Molecular cloning of the antibacterial protein of the giant African snail, <i>Achatina fulica</i> Férussac

Obara, Kuniaki; Otsuka-Fuchino, Hisako; Sattayasai, Nison; Nonomura, Yoshiaki; Tsuchiya, Takahide; Tamiya, Tôru

doi:10.1111/j.1432-1033.1992.tb17254.x

Cited by 50 publications

(41 citation statements)

References 46 publications

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“…Both proteins purportedly form large ion channels in the bacterial membrane in a manner similar to insect defensins. Furthermore, it has been shown that larger antibacterial proteins, such as aplysianin A from the sea hare (41) and achacin from the giant African snail (42), are constitutively secreted rather than secreted under stimulation (i.e. injury).…”

Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder

Cole

Weis

Diamond

1997

Journal of Biological Chemistry

463

301

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Antimicrobial peptides are found in both myeloid cells and mucosal tissues of many vertebrates and invertebrates. These peptides are predicted to operate as a first-line host defense mechanism exerting broad-spectrum activity against pathogenic bacteria, fungi, parasites, and enveloped viruses. We report the characterization of a novel 25-residue linear antimicrobial peptide found in the skin mucous secretions of the winter flounder (Pleuronectes americanus). This peptide was purified through multiple chromatographic methods to obtain a single peak by reversed-phase high performance liquid chromatography. This purified peptide, which we named pleurocidin, exhibited antimicrobial activity against Escherichia coli in a bacterial cell lysis plate assay. Mass spectrometry and amino acid sequence analysis indicated that it is 25 amino acids in length. Pleurocidin is predicted to assume an amphipathic ␣-helical conformation similar to many other linear antimicrobial peptides. There is a high degree of homology between pleurocidin and two antimicrobial peptides, ceratotoxin from the Mediterranean fruit fly and dermaseptin from the skin of a hylid frog. The minimal inhibitory concentration and minimal bactericidal concentration of pleurocidin were determined against 11 different Gram-negative and Gram-positive bacteria. Immunohistochemistry locates pleurocidin in the epithelial mucous cells of flounder skin. Pleurocidin represents a novel antimicrobial peptide found in fish and may play a role in innate host defense.

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Section: Resultsmentioning

confidence: 99%

Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder

Cole

Weis

Diamond

1997

Journal of Biological Chemistry

463

301

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“…Carp antimicrobial proteins, 27 kD and 31 kD proteins, had potent microbicidal activities (0.018−0.18 mol/L) against different strains of Gram-negative and Gram-positive bacteria (Lemaitre et al, 1996). Furthermore, it has been shown that larger antimicrobial proteins, such as aplysianin A from the sea hare (Takamatsu et al, 1995) and achacin from the giant African snail (Obara et al, 1992), have strong antimicrobial activities. Those proteins purportedly form large ion channels in the bacterial membrane in a manner similar to insect defensins.…”

Section: Discussionmentioning

confidence: 99%

Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, <I>Epinephelus fario</I>

Zhang¹,

Zou²,

RiGa³

et al. 2009

Zoological Research

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Antimicrobial proteins and peptides had been found from a wide variety of organisms in the last few years. These molecules have attracted much research interest because of their biochemical diversity, broad specificity on anti-viral, anti-bacterial, anti-fungi, anti-protozoan parasites, anti-tumoural, and wound-healing effects. Antimicrobial proteins and peptides play key roles in innate immunity. They interact directly with bacteria and kill them. The brown-spotted grouper, Epinephelus fario, is an important marine fish cultured in southern China. Recently, bacteria and virus have caused high mortality in E. fario cultures, but its endogenous antimicrobial peptides and proteins have not been explored. An antimicrobial component was found from the skin homogenate of E. fario. After the skin homogenate was digested with trypsin, its antimicrobial activity was lost, which showed that the antimicrobial component is a protein. The antimicrobial protein (Efap) was purified from the skin homogenate of E. fario by successive ion-exchange and gel filtration chromatography. Efap was demonstrated to be single protein band by SDS-PAGE, with the apparent molecular weight of 41 kD. Efap exhibited antimicrobial activity both for the Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Bacillus subtilis, and for the Gram-negative bacteria, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Aeromonas hydrophila, Eschrrichiu coli, and Pseudomonas aeruginosa. Except A. hydrophila, P. aeruginosa, and E. coli (MIC>20 mol/L), most of the tested Gram-negative bacteria were sensitive to Efap (MIC<20 mol/L). Interestingly, Efap showed potent antimicrobial activity against Gram-positive bacteria S. aureus (MIC 5-10 mol/L) but comparatively weak antimicrobial activity against M. luteus and B. subtilis. The broad antimicrobial activities of Efap suggest that it contributes to the innate host defence of E. fario.

