Open AccessA Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagii
Author(s) -
Gebhard Leopoldo G.,
Carrizo Federico U.,
Stern Ana L.,
Burgardt Noelia I.,
Faivovich Julián,
Lavilla Esteban,
Ermácora Mario R.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04127.x
Subject(s) - prolyl endopeptidase , endopeptidase , biochemistry , serine protease , chymotrypsin , protease , serine proteinase inhibitors , trypsin , biology , chemistry , enzyme , microbiology and biotechnology
Searching for bioactive peptides, we analyzed acidic extracts of Phyllomedusa sauvagii skin and found two new proteins, PSKP‐1 and PSKP‐2, of 6.7 and 6.6 kDa, respectively, which, by sequence homology, belong to the Kazal family of serine protease inhibitors. PSKP‐1 and PSKP‐2 exhibit the unprecedented feature of having proline at P 1 and P 2 positions. A gene encoding PSKP‐1 was synthesized and expressed in Escherichia coli . Recombinant PSKP‐1 was purified from inclusion bodies, oxidatively refolded to the native state, and characterized by chemical, hydrodynamic and optical studies. PSKP‐1 shows inhibitory activity against a serum prolyl endopeptidase, but is unable to inhibit trypsin, chymotrypsin, V8 protease, or proteinase K. In addition, PSKP‐1 can be rendered active against trypsin by active‐site site‐specific mutagenesis, has bactericidal activity, and induces agglutination of red cells at micromolar concentrations. PSKP‐1 might protect P. sauvagii teguments from microbial invasion, by acting as an inhibitor of an as‐yet unidentified prolyl endopeptidase or directly as a microbicidal compound.