A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity

Abstract

Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 μM. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin.Graphical Abstract

The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection

“…In the immunity category, five CDSs were upregulated by infection: one peptidoglycan receptor protein (PGRP; Ambaur-18924), two α-macroglobulins (AmbarSigP-61895 and AmbarSigP-5190), and two antimicrobial peptides (AMPs) similar to the microplusin of R. microplus (Fogaca et al, 2004) (Ambaur-69859 and Ambaur-25218) (Table 1). Moreover, one CDS of a trypsin-inhibitor like (TIL) (Ambaur-46428), a type of protease inhibitor previously reported to exhibit antimicrobial activity (Fogaca et al, 2006; Wang et al, 2015), was also upregulated by R. rickettsii . Conversely, one microplusin (AmbarSigP-22173) and two 5.3 kDa antimicrobial peptides (Ambaur-50152 and Ambaur-19862), were downregulated in infected ticks in relation to the control (Supplementary Table 1 and Table 1).…”

“…Interestingly, the knockdown of α2-macroglobulins mediated by RNAi diminished the phagocytosis of Chryseobacterium indologenes by the hemocytes of I. ricinus (Buresova et al, 2011). Additionally, the transcriptional levels of one trypsin-inhibitor like (TIL) encoding sequence (Ambaur-46428), a type of protease inhibitors that exhibit antimicrobial activity (Fogaca et al, 2006; Wang et al, 2015), was also higher in infected A. aureolatum ticks.…”

Analysis of the Salivary Gland Transcriptome of Unfed and Partially Fed Amblyomma sculptum Ticks and Descriptive Proteome of the Saliva

“…The annotation for the protein encoded by the CDS Acaj-56179 in protein domains databases (Pfam ID 01826 and UniprotKB/Swiss-Prot ID P83516 , Supplementary Table 1 ) shows that it possesses the key features of TIL domain containing proteins. TIL domain is typically composed of five disulfide bonds formed by 10 cysteine residues in a stretch of approximately 54 amino acid residues (Bania et al, 1999 ), necessary for their biological properties, which include both antimicrobial and serine protease inhibitory activities (Fogaça et al, 2006 ; Wang et al, 2015 ). The MSA analysis of the amino acid sequence deduced from the CDS Acaj-56179 with similar sequences of other arachnids (Figure 3A ) illustrated the highly conserved feature of the 10 cysteine residue positions among the sequences of hard ticks (family Ixodidae).…”

Trypsin-like Inhibitor Domain (TIL)-Harboring Protein Is Essential for Aedes aegypti Reproduction

“…In humans, a TIL domain-containing protein, Von Willebrand factor, is translated as a massive 2818-aa protein and plays a key role in blood clotting [ 30 , 31 ]. At the same time, in the frog Lepidobatrachus laevis , a small 55-aa peptide containing the same TIL domain has protease-inhibiting activity [ 32 ]. These examples highlight the fact that despite the presence of a conserved TIL domain, there is functional and spatial diversity in TIL-containing proteins.…”