Characterization and cDNA Cloning of a Cecropin-Like Antimicrobial Peptide, Papiliocin, from the Swallowtail Butterfly, Papilio xuthus

Kim, Seong Ryul; Hong, Mee Yeon; Park, Seung Won; Choi, Kwang Ho; Yun, Eun Young; Goo, Tae Won; Kang, Seok Woo; Suh, Hwa Jin; Kim, Iksoo; Hwang, Jae Sam

doi:10.1007/s10059-010-0050-y

Cited by 55 publications

(63 citation statements)

References 22 publications

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“…expression by annealing control primer (ACP)-based differential display PCR (Kim et al, 2010). Up-expressed genes in immune challenged larvae ACP-based PCR were conducted using 120 pairs of arbitrary ACPs and dT-ACP2 to synthesize the second-strand cDNA.…”

Section: Resultsmentioning

confidence: 99%

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Kim¹,

Hwang²,

Park³

et al. 2011

International Journal of Industrial Entomology

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Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, Papilio xuthus. A full length cDNA of P. xuthus attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete P. xuthus attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of P. xuthus attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of P. xuthus attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature P. xuthus attacin into the expression vector, highly expressed in E. coli BL21 cells, and its antibacterial activity was analyzed. Recombinant P. xuthus attacin evidenced considerably antibacterial activity against Gram-negative bacteria, E. coli ML 35 and Klebsiella pneumonia.

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Section: Resultsmentioning

confidence: 99%

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Kim¹,

Hwang²,

Park³

et al. 2011

International Journal of Industrial Entomology

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“…Since then, cecropins have also been isolated from several orders of insects such as lepidopteran, dipteran, coleopteran (Kim et al, 2010;Liang et al, 2006;Kylsten et al, 1990;Morishima et al, 1990). …”

Section: Reverse Transcription Pcr (Rt-pcr)mentioning

confidence: 99%

“…The sequences of cecropins have basic residues in N-terminal segments and hydrophobic residues in their C-terminal segments. They have a broad spectrum of activity againstGram-negative and Gram-positive bacteria as well as certain fungi and metazoan parasites (Chalk et al, 1995;DeLucca et al, 1997), but they have little effect on normal eukaryotic cells.Previously, a 37-residue cecropin-like peptide named papiliocin was isolated from the bacteria-immunized larvae of the swallowtail butterfly Papilio xuthus (Kim et al, 2010). This peptide was shown significant antimicrobial activities against both human pathogenic bacterial and fungal strains, and also evidenced no hemolytic activity against human red blood cells.…”

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confidence: 99%

Molecular cloning of a novel cecropin-like peptide gene from the swallowtail butterfly, Papilio xuthus

Kim¹,

Choi²,

Kim³

et al. 2015

International Journal of Industrial Entomology

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A new cecropin-like antimicrobial peptide (Px-CLP) gene was isolated from the immunechallenged larvae of the swallowtail butterfly, Papilio xuthus, by employing annealing control primer (ACP)-based GeneFishing PCR. The full-length cDNA of Px-CLP is 310 nucleotides encoding a 70 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propeptide, a presumed 37-residue mature peptide, and an uncommon 7-residue acidic pro-region at the C-terminus. The deduced amino acid sequence of Px-CLP showed significant identities with other Lepidopteran cecropin D type peptides. RT-PCR revealed that the Px-CLP transcript was detected at significant level after injection with bacterial lipopolysaccharide (LPS). The peptides with or without C-terminal acidic sequence region were synthesized on-solid phage and submitted to antibacterial activity assay. The synthetic 37-mer peptide (Px-CLPa), which removed C-terminal acidic sequence region, was showed exclusively antibacterial activity against E. coli ML35; meanwhile, a 44-mer peptide (PxCLPb) with C-terminal acidic peptide region was not active. This result suggests that Px-CLP is produced as a larger precursor containing a C-terminal pro-region that is subsequently removed by C-terminal modification. IntroductionInsect antimicrobial peptides (AMPs) accomplishes important role as key factors in systemic immune response against invading pathogens such as bacteria, fungi, and viruses (Hoffman et al., 1999). To date, a large number of antimicrobial peptides, which have a broad spectrum of antimicrobial activities, have been identified from various insects such as lepidopteran, hymenopteran, dipteran, and coleopteran insect (Bulet et al., 1999). Most of these AMPs have relatively short amino acids, positively charged residues (net charge of +2 to +9) and amphipathic properties (Jenssen et al., 2006). They are divided into five groups on the basis of their amino acid sequences and secondary structures in insects (Boman, 1995;Bulet et al., 1999). These are cecropin-like peptides, defensins, proline-rich peptides, glycine-rich peptides and lysozymes.Cecropin, which is a well-studied antimicrobial peptide in Screening of differentially expressed genes (DEGs)For screening DEGs in immune-challenged P. xuthus larvae, we used ACP-based GeneFishing PCR kit (Seegene, Korea). Briefly, polymerase chain reaction (PCR) was conducted by using 120 pairs of arbitrary ACPs and dT-ACP2 to synthesize the second-strand cDNAs under annealing conditions. PCR analysis were performed Reverse transcription PCR (RT-PCR)Total RNA were extracted from whole larvae 0 h, 12 h and 24 h post injection using Trizol reagent (Invitrogen, Carlsbad, CA) and then treated for 15 min with DNase I. The extracted total RNA samples (1 μg per sample) were reversely transcribed to cDNA using oligo (dT)primer and SuperScript III reverse transcriptase (Invitrogen, CA).PCR amplifications were performed with mixture containing cDNA, a pair of specific primers (5'-CGTCACAATCATCCTGTTCGT-3'and 5-CATCTTCGTC...

