Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding
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 <i>E. coli</i> against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own se...
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doaj-d3ecdef074734e11a2d1b3163cf176e42020-11-25T02:25:06ZengMDPI AGBiomolecules2218-273X2020-06-011092292210.3390/biom10060922Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro RefoldingGaurav Sinsinbar0Sushanth Gudlur1Kevin J Metcalf2Milan Mrksich3Madhavan Nallani4Bo Liedberg5Center for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, SingaporeCenter for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, SingaporeDepartments of Chemistry and Biomedical Engineering, Northwestern University, Evanston, IL 60208, USADepartments of Chemistry and Biomedical Engineering, Northwestern University, Evanston, IL 60208, USACenter for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, SingaporeCenter for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, SingaporeOuter 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 <i>E. coli</i> 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.https://www.mdpi.com/2218-273X/10/6/922LPSautoproteolysisOmpTheat modifiable proteinomptin familyouter membrane protease |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Gaurav Sinsinbar Sushanth Gudlur Kevin J Metcalf Milan Mrksich Madhavan Nallani Bo Liedberg |
spellingShingle |
Gaurav Sinsinbar Sushanth Gudlur Kevin J Metcalf Milan Mrksich Madhavan Nallani Bo Liedberg Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding Biomolecules LPS autoproteolysis OmpT heat modifiable protein omptin family outer membrane protease |
author_facet |
Gaurav Sinsinbar Sushanth Gudlur Kevin J Metcalf Milan Mrksich Madhavan Nallani Bo Liedberg |
author_sort |
Gaurav Sinsinbar |
title |
Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding |
title_short |
Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding |
title_full |
Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding |
title_fullStr |
Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding |
title_full_unstemmed |
Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding |
title_sort |
role of lipopolysaccharide in protecting ompt from autoproteolysis during in vitro refolding |
publisher |
MDPI AG |
series |
Biomolecules |
issn |
2218-273X |
publishDate |
2020-06-01 |
description |
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 <i>E. coli</i> 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. |
topic |
LPS autoproteolysis OmpT heat modifiable protein omptin family outer membrane protease |
url |
https://www.mdpi.com/2218-273X/10/6/922 |
work_keys_str_mv |
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