title: A novel Kazal-type trypsin inhibitor from the skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas.
abstract: The chemical complexity of the defensive skin secretion of the red-eyed leaf frog, (Agalychnis callidryas), has not been elucidated in detail. During a systematic study of the skin secretion peptidomes of phyllomedusine frogs, we discovered a novel Kazal-type protein with potent trypsin inhibitory activity (Ki = 1.9 nM) that displays the highest degree of structural similarity with Kazal proteins from bony fishes. The protein was located in reverse-phase HPLC fractions following a screen of such for trypsin inhibition and subsequent partial Edman degradation of the peak active fraction derived the sequence: ATKPR-QYIVL-PRILRPV-GT. The molecular mass of the major component in this fraction was established by MALDI-TOF MS as 5893.09 Da. This partial sequence (assuming blank cycles to be Cys residues) was used to design a degenerate primer pool that was employed successfully in RACE-PCR to clone homologous precursor-encoding cDNA that encoded a mature Kazal protein of 52 amino acid residues with a computed molecular mass of 5892.82 Da. The protein was named A. callidryas Kazal trypsin inhibitor (ACKTI). BLAST analysis revealed that ACKTI contained a canonical Kazal motif (C-x(7)-C-x(6)-Y-x(3)-C-x(2,3)-C). This novel amphibian skin Kazal trypsin inhibitor adds to the spectrum of trypsin inhibitors of Kunitz- and Bowman Birk-type reported from this amphibian source.
authors.affiliation: Natural Drug Discovery Group, School of Pharmacy, Queen's University, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
authors.forename: Renjie
authors.initials: R
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Jiang
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Mei
authors.initials: M
authors.lastname: Zhou
authors.forename: Yingqi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Tianbao
authors.initials: T
authors.lastname: Chen
authors.forename: Chris
authors.initials: C
authors.lastname: Shaw
mesh_terms: D000595:Amino Acid Sequence; D029845:Amphibian Proteins / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001001:Anura; D001483:Base Sequence; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D012867:Skin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C574625:ACKTI protein, Agalychnis callidryas; D029845:Amphibian Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin