometer. Both denaturing and nondenaturing deglycosylation of purified glycoprotein was performed and the ability of the protein moiety and carbohydrate moiety to induce HUVEC apoptosis was evaluated respectively. Activation of related caspases during A. corymbifera-induced apoptosis was analyzed by Western blot. The role of caspase-8 and -9 in HUVEC apoptosis was investigated using caspase inhibitors. Caspase inhibitors were used to stop the suppression of HUVEC viability by XTT assay. Results Flow cytometric analysis shows the total protein as well as the glycoprotein fraction of A. corymbifera may induce HUVEC apoptosis in a dose dependent manner. In contrast, similar activity was not observed in the non-glycoprotein fraction. Neither deglycosylated protein nor carbohydrate moiety is able to induce HUVEC apoptosis alone. In the apoptotic signaling pathway, caspase9, caspase-3 and cytochrome C were activated significantly, except caspase-8. Moreover, caspase-9 inhibitor, instead of caspase-8 inhibitor, completely abrogates A. corymbifera-induced HUVEC apoptosis. Caspase9 and caspase-3 inhibitors completely waived the suppression of HUVEC viability by A. corymbifera. Conclusion Glycoprotein secreted by A. corymbifera is associated with HUVEC apoptosis. Intact glycoprotein is essential for the apoptotic progress. Intrinsic apoptotic signaling pathway mediates A. corymbifera-induced HUVEC apoptosis.