Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system

Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide.

© 2018 Elsevier Inc. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/