Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

Pearson, M.; Haslam, C.; Fosberry, A.; Jones, E. J.; Reglinski, M.; Edwards, R. J.; Lawrenson, R. A.; Mossakowska, D.; Pease, J. E.; Sriskandan, S.

doi:10.1101/2021.11.14.468564

bioRxiv · 10.1101/2021.11.14.468564

Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

Pearson, M.·Haslam, C.·Fosberry, A.·Jones, E. J.·Reglinski, M.·Edwards, R. J.·Lawrenson, R. A.·Mossakowska, D.·Pease, J. E.·Sriskandan, S.

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Abstract

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2,200- fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

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Pearson, M., Haslam, C., Fosberry, A., Jones, E. J., Reglinski, M., Edwards, R. J., Lawrenson, R. A., Mossakowska, D., Pease, J. E., Sriskandan, S.. 2021-11-15. Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP. https://doi.org/10.1101/2021.11.14.468564

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