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1H nuclear magnetic resonance study of the solution conformation of an antibacterial protein, Sapecin

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Abstract

The solution conformation of an antibacterial protein sapecin has been determined by 1H nuclear magnetic resonance (NMR) and dynamical simulated annealing calculations. It has been shown that the polypeptide fold consists of one flexible loop (residues 4-12), one helix (residues 15-23), and two extended strands (residues 24-31 and 34-40). It was found that the tertiary structure of sapecin is completely different from that of rabbit neutrophil defensin NP-5, which is homologous to sapecin in the amino acid sequences and also has the antibacterial activity. The three-dimensional structure determination has revealed that a basic-residue rich region and the hydrophobic surface face each other on the surface of sapecin.

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... They may be further classified in two sub-families according to their in vitro activity against bacteria or filamentous fungi [11]: antimicrobial defensins that possesses activity against Gram-positive bacteria, including human pathogens, but are less effective against Gram-negative bacteria and fungi, and antifungal defensins that are mainly effective against filamentous fungi. Structurally, insect defensins possess an N-terminal flexible loop, a central α-helix and a C-terminal anti parallel β-sheet as has been determined by two-dimensional 1 H-NMR spectroscopy carried out on isolated Sarcophaga peregrina defensin [19] and on a recombinant Phormia terranovae defensin [20]. The antimicrobial defensins contain six cysteine residues engaged in a characteristic conserved motif of three intramolecular disulfide bridges connected in a Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 pattern. ...
... The tertiary structure of lucifensin determined using NMR [36] showed a high degree of similarity to the structure of other insect defensins: sapecin [19] and insect defensin A [20]. Lucifensin adopts a characteristic insect defensin structure that includes an N-terminal loop (residues 1-12), followed by an α-helix (residues 13-23), which is linked by a turn to a pair of β-strands (residues 28-31 and 34-38) folded into an antiparallel β-sheet (Figure 1). ...
... The Cys3-Cys30 disulfide bridge connects the N-terminal loop with the first β-strand and the other two bridges (Cys16-Cys36, Cys20-Cys38) link the α-helix and second β-strand [36]. The α-helix and β-structure connected by two disulfide bridges form a common structural element typical for insect defensins, known as the cysteine-stabilised αβ (CS αβ) motif, which is essential for their antimicrobial activity [19,20]. ...
... We expected that the omission of the other two bridges would certainly negatively affect the antimicrobial activity of lucifensin. We supposed that the three-dimensional structure of lucifensin is similar to that of sapecin, [30] because the primary sequence of sapecin (40 residues) differs from the sequence of lucifensin by only four amino acid residues (Ile11, Asn12, Lys33, Val35). The conformation of sapecin was determined by 1 H NMR spectroscopy and simulated annealing calculations, [30] resulting in a structure that includes an N-terminal flexible loop (residues 4-12), a helical part (residues 15-23), and an antiparallel b-structure (residues 24-31 and 34-40). ...
... We supposed that the three-dimensional structure of lucifensin is similar to that of sapecin, [30] because the primary sequence of sapecin (40 residues) differs from the sequence of lucifensin by only four amino acid residues (Ile11, Asn12, Lys33, Val35). The conformation of sapecin was determined by 1 H NMR spectroscopy and simulated annealing calculations, [30] resulting in a structure that includes an N-terminal flexible loop (residues 4-12), a helical part (residues 15-23), and an antiparallel b-structure (residues 24-31 and 34-40). Significant antimicrobial activity of the Luc[C16-C36] analogue against the most sensitive bacterium M. luteus (Figure 7 A) in comparison with the other two analogues containing one disulfide bridge clearly indicates that the disulfide bond between Cys16 and Cys36, which connects the 15-23 helical part of the molecule with the 34-40 strand is the most important one for maintaining the ordered structure of lucifensin. ...
... The a-helix and bstructure connected by two disulfide bridges form a common structural element typical for insect defensins known as the cysteine-stabilized ab (CSab) motif and which is important for antimicrobial activity. [30,31] The percentage of a-helical fraction in lucifensin and its analogues that was calculated from CD spectra (25 %) measured in the presence of TFE is in good agreement with the secondary structure of sapecin that was determined from NMR spectroscopic data, just as the presence of b-sheet conformations assumed from lucifensin CD spectra when measured in water or a membrane-mimicking environment. ...
