Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis

In this study, an amphibian (Odorrana hejiangensis) skin extract was fractionated by reverse phase HPLC and fractions were screened for trypsin inhibitory activity. Using this initial approach, a novel trypsin inhibitory peptide was detected with an apparent protonated molecular mass of 1804.83 Da, as determined by MALDI-TOF mass spectrometry. It was named Hejiang trypsin inhibitor (HJTI) in accordance. The primary structure of the biosynthetic precursor of HJTI was deduced from a cDNA sequence cloned from a skin-derived cDNA library. The primary structure of the encoded predicted mature active peptide was established as: GAPKGCWTKSYPPQPCS (non-protonated monoisotopic molecular mass – 1802.81 Da). On the basis of this unequivocal amino acid sequence, a synthetic replicate was synthesized by solid phase Fmoc chemistry. This replicate displayed a moderately potent trypsin inhibition with a Ki of 388 nM. Bioinformatic analysis of the primary structure of this peptide indicated that it was a member of the Bowman-Birk family of protease inhibitors. The substitutions of Gln-14 and Ser-17 by Lys, resulted in an increase in cationicity and a small increase in potency to a Ki value of 218 nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.

► The manuscript describes a novel Bowman Birk type trypsin inhibitor from the skin of a rare Chinese frog.
► This peptide was devoid of antimicrobial activity and an enhanced cationic analog was only weakly active against E. coli.
► Antimicrobial experiments were carried out in two different labs and the data were in accordance.
► Amphibian skin Bowman Birk protease inhibitors are the first of their class to be found outside the Plant Kingdom.