The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Transmembrane protease serine (TMPRSS)2 is a cell surface host protease, which plays a decisive role in viral infections. This trypsin-like enzyme cleaves the spike protein of coronaviruses [e.g. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] and the hemagglutinin protein of influenza viruses, enabling viral membrane fusion and subsequent viral cell entry. Consequently, TMPRSS2 is an attractive therapeutic target, and it is of utmost importance to produce wildtype, enzymatically active recombinant TMPRSS2 for drug development purposes. We present the first successful strategy for the expression of wildtype and proteolytically fully active ectodomain of human and Syrian hamster TMPRSS2 in mammalian cells. To achieve that, we co-expressed the TMPRSS2 ectodomain with its natural inhibitor, hepatocyte growth factor activator inhibitor-2, which yielded substantial amounts of secreted, native protease. Most of the recombinant TMPRSS2 was secreted as zymogen, which was activated during purification. The purified activated recombinant TMPRSS2 ectodomain cleaved synthetic and protein substrates with high efficiency (kcat/KM in the 104–106 M-1s-1 range). To study the mechanism of auto-activation, we expressed zymogen TMPRSS2 mutants as well. We found that the zymogen is an ideal substrate for the active protease as it cleaves it extremely efficiently. We also showed that zymogen TMPRSS2 itself has a weak proteolytic activity, which can initiate the auto-activation process. We demonstrated that a related cell surface protease, TMPRSS13, is also able to activate zymogen TMPRSS2. Our findings prove both a zymogen trans(auto)-activation as well as a TMPRSS13-based cross-activation mechanism, the latter supporting that type II transmembrane serine proteases can form a pericellular proteolytic cascade.