Kallikrein 13: a new player in coronaviral infections

Aleksandra Milewska; Katherine Falkowski; Magdalena Kalinska; Ewa Bielecka; Antonina Naskalska; Pawel Mak; Adam Lesner; Marek Ochman; Maciej Urlik; Jan Potempa; Tomasz Kantyka; Krzysztof Pyrc

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.03.01.971499 doi: bioRxiv preprint KLK13 primes the HCoV-HKU1 S protein 227 Expression of KLK13 by cells previously resistant to HCoV-HKU1 renders them 228 susceptible; therefore, we asked whether this is due to proteolytic activation of the S protein. 229 We tested this using the CleavEx method, in which a peptide of interest is exposed in the KLK13. Subsequently, proteins were resolved by SDS-PAGE and detected by western blotting 238 with antibodies specific for His-tagged proteins. The analysis showed that, in the presence of 239 500 nM KLK13, the CleavEx protein harboring the S1/S2 cleavage site was degraded; however, 240 the CleavEx protein harboring the S2/S2' site remained intact. Because KLKs are produced as 241 pro-forms that undergo self-activation, an additional band of His-tagged purified pro-KLK13 242 (HisTag-pro-KLK13) was observed after treatment with 500 nM KLK13 (Fig. 6A) . The product 243 of the S1/S2 cleavage was further sequenced by N-terminal Edman degradation showing the 244 following sequence: R↓SISA, which corresponds to the S1/S2 site. This result shows clearly 245 that the S1/S2 region of the HCoV-HKU1 S protein is prone to KLK13-mediated cleavage. 246 Furthermore, we aimed to confirm the cleavage using a full-length Spike protein of HCoV-HKU1 (HKU1-S). For this, we expressed the HKU1-S in 293T cells, purified the protein 248 using 6 × His tag, and incubated for 3 h at 37°C with increasing concentrations of purified 249 KLK13. Subsequently, HKU-S or mock proteins were resolved by SDS-PAGE and detected by 250 author/funder. All rights reserved. No reuse allowed without permission.