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 DH5␣ 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°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. LineweaverBurk plot analysis indicated increased K m 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.Metalloproteases, in which zinc is an essential metal ion for catalytic activity, are elaborated by various human pathogenic bacteria, as well as by nonpathogenic ones (5). They can be classified into three families: thermolysins, serralysins, and neurotoxins (7). The members of the thermolysin family are synthesized as inactive precursors, with a large N-terminal propeptide acting as a specific inhibitor for the mature protease and/or an intramolecular chaperone to control the folding of the protease (10,14,20,24). Since the N-terminal propeptide is cleaved by autoproteolysis (9, 13, 24), a transformant carrying a cloned metalloprotease gene also produces a correctly processed metalloprotease (2, 13). On the other hand, studies of metalloproteases from vibrios (4,6,15) have demonstrated that, although these also belong to the thermolysin family, their precursors have C-terminal propeptides as well as N-terminal ones. The biochemical function of the Cterminal propeptide is currently unknown.Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia (8,22). Like other vibrios, this pathogen secretes the 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) since it belongs to the thermolysin family (18). VVP has been reported to have many biological functions: proteolytic degradation of a wide variety of host proteins, such as plasma proteins, involved in coagulation or complement action, induction of hemorrhagic tissue damage, and enhancement of vascular permeability through the generation of inflammatory mediators (18). Recently, ...

-S-V-S-A-N-N-V-T-N-N-N-E-T.This sequence is identical from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. mimicus hemolysin.

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