Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Stx2a Production and Purification
2.2. Sampling and Fractioning of Stool
2.3. Cleavage of Stx2a A Subunit by Trypsin or Furin
2.4. Incubation of Stx2a with Human Stool Supernatant or Its Fractions with or without Protease Inhibitors
2.5. Incubation of Stx2a with Recombinant CELA3B
2.6. Determination of the Structure of Stx2a A Subunit by Immunoblotting
2.7. Identification of the Cleavage Position of the A Subunit of Stx2a by Components Present in Human Stool
2.8. Detection of Chymotrypsin-like Elastase 3B (CELA3B) by Immunoblotting
3. Results
3.1. Enzymes Present in Human Stool Cleave the A Subunit of Stx2a In Vitro
3.2. Serine Protease(s) in Human Stool Are Responsible for the Cleavage of the A Subunit of Stx2a
3.3. Localization of the Cleavage Position of the A subunit of Stx2a Induced by Different Purified Enzymes or Components Present in Stool from Healthy Human Donors
3.4. Chymotrypsin-like Elastase 3B (CELA3B) Induces Partial Cleavage of A Subunit of Stx2a In Vitro
3.5. Stool of Healthy Donors Appear to Contain CELA3B
3.6. Stool from EHEC-Infected Patients Show a Similar Stx2a Cleavage Pattern
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kellnerová, S.; Huber, S.; Massri, M.; Fleischer, V.; Losso, K.; Sarg, B.; Kremser, L.; Talasz, H.; He, X.; Varrone, E.; et al. Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers. Microorganisms 2023, 11, 2487. https://doi.org/10.3390/microorganisms11102487
Kellnerová S, Huber S, Massri M, Fleischer V, Losso K, Sarg B, Kremser L, Talasz H, He X, Varrone E, et al. Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers. Microorganisms. 2023; 11(10):2487. https://doi.org/10.3390/microorganisms11102487
Chicago/Turabian StyleKellnerová, Sára, Silke Huber, Mariam Massri, Verena Fleischer, Klemens Losso, Bettina Sarg, Leopold Kremser, Heribert Talasz, Xiaohua He, Elisa Varrone, and et al. 2023. "Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers" Microorganisms 11, no. 10: 2487. https://doi.org/10.3390/microorganisms11102487
APA StyleKellnerová, S., Huber, S., Massri, M., Fleischer, V., Losso, K., Sarg, B., Kremser, L., Talasz, H., He, X., Varrone, E., Brigotti, M., Ardissino, G., Orth-Höller, D., & Würzner, R. (2023). Enzymatic Cleavage of Stx2a in the Gut and Identification of Pancreatic Elastase and Trypsin as Possible Main Cleavers. Microorganisms, 11(10), 2487. https://doi.org/10.3390/microorganisms11102487