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Functional domains of a zinc metalloprotease from Vibrio vulnificus
S Miyoshi1, H Wakae, K Tomochika
1Faculty of Pharmaceutical Sciences, Okayama University, Tsushima-Naka, Japan. miyoshi@pheasant.pharm.okayama-u.ac.jp
Abstract:
Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, secretes a 45-kDa metalloprotease (V. vulnificus protease; VVP). A plasmid which carries the entire vvp gene subcloned into pBluescriptIIKS+ was transformed into Escherichia coli DH5alpha for overproduction of the protease. The 45-kDa recombinant protease (rVVP) was isolated from the periplasmic fraction of the transformant by ammonium sulfate precipitation followed by column chromatography on phenyl Sepharose. Biochemical characterization of the isolated rVVP showed that the recombinant protease was identical to that produced by V. vulnificus. When rVVP was incubated at 37 degrees C, a 35-kDa fragment was generated through autoproteolytic removal of the C-terminal peptide. This 35-kDa fragment (rVVP-N) was found to have sufficient proteolytic activity toward oligopeptides and soluble proteins but had markedly reduced activity toward insoluble proteins. Lineweaver-Burk plot analysis indicated increased Km values of rVVP-N for all of the protein substrates. rVVP, but not rVVP-N, was shown to agglutinate rabbit erythrocytes, bind to the erythrocyte ghosts, and digest the ghost membrane proteins. These results strongly suggest that rVVP (and VVP) consists of at least two functional domains: an N-terminal 35-kDa polypeptide mediating proteolysis and a C-terminal 10-kDa polypeptide which may be essential for efficient attachment to protein substrates and erythrocyte membranes.
Insights
Vibrio vulnificus protease (VVP) has two functional domains. The N-terminal domain mediates proteolysis, while the C-terminal domain aids in substrate attachment and erythrocyte membrane interaction.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Vibrio vulnificus is an opportunistic pathogen causing severe human infections.
- V. vulnificus secretes a 45-kDa metalloprotease (VVP) crucial for virulence.
- Understanding VVP's structure-function relationship is key to combating V. vulnificus infections.
Purpose of the Study:
- To overproduce and characterize the V. vulnificus protease (VVP).
- To investigate the functional domains of VVP and their roles in proteolytic activity and substrate interaction.
Main Methods:
- Subcloning the vvp gene into a pBluescriptIIKS+ plasmid for expression in Escherichia coli.
- Isolation and purification of recombinant VVP (rVVP) using ammonium sulfate precipitation and phenyl Sepharose chromatography.
- Biochemical characterization of rVVP, including autoproteolysis studies and kinetic analysis (Lineweaver-Burk plots).
- Assays for erythrocyte agglutination, binding to erythrocyte ghosts, and digestion of ghost membrane proteins.
Main Results:
- Recombinant VVP (rVVP) was successfully produced and purified, exhibiting identical biochemical properties to native VVP.
- Autoproteolysis generated a 35-kDa N-terminal fragment (rVVP-N) with retained proteolytic activity.
- rVVP-N showed reduced activity against insoluble proteins and increased Km values, indicating altered substrate specificity.
- rVVP, but not rVVP-N, agglutinated erythrocytes, bound to erythrocyte ghosts, and degraded ghost membrane proteins.
Conclusions:
- VVP comprises at least two functional domains: a 35-kDa N-terminal domain for proteolysis and a 10-kDa C-terminal domain for substrate/membrane interaction.
- The C-terminal domain is essential for efficient binding to protein substrates and erythrocyte membranes, contributing to VVP's pathogenic mechanisms.