Molecular identification, characterization, and expression analysis of a novel trypsin inhibitor-like cysteine-rich peptide from the cotton bollworm, Helicoverpa armigera (H\xfcbner) (Lepidoptera: Noctuidae)

Muhammad Shakeel,Junaid Zafar

doi:10.1186/s41938-020-0208-7

Muhammad Shakeel, Junaid Zafar

Open Access

https://doi.org/10.1186/s41938-020-0208-7

Copy DOI

Abstract

Trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors have been reported to inhibit proteases such as trypsin, cathepsin, elastase, and chymotrypsin, and thus play a critical role in several physiological processes. However, the information about TIL peptides in insects is limited. In the present study, a novel cysteine-rich trypsin inhibitor-like protease, designated as HaTIL2, was isolated from the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). The cDNA sequence of HaTIL2 is 470 nucleotides long, with 240 nucleotides open reading frame encoding 80 amino acid residues. The analysis of genomic DNA revealed that the full-length genomic DNA sequence of HaTIL2 was 574 bp with two exons and one intron. The predicted molecular weight of HaTIL2 is 8.632 kDa, with an isoelectric point of 4.41. The results of neighbor-joining tree demonstrated that HaTIL2 was closely related to H. armigera TIL3, DmCEI, and DsCtAPI followed by TcIMI and MdCEI. The mRNA of HaTIL2 was constitutively expressed at different levels in different stages of H. armigera. The HaTIL2 showed a high expression on different days of the pupal stage, which revealed that HaTIL2 might play a vital role during the pupal stage. Although the detailed function of HaTIL2 needs to be elucidated, the obtained results are of particular importance to open up new avenues of research into the functional studies of insect peptidase inhibitors.

Highlights

Protease inhibitors are classified either by the type of proteases they inhibit, such as serpins, cystatins, and metalloproteases, or according to the presence of structural motifs such as Kunitz, Kasal, or Bowman-Birk (Song et al 2008)
Considering the importance of trypsin inhibitorlike domain (TIL) domain cysteine-rich peptides in physiological processes, as exhibited by previous reports, the present study aimed to identify and describe the bioinformatic analysis of a cysteine-rich peptide of H. armigera including cloning, isolation of genomic Deoxyribonucleic acid (DNA), and investigation of the expression pattern in the fat body of H. armigera, especially at the pupal stage
Identification of HaTIL2 Complementary deoxyribonucleic acid (cDNA) and nucleotide sequencing The sequence-specific primers were designed in accordance with the length of the cDNA sequence of HaTIL2 to amplify the open reading frame (ORF) (Table 1)

Summary

Introduction

Protease inhibitors are classified either by the type of proteases they inhibit, such as serpins, cystatins, and metalloproteases, or according to the presence of structural motifs such as Kunitz, Kasal, or Bowman-Birk (Song et al 2008). Peptides containing trypsin inhibitorlike domain (TIL) are usually comprised of 56–84 amino residues and known to play a key role in the various physiological processes by inhibiting proteinases (Nie et al 2012). TIL-type protease contains a single TIL domain with 10 cysteine residues, which form 5. Lepidobatrachus laevis, a TIL domain cysteine-rich peptide was isolated from the skin and showed inhibitory activity against trypsin (Wang et al 2015). In A. cerana, it was identified that the recombinant AcCI demonstrated inhibitory activity against chymotrypsin (Kim et al 2013). It has been reported that in Bombyx mori, mutations at the second and sixth cysteine residue dramatically reduced the activity of inhibition against microbial proteases (Li et al 2016). Though a couple of studies are available on TIL domain-containing cysteinerich peptides from insects, until now, to the best of our knowledge, there is no information available for such kind of peptides in H. armigera

Methods

Results

Conclusion