Novel Kazal-type Protease inhibitors from the skin secretion of the Splendid leaf frog, Cruziohyla calcarifer

Protease inhibitors have an important role controlling a variety of biological processes. Here, we employed a peptidomic approach including molecular cloning, tandem mass spectrometry and enzymatic assays to reveal 7 Kazal-type proteinase inhibitors (CCKPs) (18 variants) in the skin secretion of the unexplored frog, Cruziohyla calcarifer. All 18 proteins shared the Kazal pattern (CX(6-7)-CX(6-7)-Y-X(2-3)-CX(2)-C) and 3 disulphide bridges. Based on structural comparative analysis, we deemed trypsin and chymotrypsin inhibitory activity in CCKP-1, 4 and CCKP 2, 5, 7, respectively. These protease inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions.