title: Identification and molecular cloning of a novel amphibian Bowman Birk-type trypsin inhibitor from the skin of the Hejiang Odorous Frog; Odorrana hejiangensis.
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.
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Medical Biology Center, Queen's University Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Tianbao
authors.initials: T
authors.lastname: Chen
authors.forename: Brian
authors.initials: B
authors.lastname: Walker
authors.forename: Mei
authors.initials: M
authors.lastname: Zhou
authors.forename: Dayuan
authors.initials: D
authors.lastname: Sui
authors.forename: J Michael
authors.initials: JM
authors.lastname: Conlon
authors.forename: Chris
authors.initials: C
authors.lastname: Shaw
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011898:Ranidae; D020539:Sequence Analysis, Protein; D012867:Skin / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D029845:Amphibian Proteins; D000900:Anti-Bacterial Agents; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D014357:Trypsin