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pLR ‐ HL : A Novel Amphibian Bowman–Birk‐type Trypsin Inhibitor from the Skin Secretion of the Broad‐folded Frog, Hylarana latouchii
Author(s) -
Lin Yan,
Hang Haiying,
Chen Tianbao,
Zhou Mei,
Wang Lei,
Shaw Chris
Publication year - 2016
Publication title -
chemical biology and drug design
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.59
H-Index - 77
eISSN - 1747-0285
pISSN - 1747-0277
DOI - 10.1111/cbdd.12626
Subject(s) - trypsin , chemistry , protease , chymotrypsin , peptide sequence , stereochemistry , biochemistry , complementary dna , moiety , microbiology and biotechnology , enzyme , biology , gene
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 K i of 143 n m . 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 K i of 2.141 μ m . 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.