pLR-HL: A Novel Amphibian Bowman-Birk-type Trypsin Inhibitor from the Skin Secretion of the Broad-folded Frog, Hylarana latouchii.
Chem Biol Drug Des
; 87(1): 91-100, 2016 Jan.
Article
en En
| MEDLINE
| ID: mdl-26228512
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ranidae
/
Piel
/
Inhibidores de Tripsina
Límite:
Animals
Idioma:
En
Revista:
Chem Biol Drug Des
Asunto de la revista:
BIOQUIMICA
/
FARMACIA
/
FARMACOLOGIA
Año:
2016
Tipo del documento:
Article
País de afiliación:
Reino Unido