Next Article in Journal
Antioxidant-Based Medicinal Properties of Stingless Bee Products: Recent Progress and Future Directions
Previous Article in Journal
Preclinical Efficacy and Safety of an Anti-Human VEGFA and Anti-Human NRP1 Dual-Targeting Bispecific Antibody (IDB0076)
Open AccessArticle

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

1
Center for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, Singapore
2
Departments of Chemistry and Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA
*
Author to whom correspondence should be addressed.
G.S. and S.G. contributed equally to this work.
Biomolecules 2020, 10(6), 922; https://doi.org/10.3390/biom10060922
Received: 8 May 2020 / Revised: 6 June 2020 / Accepted: 14 June 2020 / Published: 18 June 2020
Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed. View Full-Text
Keywords: LPS; autoproteolysis; OmpT; heat modifiable protein; omptin family; outer membrane protease; E. coli LPS; autoproteolysis; OmpT; heat modifiable protein; omptin family; outer membrane protease; E. coli
Show Figures

Figure 1

MDPI and ACS Style

Sinsinbar, G.; Gudlur, S.; Metcalf, K.J.; Mrksich, M.; Nallani, M.; Liedberg, B. Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding. Biomolecules 2020, 10, 922.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Search more from Scilit
 
Search
Back to TopTop