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

Biomolecules

Sushanth Gudlur¹  Gaurav Sinsinbar¹  Bo Liedberg¹  Madhavan Nallani¹  Milan Mrksich²  KevinJ Metcalf² 

[1] Center for Biomimetic Sensor Science, School of Materials Science and Engineering, Nanyang Technological University, Singapore 637553, Singapore;Departments of Chemistry and Biomedical Engineering, Northwestern University, Evanston, IL 60208, USA;

关键词: LPS;    autoproteolysis;    OmpT;    heat modifiable protein;    omptin family;    outer membrane protease;   

DOI  :  10.3390/biom10060922

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown