Cloning, expression of porcine GSDME and identification of its site cleaved by caspase-3

• Published in: Biochemical and biophysical research communications 2023-08, Vol.669, p.61-67
• Main Authors: Li, Chenyu, Pang, Yu, Wang, Yuchen, Zhou, Yanrong, Fang, Liurong, Xiao, Shaobo, Qiu, Dexin
• Format: Article
• Language: English
• Subjects: Animals; Caspase 3 - genetics; Caspase 3 - metabolism; Caspase-3 cleavage site; Cattle; Cisplatin; Cloning, Molecular; Gasdermins; Humans; Mammals - metabolism; Mice; Paclitaxel and cisplatin; Polyclonal antibody; Porcine GSDME; Pyroptosis - physiology; Rabbits; Recombinant protein; Swine; Tissue distribution
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2023.05.076

As a member of the gasdermin family, gasdermin E (GSDME) is specifically cleaved by caspase-3, resulting in pyroptosis. To date, the biological characteristics and functions of human and mouse GSDME have been extensively studied; however, little is known of porcine GSDME (pGSDME). In this study, the full-length pGSDME-FL was cloned, which encodes 495 amino acids (aa) that have closely evolutionary relationships to the homolog of camelus, aquatic mammals, cattle and goat. Moreover, pGSDME was detected at different levels of expression in 21 tissues and 5 pig-derived cell lines tested by qRT-PCR, with the highest expression levels in mesenteric lymph nodes and PK-15 cell lines. Anti-pGSDME polyclonal antibody (pAb) with good specificity was generated by expressing the truncated recombinant protein pGSDME-1-208 and immunizing the rabbits. By western blot analysis using highly specific anti-pGSDME polyclonal antibody (pAb) prepared as primary antibody, it was not only confirmed that paclitaxel and cisplatin were positive stimuli to pGSDME cleavage and caspase-3 activation, but also identified the aspartate (D268) at position 268th of pGSDME as a cleavage site of caspase-3, and the overexpressed pGSDME-1-268 possesses cytotoxicity to HEK-293T cells, indicating that pGSDME-1-268 may contain active domains and involve pGSDME-mediated pyroptosis. These results lay a foundation for further investigating the function of pGSDME, especially its role in pyroptosis and its interaction with pathogens. •Porcine gasdermin E (pGSDME) is expressed in various tissues and pig-derived cell lines.•Anti-pGSDME polyclonal antibody with good specificity is generated.•Paclitaxel and cisplatin are positive stimuli to pGSDME cleavage and caspase-3 activation.•The aspartate at position 268th of pGSDME as a cleavage site of caspase-3.