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“…[5][6][7] Studies of innate immune systems of insects have shown the importance of AMPs for the defense of these organisms against bacteria. 7,8 In insects, over 200 AMPs have been identified. Papiliocin, with 37-residues (RWKIFKKIEKVGRNVRDGIIKAGPA VAVVGQAATVVK-NH2) has been isolated from the larvae of the swallowtail butterfly, Papilio xuthus, and it belongs to a cecropin family that have strong antibacterial activities against Gram-negative bacteria.…”

Section: Introductionmentioning

confidence: 99%

“…Papiliocin, with 37-residues (RWKIFKKIEKVGRNVRDGIIKAGPA VAVVGQAATVVK-NH2) has been isolated from the larvae of the swallowtail butterfly, Papilio xuthus, and it belongs to a cecropin family that have strong antibacterial activities against Gram-negative bacteria. 8 We studied the structure of papiliocin by NMR spectroscopy and the structure shows that papiliocin has an α-helical structure from Lys 3 to Lys 21 and from Ala 25 to Val 35 , linked by a hinge region, in 300 mM dodecylphosphocholine (DPC) micelles. 9 In order to understand the structural requirements for papiliocin function and to design shorter and potent peptide antibiotics, we designed papiliocin analog, PapN (residues Arg 1 -Ala 22 from the N-terminal amphipathic helix).…”

Section: Introductionmentioning

confidence: 99%

Structure-Activity Relationship of the N-terminal Helix Analog of Papiliocin, PapN

Jeon¹,

Jeong²,

Kim³

et al. 2015

Journal of the Korean Magnetic Resonance Society

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Papiliocin, from the swallowtail butterfly, Papilio xuthus, shows high bacterial cell selectivity against Gram-negative bacteria. Recently, we designed a 22mer analog with N-terminal helix from Lys 3 to Ala 22 , PapN. It shows outstanding antimicrobial activity against Gram-negative bacteria with low toxicity against mammalian cells. In this study, we determined the 3-D structure of PapN in 300 mM DPC micelle using NMR spectroscopy and investigated the interactions between PapN and DPC micelles. The results showed that PapN has an amphipathic α-helical structure from Lys 3 to Lys 21 .STD-NMR and DOSY experiment showed that this helix is important in binding to the bacterial cell membrane. Furthermore, we tested antibacterial activities of PapN in the presence of salt for therapeutic application. PapN was calcium-and magnesium-resistant in a physiological condition, especially against Gram-negative bacteria, implying that it can be a potent candidate as peptide antibiotics.

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Characterization and cDNA Cloning of a Cecropin-Like Antimicrobial Peptide, Papiliocin, from the Swallowtail Butterfly, Papilio xuthus

Cited by 55 publications

References 22 publications

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Molecular Cloning and Characterization of Attacin from the Swallowtail Butterfly, Papilio xuthus

Molecular cloning of a novel cecropin-like peptide gene from the swallowtail butterfly, Papilio xuthus

Structure-Activity Relationship of the N-terminal Helix Analog of Papiliocin, PapN

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