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Article

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches

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Bioanalytical Research Labs, Department of Biosciences, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria
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Structural Biology, Department of Biosciences, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria
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Molecular Immunology & Signal Transduction, Department of Biosciences, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria
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Cancer Cluster Salzburg, Department of Biosciences, University of Salzburg, Hellbrunner Strasse 34, 5020 Salzburg, Austria
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Department of Structural Biology, Molecular Biology Institute of Barcelona, CSIC, Barcelona Science Park, Baldiri Reixac, 15-21, 08028 Barcelona, Spain
*
Author to whom correspondence should be addressed.
Academic Editors: Marcello Manfredi and Elettra Barberis
Int. J. Mol. Sci. 2022, 23(1), 412; https://doi.org/10.3390/ijms23010412
Received: 13 December 2021 / Revised: 27 December 2021 / Accepted: 29 December 2021 / Published: 30 December 2021
Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection. View Full-Text
Keywords: streptococcal cysteine protease; SCP; streptopain; protease degradomics; HUNTER; N-terminomics; positional proteomics; sequence specificity; sequence logo; IgG subclasses streptococcal cysteine protease; SCP; streptopain; protease degradomics; HUNTER; N-terminomics; positional proteomics; sequence specificity; sequence logo; IgG subclasses
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MDPI and ACS Style

Blöchl, C.; Holzner, C.; Luciano, M.; Bauer, R.; Horejs-Hoeck, J.; Eckhard, U.; Brandstetter, H.; Huber, C.G. Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches. Int. J. Mol. Sci. 2022, 23, 412. https://doi.org/10.3390/ijms23010412

AMA Style

Blöchl C, Holzner C, Luciano M, Bauer R, Horejs-Hoeck J, Eckhard U, Brandstetter H, Huber CG. Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches. International Journal of Molecular Sciences. 2022; 23(1):412. https://doi.org/10.3390/ijms23010412

Chicago/Turabian Style

Blöchl, Constantin, Christoph Holzner, Michela Luciano, Renate Bauer, Jutta Horejs-Hoeck, Ulrich Eckhard, Hans Brandstetter, and Christian G. Huber. 2022. "Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches" International Journal of Molecular Sciences 23, no. 1: 412. https://doi.org/10.3390/ijms23010412

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