Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis |
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Authors: | Wang Min Wang Lei Chen Tianbao Walker Brian Zhou Mei Sui Dayuan Conlon J Michael Shaw Chris |
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Institution: | Natural Drug Discovery Group, School of Pharmacy, Medical Biology Center, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK. |
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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--1802.81Da). 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 218nM. Neither HJTI nor its synthetic analog, possessed any significant antimicrobial activity. |
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Keywords: | Amphibian Skin Peptide Protease Inhibitor Cloning |
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