title: pLR-HL: A Novel Amphibian Bowman-Birk-type Trypsin Inhibitor from the Skin Secretion of the Broad-folded Frog, Hylarana latouchii.
abstract: In this study, we report a novel heptadecapeptide (LIGGCWTKSIPPKPCLV) of the pLR/ranacyclin family, named pLR-HL, whose structure was deduced from its biosynthetic precursor-encoding cDNA cloned from the skin secretion-derived cDNA library of the broad-folded frog, Hylarana latouchii, by employing a 'shotgun' cloning technique. It contains a disulphide loop between Cys(5) and Cys(15) which is consistent with Bowman-Birk-type protease inhibitors. The primary structure of pLR-HL deduced from the cDNA sequence was confirmed by fractionating the skin secretion using reverse-phase HPLC and subsequent analysis using MALDI-TOF mass spectrometry and LC/MS/MS fragmentation sequencing. On the basis of the establishment of unequivocal amino acid sequence, a synthetic replicate was synthesized by solid-phase Fmoc chemistry, and it displayed a moderately potent trypsin inhibition with a Ki of 143 nm. The substitution of Lys-8 by Phe (Phe(8) -pLR-HL) resulted in abolition of trypsin inhibition but generation of modest inhibition on chymotrypsin with a Ki of 2.141 μm. Additionally, both the disulphide loops of pLR-HL and Phe(8) -pLR-HL were synthesized and tested. Both of the catalytic loops retained similar inhibitory potencies towards trypsin or chymotrypsin in comparison with the original intact molecules. Thus, the replacement of reactive site residues could alter the specificity of these protease inhibitors, while the canonical reactive loop alone can independently constitute biologically active moiety.
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, Belfast, Northern Ireland, BT9 7BL, UK.|Key Laboratory for Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Haiying
authors.initials: H
authors.lastname: Hang
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, Belfast, Northern Ireland, BT9 7BL, UK.
authors.forename: Tianbao
authors.initials: T
authors.lastname: Chen
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, Belfast, Northern Ireland, BT9 7BL, UK.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhou
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, Belfast, Northern Ireland, BT9 7BL, UK.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, Belfast, Northern Ireland, BT9 7BL, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D016366:Open Reading Frames; D011898:Ranidae / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D014361:Trypsin Inhibitors
keywords: amphibian; mass spectrometry; molecular cloning; peptide; protease inhibitor