Article
Recently, we identified a new insect defensin, named lucifensin that is secreted/excreted by the blowfly Lucilia sericata larvae into a wound as a disinfectant during the medicinal process known as maggot therapy. Here, we report the total chemical synthesis of this peptide of 40 amino acid residues and three intramolecular disulfide bridges by using three different protocols. Oxidative folding of linear peptide yielded a peptide with a pattern of disulfide bridges identical to that of native lucifensin. The synthetic lucifensin was active against Gram-positive bacteria and was not hemolytic. We synthesized three lucifensin analogues that are cyclized through one native disulfide bridge in different positions and having the remaining four cysteines substituted by alanine. Only the analogue cyclized through a Cys16-Cys36 disulfide bridge showed weak antimicrobial activity. Truncating lucifensin at the N-terminal by ten amino acid residues resulted in a drop in antimicrobial activity. Linear lucifensin having all six cysteine residues alkylated was inactive. Circular dichroism spectra measured in the presence of α-helix-promoting compounds showed different patterns for lucifensin and its analogues. Transmission electron microscopy revealed that Bacillus subtilis treatment with lucifensin induced significant changes in its envelope.
... They may be further classified in two sub-families according to their in vitro activity against bacteria or filamentous fungi [11]: antimicrobial defensins that possesses activity against Gram-positive bacteria, including human pathogens, but are less effective against Gram-negative bacteria and fungi, and antifungal defensins that are mainly effective against filamentous fungi. Structurally, insect defensins possess an N-terminal flexible loop, a central α-helix and a C-terminal anti parallel β-sheet as has been determined by two-dimensional 1 H-NMR spectroscopy carried out on isolated Sarcophaga peregrina defensin [19] and on a recombinant Phormia terranovae defensin [20]. The antimicrobial defensins contain six cysteine residues engaged in a characteristic conserved motif of three intramolecular disulfide bridges connected in a Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 pattern. ...
... The tertiary structure of lucifensin determined using NMR [36] showed a high degree of similarity to the structure of other insect defensins: sapecin [19] and insect defensin A [20]. Lucifensin adopts a characteristic insect defensin structure that includes an N-terminal loop (residues 1-12), followed by an α-helix (residues 13-23), which is linked by a turn to a pair of β-strands (residues 28-31 and 34-38) folded into an antiparallel β-sheet (Figure 1). ...
... The Cys3-Cys30 disulfide bridge connects the N-terminal loop with the first β-strand and the other two bridges (Cys16-Cys36, Cys20-Cys38) link the α-helix and second β-strand [36]. The α-helix and β-structure connected by two disulfide bridges form a common structural element typical for insect defensins, known as the cysteine-stabilised αβ (CS αβ) motif, which is essential for their antimicrobial activity [19,20]. ...
Article
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Defensins are the most widespread antimicrobial peptides characterised in insects. These cyclic peptides, 4-6 kDa in size, are folded into α-helical/β-sheet mixed structures and have a common conserved motif of three intramolecular disulfide bridges with a Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6 connectivity. They have the ability to kill especially Gram-positive bacteria and some fungi, but Gram-negative bacteria are more resistant against them. Among them are the medicinally important compounds lucifensin and lucifensin II, which have recently been identified in the medicinal larvae of the blowflies Lucilia sericata and Lucilia cuprina, respectively. These defensins contribute to wound healing during a procedure known as maggot debridement therapy (MDT) which is routinely used at hospitals worldwide. Here we discuss the decades-long story of the effort to isolate and characterise these two defensins from the bodies of medicinal larvae or from their secretions/excretions. Furthermore, our previous studies showed that the free-range larvae of L. sericata acutely eliminated most of the Gram-positive strains of bacteria and some Gram-negative strains in patients with infected diabetic foot ulcers, but MDT was ineffective during the healing of wounds infected with Pseudomonas sp. and Acinetobacter sp. The bactericidal role of lucifensins secreted into the infected wound by larvae during MDT and its ability to enhance host immunity by functioning as immunomodulator is also discussed.
... The kinetics of killing indicates that insect defensins act rapidly [11,32]. The three dimensional structures of few insect defensins have been determined by nuclear magnetic resonance spectroscopy [6,13,20]. The common structural feature observed is an amino-terminal loop, an a-helix and a two-stranded antiparallel b-sheet. ...
