Purification and characterization of a novel defensin from the salivary glands of the black fly, Simulium bannaense

Lin, Wei; Mu, Lingli; Wang, Yipeng; Bian, Hui; Li, Jun; Lu, Yiling; Han, Yamei; Liu, Tong; Lv, Jing; Feng, Cui-Ping; Wu, Jing; Yang, Hailong

doi:10.1186/s13071-015-0669-9

Cited by 17 publications

(14 citation statements)

References 42 publications

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“…The solution NMR structures of papiliocin (59 % identity, PDB entry code 2LA2) from swallowtail butterfly of Papilio xuthus was selected as the template for homology modeling. The comparative 3D structure model of cecropin-TY1 was optimized using MODELLER and visualized using PYMOL software ( http://www.pymol.org/ ) [ 7 ].…”

Section: Methodsmentioning

confidence: 99%

A potent anti-inflammatory peptide from the salivary glands of horsefly

et al. 2015

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BackgroundA diverse group of physiologically active peptides/proteins are present in the salivary glands of horsefly Tabanus yao (Diptera, Tabanidae) that facilitate acquisition of blood meal. However, their roles in the regulation of local inflammation remains poorly understood.MethodsInduction expression profiles of immune-related molecules in the salivary glands of T. yao was analyzed by quantitative PCR (qPCR) after bacterial feeding. A significantly up-regulated molecule (cecropin-TY1) was selected for anti-inflammatory assay in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages. The transcription levels of inducible NO synthase (iNOS) and pro-inflammatory cytokines were quantified by qPCR. Nitric oxide (NO) production was determined by Griess reagent. Pro-inflammatory cytokine production was determined by an enzyme-linked immunosorbent assay (ELISA). The inflammatory signals were assayed by Western blotting analysis. The secondary structure of cecropin-TY1 was measured by Circular dichroism (CD) spectroscopy. Interaction of cecropin-TY1 with LPS was evaluated by the dissociation of fluorescein isothiocyanate (FITC)-conjugated LPS aggregates and neutralization of LPS determined by a quantitative Chromogenic End-point Tachypleus amebocyte lysate (TAL) assay kit. Homology modeled structure analysis and mutation of key residues/structures were performed to understand its structure-activity relationship.ResultsCecropin-TY1 was demonstrated to possess high anti-inflammatory activity and low cytotoxicity toward mouse macrophages. In LPS-stimulated mouse peritoneal macrophage, addition of cecropin-TY1 significantly inhibited the production of nitric oxide (NO) and pro-inflammatory cytokines. Further study revealed that cecropin-TY1 inhibited inflammatory cytokine production by blocking activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signals. Cecropin-TY1 even interacted with LPS and neutralized LPS. The secondary structure analysis revealed that cecropin-TY1 adopted unordered structures in hydrophobic environment but converted to α-helical confirmation in membrane mimetic environments. Homology modeled structure analysis demonstrated that cecropin-TY1 adopted two α-helices (Leu3-Thr24, Ile27-Leu38) linked by a hinge (Leu25-Pro26) and the structure surface was partly positively charged. Structure-activity relationship analysis indicated that several key residues/structures are crucial for its anti-inflammatory activity including α-helices, aromatic residue Trp2, positively charged residues Lys and Arg, hinge residue Pro26 and N-terminal amidation.ConclusionsWe found a novel anti-inflammatory function of horsefly-derived cecropin-TY1 peptide, laying groundwork for better understanding the ectoparasite-host interaction of horsefly with host and highlighting its potency in anti-inflammatory therapy for sepsis and endotoxin shock caused by Gram-negative bacterial infections.Electronic supplementary materialThe online version of this article (doi:10.1186...

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Section: Methodsmentioning

confidence: 99%

A potent anti-inflammatory peptide from the salivary glands of horsefly

et al. 2015

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“…Recent research has focused on the biodefense mechanisms of insects to identify novel AMPs (Kaushal et al 2016;Wei et al 2015;Choi et al 2014). We induced and purified a novel AMP from the immunized hemolymph of Hermetia illucens and evaluated its structural characteristics, cDNA sequence, and antimicrobial activity against various Gram-positive and -negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

A novel cecropin‐like peptide from black soldier fly, Hermetia illucens: Isolation, structural and functional characterization

Park

Yoe

2017

Entomological Research

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Cecropins are basic antibacterial peptides that have potent antimicrobial activities. We induced and purified a novel antimicrobial peptide exhibiting activity against Gram‐negative bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel cecropin‐like peptide 1 (CLP1) was purified using solid‐phase extraction and reverse‐phase chromatography. The purified CLP1 demonstrated a molecular weight of 4,840 Da, as determined by matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF). From analysis of CLP1 by N‐terminal amino acid sequencing using Edman degradation, combined with MALDI‐TOF and rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR), the amino acid sequence of the mature peptide was determined to be GWRKRVFKPVEKFGQRVRDAGVQGIAIAQQGANVLATARGGP PQQG. In NCBI BLAST, the amino acid sequence of CLP1 was found to be 60 % identical to the Drosophila melanogaster cecropin C. In silico analysis revealed that CLP1 was suggested to be part of the cecropin superfamily of AMPs characterized as cationic, linear, α‐helical, and amphipathic polypeptides. Analysis of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that CLP1 exerted antibacterial effects against Gram‐negative bacteria. The expression of CLP1 transcripts in several tissues after bacterial challenge was measured by quantitative real‐time PCR. CLP1 expression was negligible throughout the body before immunization, and was mostly evident in the fat body after immunization.

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“…Recently, research has been actively underway to explore the biodefense mechanisms of insects in order to identify novel AMPs (Dang et al, 2010;Wei et al, 2015). However, research on AMP extracted from H. illucens has not yet been conducted.…”

Section: Introductionmentioning

confidence: 99%