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

Abstract Peptidase 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 C-X (7) -C-X (6,7) -C-X (6,7) -Y-X (3) -C-X (2) -C-X (15-21) -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 peptidase inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions.