Byung Rae Jin

Molecular cloning and characterization of a short peptidoglycan recognition protein (PGRP-S) with antibacterial activity from the bumblebee Bombus ignitus

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique bacterial cell wall component. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus (BiPGRP-S). The BiPGRP-S gene consists of four exons that encode 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares a high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in both the fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that the BiPGRP-S gene is highly induced in both the fat body and the epidermis after an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to Bacillus megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. After B. thuringiensis injection, the expression profiles of four antibacterial peptide genes in the fat body of RNA interference (RNAi)-mediated BiPGRP-S-knock-down B. ignitus worker bees was similar to that of control worker bees, indicating that BiPGRP-S does not affect the activation of antibacterial peptide gene expression. These results indicate that BiPGRP-S is an inducible protein that may function as an amidase-type PGRP-S during the immune response against Bacillus bacteria.

Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme

In this study, we describe the cloning and characterization of a Prx from the common cutworm Spodoptera litura (SlPrx5). The SlPrx5 cDNA contains an open reading frame of 477 bp encoding a predicted protein of 159 amino acid residues, 16.902 kDa, and an isoelectric point of 7.68. Furthermore, the deduced amino acid sequence of the SlPrx5 cDNA showed 86% identity to Papilio xuthus Prx5, 72% to Aedes aegypti Prx5, and 64-67% to other insect Prxs. A phylogenetic analysis further revealed that the deduced amino acid sequence of SlPrx5 groups within the atypical 2-Cys Prx cluster. Recombinant SlPrx5 (20 kDa) purified from baculovirus-infected insect cells was found to reduce H2O2 in the presence of electrons donated by dithiothreitol and protect super-coiled DNA from damage by metal-catalyzed oxidation in vitro. During S. litura development, SlPrx5 is constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the fat body and midgut. Additionally, SlPrx5 mRNA expression was up-regulated after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. A disc diffusion assay indicated that recombinant SlPrx5 can play a functional role in protecting cells from oxidative stress in vivo. These results provide insight into the role of SlPrx5 during development and the oxidative stress response of S. litura.

Identification and functional characterization of an epsilon glutathione S-transferase from the beet armyworm (Spodoptera exigua)

Abstract In the present study, an epsilon glutathione S -transferase from the beet armyworm Spodoptera exigua (SeGSTe) was cloned and characterized. The SeGSTe gene consists of 746xa0bp full-length cDNA with a 669xa0bp open reading frame encoding a predicted protein of 223 amino acid residues weighing 24.517xa0kDa and an isoelectric point of 6.44. Furthermore, sequence analysis of SeGSTe showed 90% sequence identity to Spodoptera litura GSTe2 and 47–36% to other insect GSTs. Phylogenetic relationship analysis further revealed that SeGSTe is a novel member of the Epsilon-class of GSTs. During S. exigua development, SeGSTe is expressed in the midgut of the prepupa stage but not in the epidermis and fat body. Recombinant SeGSTe (25xa0kDa) purified from baculovirus-infected insect cells was able to protect super-coiled DNA from oxidative damage. It was also able to catalyze the conjugation of both 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB), but not 4-nitrobenzyl chloride (4-NBC) and 4-nitrophenethyl bromide (4-NPB), to glutathione (GSH). Additionally, recombinant SeGSTe exhibited an optimal pH of 6.0 and an optimal temperature of 40xa0°C. The enzyme activity of recombinant SeGSTe was sensitive to heavy metals Cu 2xa0+ and Cd 2xa0+ and was significantly inhibited by various kinds of pesticides including insecticide, herbicide, and fungicide. Taken together, these findings suggest that the SeGSTe may be involved in the detoxification of pesticides and protecting S. exigua against oxidative stress, which provides a molecular basis for understanding the mechanisms of insecticide resistance in S. exigua .

A spider (Araneus ventricosus) chymotrypsin inhibitor that acts as an elastase inhibitor and a microbial serine protease inhibitor

Spider-derived Kunitz-type serine protease inhibitors have been shown to exhibit plasmin and elastase inhibition activity and potassium channel blocking activity, but thus far, no additional roles for spider-derived chymotrypsin inhibitors have been elucidated. In this study, a spider (Araneus ventricosus) chymotrypsin inhibitor (AvCI) that acts as an elastase inhibitor and a microbial serine protease inhibitor was identified. AvCI is a 70-amino acid mature peptide that displays eight conserved cysteine residues and a P1 lysine residue. Recombinant AvCI expressed in baculovirus-infected insect cells demonstrated inhibitory activity against chymotrypsin (Ki 49.85 nM), but not trypsin, which defines a role for AvCI as a spider-derived chymotrypsin inhibitor. AvCI also exhibited inhibitory activity against microbial serine proteases such as subtilisin A (Ki 20.51 nM) and proteinase K (Ki 65.42 nM). Furthermore, AvCI exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or plasmin; however, AvCI strongly inhibited human neutrophil elastase (Ki 8.74 nM) and porcine pancreatic elastase (Ki 11.32 nM), indicating that AvCI acts as an anti-elastolytic factor. These findings constitute molecular evidence that AvCI acts as an inhibitor against chymotrypsin, microbial serine proteases, and elastases. This paper provides a novel view of the functions of a spider-derived chymotrypsin inhibitor.

Developmental regulation and antifungal activity of a growth-blocking peptide from the beet armyworm Spodoptera exigua

Insect cytokine growth-blocking peptides (GBPs) are involved in growth regulation and the innate immune response. However, the microbial binding and antimicrobial activities of GBPs remain unclear. Here, we investigate the developmental role and antifungal activity of a GBP from the beet armyworm Spodoptera exigua (SeGBP). Sequence analysis predicted that mature SeGBP consists of 24 amino acid residues, including 2 cysteine residues. During S. exigua development, SeGBP is constitutively expressed in the fat body during the larval and adult stages but not in pupae. SeGBP expression is up-regulated by 20-hydroxyecdysone and down-regulated by juvenile hormone analog. Recombinant SeGBP purified from baculovirus-infected insect cells retards the growth of S. exigua larvae. Additionally, SeGBP expression is acutely induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. Recombinant SeGBP can bind to B. bassiana but not to E. coli or B. thuringiensis. Consistent with these findings, SeGBP shows antifungal activity against B. bassiana. Therefore, these results provide insight into the role of SeGBP during the innate immune response following microbial infection, and furthermore, they suggest a novel function for SeGBP as a direct antifungal agent against entomopathogenic fungi, such as B. bassiana.

Comparative study of two thioredoxins from common cutworm (Spodoptera litura): cloning, expression, and functional characterization.

Thioredoxins (Trxs) are a ubiquitous family of antioxidant enzymes that are involved in protecting organisms against various oxidative stresses. Here, we cloned and characterized two thioredoxins, named SlTrx1 and SlTrx2, from the common cutworm Spodoptera litura. SlTrx1 and SlTrx2, respectively, consist of 988 and 606 bp full-length cDNA with 318 and 447 bp open reading frames encoding 106 and 149 amino acid residues. Furthermore, the N-terminal region of SlTrx2 contains a predicted mitochondrial localization signal (33 amino acids). A phylogenetic relationship analysis revealed that SlTrx1 is in the cytosolic thioredoxin Trx1 cluster, whereas SlTrx2 is in the mitochondrial thioredoxin Trx2 cluster. Recombinant SlTrx1 (14 kDa) and SlTrx2 (16 kDa), expressed in baculovirus-infected insect Sf9 cells, demonstrated insulin disulfide reductase activity at the same optimum temperature and pH value of 35 °C and 7.0, respectively, in vitro. During S. litura development, we found that SlTrx1 and SlTrx2 had similar transcript expression patterns and were constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the epidermis and midgut. In addition, both SlTrx1 and SlTrx2 mRNA were up-regulated in S. litura after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. These results suggest that SlTrx1 and SlTrx2 function as potent antioxidant enzymes, and provide a molecular basis for the roles SlTrx1 and SlTrx2 during development and the oxidative stress response of S. litura.