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“…No approach to investigate the antibacterial action mechanism was proposed. Nevertheless, in amphibians (Gibson, 1991;Mor et al, 1991;Simmaco et al, 1993;Douglas et al, 1994) and mollusks (Takamatsu et al, 1995 ;Obara et al, 1992), substances are secreted at the body surface that are involved as natural defense factors. These substances have been shown to belong to the defensin family, the members of which are well known for their antibacterial activities as well as their channelforming properties.…”

Section: Discussionmentioning

confidence: 99%

“…Fouz et al (1990) have already pointed out the presence of a glycoprotein in the mucus secretions of the turbot. The molecular cloning of achacin from the giant African snail (Obara et al, 1992) and aplysianin A from the sea hare (Takamatsu et al, 1995) revealed several glycosylation consensus sites in the deduced amino acid sequence. Nevertheless, the presence of a glycosylation site in the sequence is not necessary for inducing ion channels in planar lipid membranes or antibacterial activities since the 31-kDa protein, which is not glycosylated, exhibits this behavior.…”

Section: Discussionmentioning

confidence: 99%

“…Almost all of these antibacterial factors are peptides and some of their primary structures have been elucidated. Nevertheless, antibacterial proteins have been discovered in invertebrates from the mucus of mollusks (aplysianin A from the sea hare, Takamatsu et al, 1995; achacin from the giant African snail, Obara et al, 1992) that have high molecular masses (320 kDa and 63 kDa, respectively) and also subunit structures. These proteins are always being secreted, even without stimulation, while the antibacterial polypeptides from insects are generally secreted after some stimulation, such as an injury.…”

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confidence: 99%

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Characterization and Ion Channel Activities of Novel Antibacterial Proteins from the Skin Mucosa of Carp (Cyprinus carpio)

Lemaître

Orange²,

Saglio³

et al. 1996

European Journal of Biochemistry

106

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A detergent-solubilized fraction of skin mucus of carp (Cyprinus carpio) induced ion channels after reconstitution into planar lipid bilayers. A differential extraction using a non-ionic detergent followed by electrophoretic separation led to the isolation of two hydrophobic 31 -kDa and 27-kDa proteins. In contrast to the 27-kDa protein, which was glycosylated, the 31-kDa did not bind to concanavalin A. The reconstitution of these proteins into a planar lipid bilayer restored the ionophore behavior already observed with the crude mucus. The main unit conductance levels were about 900 pS for the 27-kDa protein and 500 pS for the 31-kDa protein, and selectivity measurements gave P J P , ratios of 0.6 and 1.0, respectively. These proteins had large potent microbicidal activities (0.018 -0.1 8 pM) against different strains of gramnegative and gram-positive bacteria. This behavior can be compared with insect defensins that are known to form large ion channels in the bacterial membrane. To exclude the eventuality of bacterial origin, the bacterial flora of the crude mucus were analysed and the following were identified: Pseudomonas cepacia ; Micrococcus luteus; Micrococcus roseus; Flavobacterium sp. ; Aeromonas hydrophila. Antibacterial assays with both proteins were performed against these specific strains and revealed good growth inhibition activities. Furthermore, microsequencing analysis showed that the 31-kDa protein was protected on its N-terminal extremity in contrast to the 27-kDa protein, which had a 19-amino-acid sequence. This last sequence, when compared with sequences in protein data banks, did not reveal any significant similarities to other proteins. These results suggest that these novel proteins could be involved in antibacterial defense processes in fish.

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Molecular cloning of the antibacterial protein of the giant African snail, Achatina fulica Férussac

Cited by 50 publications

References 46 publications

Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder

Isolation and Characterization of Pleurocidin, an Antimicrobial Peptide in the Skin Secretions of Winter Flounder

Purification and Antimicrobial Activity of Antimicrobial Protein from Brown-spotted Grouper, <I>Epinephelus fario</I>

Characterization and Ion Channel Activities of Novel Antibacterial Proteins from the Skin Mucosa of Carp (Cyprinus carpio)

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