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

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. Key words: Mucorales; Absidia corymbifera; Glycoprotein; Human umbilical vein endothelial cell; Apoptosis