SeGSTo, a novel glutathione S-transferase from the beet armyworm (Spodoptera exigua), involved in detoxification and oxidative stress

Members of the glutathione S-transferase superfamily can protect organisms against oxidative stress. In this study, we characterized an omega glutathione S-transferase from Spodoptera exigua (SeGSTo). The SeGSTo gene contains an open reading frame (ORF) of 744 nucleotides encoding a 248-amino acid polypeptide. The predicted molecular mass and isoelectric point of SeGSTo are 29007xa0Da and 7.74, respectively. Multiple amino acid sequence alignment analysis shows that the SeGSTo sequence is closely related to the class 4 GSTo of Bombyx mori BmGSTo4 (77xa0% protein sequence similarity). Homologous modeling and molecular docking reveal that Cys35 may play an essential role in the catalytic process. Additionally, the phylogenetic tree indicates that SeGSTo belongs to the omega group of the GST superfamily. During S. exigua development, SeGSTo is expressed in the midgut of the fifth instar larval stage, but not in the epidermis or fat body. Identification of recombinant SeGSTo via SDS-PAGE and Western blot shows that its molecular mass is 30xa0kDa. The recombinant SeGSTo was able to protect super-coiled DNA from damage in a metal-catalyzed oxidation (MCO) system and catalyze the 1-chloro-2,4-dinitrobenzene (CDNB), but not 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenethyl bromide (4-NPB), or 4-nitrobenzyl chloride (4-NBC). The optimal reaction pH and temperature were 8 and 50xa0°C, respectively, in the catalysis of CDNB by recombinant SeGSTo. The mRNA expression of SeGSTo was up-regulated by various oxidative stresses, such as CdCl2, CuSO4, and isoprocarb, and the catalytic activity of recombinant SeGSTo was noticeably inhibited by heavy metals (Cu2+ and Cd2+) and various pesticides. Taken together, these results indicate that SeGSTo plays an important role in the antioxidation and detoxification of pesticides.

Functional characterization of the aspartic proteinase cathepsin D in the beet armyworm (Spodoptera exigua)

In insects, proteolytic enzymes are involved in food digestion and the metamorphosis process. In the present study, the full-length cDNA of an aspartic proteinase, Spodoptera exigua cathepsin D (SeCatD), was cloned, and its functions in metamorphosis were characterized. SeCatD contains an open reading frame of 1152 nucleotides, encoding a 384-amino acid polypeptide including a signal peptide and two functional domains (family A1 propeptide of amino acids (19-45) and a cathepsin D-like domain of 327 amino acids (55-381)). Three-dimensional structure analysis indicated that Asp66 and Asp251 may play important role in hydrolysis. Recombinant SeCatD was expressed in Sf9 insect cells and verified via SDS-PAGE and Western blot, the molecular mass of the expressed SeCatD was approximately 42kDa. The enzyme had an optimal pH value of 3 for activity. In addition, the tissue expression profile of SeCatD during metamorphosis was obtained, and the data demonstrated that SeCatD was expressed increasingly in the fat body and midgut, but not in the epidermis. Finally, injection of dsRNA-SeCatD into the fifth-instar larvae significantly reduced SeCatD expression and larvae survival rate compared to a dsRNA-GFP treatment. These data imply that SeCatD may function during metamorphosis and may represent a target for insect control.

A rapid method for titration of ascovirus infectivity

Ascoviruses are a recently described family and the traditional plaque assay and end-point PCR assay have been used for their titration. However, these two methods are time-consuming and inaccurate to titrate ascoviruses. In the present study, a quick method for the determination of the titer of ascovirus stocks was developed based on ascovirus-induced apoptosis in infected insect cells. Briefly, cells infected with serial dilutions of virus (10-2-10-10) for 24u202fh were stained with trypan blue. The stained cells were counted, and the percentage of nonviable cells was calculated. The stained cell rate was compared between virus-infected and control cells. The minimum-dilution group that had a significant difference compared with control and the maximum-dilution group that had no significant difference were selected and then compared each well of the two groups with the average stained cell rate of control. The well was marked as positive well if the stained cell rate was higher than the average stained cell rate of control wells; otherwise, the well was marked as negative wells. The percentage of positive wells were calculated according to the number of positive. Subsequently, the virus titer was calculated through the method of Reed and Muench. This novel method is rapid, simple, reproducible, accurate, and less material-consuming and eliminates the subjectivity of the other procedures for titrating ascoviruses.