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

Min Wang, Lei Wang, Tianbao Chen, Brian Walker, Mei Zhou, Dayuan Sui, J. Michael Conlon, Chris Shaw

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)

Abstract

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 K i 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 K i value of 218 nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity.

Original languageEnglish
Pages (from-to)245-250
Number of pages6
JournalPeptides
Volume33
Issue number2
DOIs
Publication statusPublished - Feb 2012
Externally publishedYes

Keywords

  • Amphibian
  • Cloning
  • Inhibitor
  • Peptide
  • Protease
  • Skin

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Cellular and Molecular Neuroscience

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