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.

Identification and pharmaceutical evaluation of novel frog skin-derived serine proteinase inhibitor peptide–PE-BBI (Pelophylax esculentus Bowman-Birk inhibitor) for the potential treatment of cancer

“…Interestingly, these peptides have highly similar primary structure but distinct bio-functions. Although many structurally similar trypsin inhibitor peptides have been found in the past years, until now, none of them showed any anticancer activity 28 , 30 – 32 . Leguminous BBIs have been identified with anti-cancer activity against human prostate, colon, breast, and skin cancer cell lines 33 .…”

“…In the complex form, BTCI can inhibit the trypsin-like proteolytic activity of the 20S proteasome, thereby inducing cytostatic and cytotoxic effects in MCF-7 breast cancer cells by induction of apoptosis 35 .

Figure 6 Comparison of different frog skin derived Bowman-Birk-type trypsin inhibitors 18 , 28 – 30 , 32 . “-” means no effect.

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“… Comparison of different frog skin derived Bowman-Birk-type trypsin inhibitors 18 , 28 – 30 , 32 . “-” means no effect.…”

“…Comparing the primary structure between PE-BBI and other frog skin-derived proteinase inhibitor peptides (Fig. 6 ) 18 , 28 – 30 , 32 , all of these peptides contained the same disulphide-bridged loop structure but differ entirely at the N-terminal and C-terminal extension amino acid residues. PE-BBI contains more lysine (K) and amidated C-terminal, which means PE-BBI has a more positive charge than pLR-HL and HV-BBI.…”

“…Each sample was carried out in triplicate and repeated at least three times. The fluorescent strength values were used to calculate the initial rate (vi) and inhibition constants (K i ) 32 .…”