Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation

Ting Gao; Mingdong Hu; Xiaopeng Zhang; Hongzhen Li; Lin Zhu; Hainan Liu; Qincai Dong; Zhang Zhang; Zhongyi Wang; Yong Hu; Yangbo Fu; Yanwen Jin; Kaitong Li; Songtao Zhao; Yongjiu Xiao; Shuping Luo; Lufeng Li; Lingfang Zhao; Junli Liu; Huailong Zhao; Yue Liu; Weihong Yang; Jing Peng; Xiaoyu Chen; Ping Li; Yaoning Liu; Yonghong Xie; Jibo Song; Lu Zhang; Qingjun Ma; Xiuwu Bian; Wei Chen; Xuan Liu; Qing Mao; Cheng Cao

The C4b deposition assay was performed using a human MBL/MASP-2 assay kit (Hycult 25 biotech) (42). In brief, diluted serum was incubated in mannan-coated plates with high salt binding buffer overnight at 4°C and removed by washing, and the MBL-MASP-2 complex was captured. Purified C4 and N protein were added and incubated for 1.5 hr, and the deposited C4b was detected following standard protocols. The functional activity of LP and AP was assessed by ELISA as previously described (43) . Nunc Maxisorb plates were coated with 10 μg mannan per 30 well in 100 mM Na 2 CO 3 /NaHCO 3 (pH 9.6) at room temperature overnight. After each step, plates were washed three times with PBST (300 μl/well). Residual binding sites were blocked by incubation with 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 2% HSA for 2-3 hr at room temperature. Serum samples were diluted 1:80 in 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 0.5 mM MgCl 2 , 0.05% Tween-20, and 0.1% gelatin with or without 2 mM CaCl 2 and N 35 protein. All samples and buffers were prepared on ice. The plates were then sequentially incubated for 1 hr at 4°C and for 1.5 hr at 37°C followed by washing. All incubation volumes were 100 μl. Complement binding was detected using antibodies followed by washing. Detection of C4, activated C3, and C5b-9 was performed using anti-C4α chain antibody (Santa Cruz), antiactivated C3 antibody (Santa Cruz), and anti-C5b-9 antibody (Calbiochem), respectively.