Bi-functional peptides with both trypsin-inhibitory and antimicrobial activities are frequent defensive molecules in Ranidae amphibian skins

Abstract

Amphibian skins act as the first line against noxious aggression by microorganisms, parasites, and predators. Anti-microorganism activity is an important task of amphibian skins. A large amount of gene-encoded antimicrobial peptides (AMPs) has been identified from amphibian skins. Only a few of small protease inhibitors have been found in amphibian skins. From skin secretions of 5 species (Odorrana livida, Hylarana nigrovittata, Limnonectes kuhlii, Odorrana grahami, and Amolops loloensis) of Ranidae frogs, 16 small serine protease inhibitor peptides have been purified and characterized. They have lengths of 17-20 amino acid residues (aa). All of them are encoded by precursors with length of 65-70 aa. These small peptides show strong trypsin-inhibitory abilities. Some of them can exert antimicrobial activities. They share the conserved GCWTKSXXPKPC fragment in their primary structures, suggesting they belong to the same families of peptide. Signal peptides of precursors encoding these serine protease inhibitors share obvious sequence similarity with those of precursors encoding AMPs from Ranidae frogs. The current results suggest that these small serine protease inhibitors are the common defensive compounds in frog skin of Ranidae as amphibian skin AMPs.

pLR‐HL: A Novel Amphibian Bowman–Birk‐type Trypsin Inhibitor from the Skin Secretion of the Broad‐folded Frog, Hylarana latouchii

“…The majority of BBI inhibitors are characterized by possession of a highly conserved canonical disulphide loop consisting of nine residues (CTP 1 SXPPXC, P 1 represents the reactive site residue) forming a short β ‐sheet . So far, several BBI‐like inhibitors of proteases have been identified in some species of ranid frogs such as Odorrana livida , Hylarana nigrovittata , Limnonectes kuhlii , Odorrana grahami and Amolops loloensis . Some of the BBI‐like inhibitors, like ORB from the skin secretion of Odorrana grahami and Ranacyclin‐T found in Rana temporaria , possessed bifunctions with both trypsin inhibitory and antimicrobial activities, while some of them, such as ORB2‐K (analogue of ORB) and pLR‐HL discovered in this study, displayed only trypsin inhibitory activity (Table ).…”

Atomic resolution crystal structure of HV-BBI protease inhibitor from amphibian skin in complex with bovine trypsin

“…We (as well as others) have recently demonstrated that certain inhibitors derived from amphibian skin secretions have a dual role possessing both antimicrobial and protease inhibitory activities . Since such compounds can be regarded as promising candidates for novel antibiotics, the further characterization of their properties is most interesting.…”

“…Since such compounds can be regarded as promising candidates for novel antibiotics, the further characterization of their properties is most interesting. It was demonstrated that synthetic disulfide‐bridged protease‐binding loops designed based on an inhibitor secreted by an oriental frog Odorrana graham (ORB) retain both the antimicrobial and trypsin inhibitory activities . Based on these findings, it became evident that the inhibitor binding loop plays an essential role in both functions.…”

“…We (as well as others) have recently demonstrated that certain inhibitors derived from amphibian skin secretions have a dual role possessing both antimicrobial and protease inhibitory activities. 9, [15][16][17] Since such compounds can be regarded as promising candidates for novel antibiotics, the further characterization of their properties is most interesting. It was demonstrated that synthetic disulfide-bridged protease-binding loops designed based on an inhibitor secreted by an oriental frog Odorrana graham (ORB) retain both the antimicrobial and trypsin inhibitory activities.…”

“…We have focused on the inhibitory and antimicrobial activities of the HV-BBI peptide from the Chinese Bamboo odorous frog Huia versabilis, demonstrating that the inhibitory activity is retained in truncated variants of the peptide. 17 To further analyze the protease inhibitory properties of the peptide, in this article we describe the crystal structure of a truncated variant of HV-BBI (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in complex with trypsin at 1 Å resolution.…”