A Kazal prolyl endopeptidase inhibitor isolated from the skin of Phyllomedusa sauvagii.
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.
Gebhard
Leopoldo G
LG
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 180 (B1876BXD), Bernal, Argentina.
Carrizo
Federico U
FU
Stern
Ana L
AL
Burgardt
Noelia I
NI
Faivovich
Julián
J
Lavilla
Esteban
E
Ermácora
Mario R
MR
D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology; D001001:Anura; D001483:Base Sequence; D002942:Circular Dichroism; D019295:Computational Biology; D008969:Molecular Sequence Data; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D012867:Skin / Q000737:chemistry; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry
D029845:Amphibian Proteins; D000900:Anti-Bacterial Agents; D015842:Serine Proteinase Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic