The study is aimed at investigating the role and mechanism of LpqH of in the activation of NLRP3 inflammasome in mouse Ana-1 macrophages. ExPASy-ProtParam, PHYRE2, ABCpred, and SYFPEITHI were used to predict and analyze the physicochemical properties, protein structure, and B cell/T cell-associated epitopes of LpqH protein. The recombinant LpqH protein was purified, and its immunoreactivity was analyzed with western blot. The LPS-treated mouse Ana-1 macrophages were incubated with purified LpqH protein directly. The expression of NLRP3, ASC, and caspase-1 protein was detected by western blot. The secretion of IL-1 was detected by ELISA, and LDH was detected by a kit. Cell death was detected by flow cytometry. ... More
The study is aimed at investigating the role and mechanism of LpqH of in the activation of NLRP3 inflammasome in mouse Ana-1 macrophages. ExPASy-ProtParam, PHYRE2, ABCpred, and SYFPEITHI were used to predict and analyze the physicochemical properties, protein structure, and B cell/T cell-associated epitopes of LpqH protein. The recombinant LpqH protein was purified, and its immunoreactivity was analyzed with western blot. The LPS-treated mouse Ana-1 macrophages were incubated with purified LpqH protein directly. The expression of NLRP3, ASC, and caspase-1 protein was detected by western blot. The secretion of IL-1 was detected by ELISA, and LDH was detected by a kit. Cell death was detected by flow cytometry. LpqH consisted of 159 amino acids and was a hydrophobic protein with stable properties. Its secondary structure contained 47% random coils, 53% -sheets, and 3% -helix. The tertiary structure showed a relatively loose spatial conformation. Additionally, it had 8 B cell epitopes (score > 0.8) and 10 CTL cell epitopes (score ≥ 20). The recombinant LpqH, which had strong immunoreactivity, significantly increased the levels of NLRP3, ASC, and caspase-1 p20 ( < 0.01) and promoted the secretion of IL-1 by the cells ( < 0.01). In addition, high concentration of KCl significantly inhibited the effect of LpqH on mouse Ana-1 macrophages and reduced the expression of NLRP3, ASC, and caspase-1 p20 ( < 0.01). However, there was no significant change in LDH ( > 0.05). Meanwhile, LpqH protein did not cause additional cell death ( > 0.05). LpqH protein has good immunogenicity and can activate the NLRP3 inflammasome through the potassium efflux pathway without causing cell death.