... The entire sequence has been divided into amino (N) and carboxy(C) terminal segments. The regions marked as a-helix and b-sheet are based on structure deduced by nuclear magnetic resonance spectroscopy studies on insect defensins [6,13,20]. The species in which the sequences are present are: Aa, Aedes aegypti (defensin B, accession number AAB35030; defensin C, accession number AAB35031); Ac, Aeshna cyanea (accession number P80154); Acp, Anomala cuprea (defensin A, accession number P83669; defensin B, accession number P83668); Ad, Allomyrina dichotoma (accession number AAB36306); Adn, Archaeoprepona demophon (accession number P84156); Ag, Anopheles gambiae (accession number AAC18575); Al, Acalolepta luxuriosa (defensin 1, accession number AAK35160); Am, Apis mellifera (accession number NP_001011616); As, Androctonus australis (accession number P56686); Bp, Bombus pascuorum (accession number P81462); Cpp, Culex pipiens pipiens (accession number AAO38519); Dm, Drosophila melanogaster (accession number P36192); Gm, Galleria mellonella (accession number AAS19170); Hd, Holotrichia diomphalia (accession number Q7M426); Hv, Heliothis virescens (accession number P81544); Lq, Leiurus quinquestriatus (accession number AAB27538); Mg, Mytilus galloprovincialis (defensin 1, accession number P80571); Om, Ornithodoros moubata (defensin A, accession number BAB41028; defensin B, accession number BAB41027; defensin C, accession number BAC22074; defensin D, accession number BAC22073); Pa, Pyrrhocoris apterus (accession number P37364); Pp, Palomena prasina (accession number P80407); Pt, Phormia terranovae (defensin A, accession number P10891); Sc, Stomoxys calcitrans (defensin 1, accession number O16136; defensin 2, accession number O16137); Tm, Tenebor molitor (accession number Q27023); Za, Zophobas atratus (defensin B, accession number AAB20745; defensin C, accession number AAB20746). ...
... The carboxy-terminal segments in all the peptides except Sc-1 have net positive charge whereas the net charge in the amino-terminal segment is negative or zero. The helix and b-strands observed in insect defensins from Phormia terranovae and Sarcophaga peregrina as determined by nuclear magnetic resonance spectroscopy [6,13,20] are indicated in the figure. The defensin Pa from the sap-sucking bug Pyrrhocoris apterus is inducible and was found to exhibit activity against Gram-positive and Gram-negative bacteria but with greater potency against Gram-positive bacteria [12]. ...
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The aggregation behavior of peptides Ac-VQIVYK-amide (AcPHF6) and Ac-QIVYK-amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self-association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly beta-conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for beta-structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and beta-structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides.
... Coprisin, similar to other defensins, adopts structure in water because it is larger than many other AMP and has disulfides which stabilize the structure. Therefore, we determined the tertiary structure of coprisin in an aqueous solution using NMR spectroscopy similar to the studies of structures of other insect defensins [54][55][56][57][58]. ...
... According to CD spectra, coprisin has a CSαβ structural motif, which is the characteristic folding pattern of other insect defensins [53][54][55]61]. Electropositive regions composed of Arg 28 , Lys 29 , Lys 30 , and Arg 42 are positioned at the end of the helix, turns between the helix and the first strand of the sheet, and the C-terminus (Fig. 7C). ...
... The NMR structures of three insect defensinscoprisin, def-AAA from Anopheles gambiae, and defensin A from Phormia terraenovae, sapecin from S. peregrina-which have basic residues located on one side of the molecule, are shown in Fig. 8A-D. These four defensins have an electropositive area consisting of two or three basic residues at turn I (between the helix and the first strand of the sheet) and one Arg residue at the C-terminus [54][55][56]61]. These insect defensins also have an amphipathic α-helical region. ...
Article
The novel 43-residue, insect defensin-like peptide coprisin, isolated from the dung beetle, Copris tripartitus, is a potent antibiotic with bacterial cell selectivity, exhibiting antimicrobial activities against Gram-positive and Gram-negative bacteria without exerting hemolytic activity against human erythrocytes. Tests against Staphylococcus aureus using fluorescent dye leakage and depolarization measurements showed that coprisin targets the bacterial cell membrane. To understand structure-activity relationships, we determined the three-dimensional structure of coprisin in aqueous solution by nuclear magnetic resonance spectroscopy, which showed that coprisin has an amphipathic α-helical structure from Ala(19) to Arg(28), and β-sheets from Gly(31) to Gln(35) and Val(38) to Arg(42). Coprisin has electropositive regions formed by Arg(28), Lys(29), Lys(30), and Arg(42) and ITC results proved that coprisin and LPS has electrostatically driven interactions. Using measurements of nitric oxide release and inflammatory cytokine production, we providethe first verification of the anti-inflammatory activity and associated mechanism of an insect defensin, demonstrating that the anti-inflammatory actions of the defensin-like peptide, coprisin, are initiated by suppressing the binding of LPS to toll-like receptor 4, and subsequently inhibiting the phosphorylation of p38 mitogen-activated protein kinase and nuclear translocation of NF-kB. In conclusion, we have demonstrated that an amphipathic helix and an electropositive surface in coprisin may play important roles in its effective interaction with bacterial cell membranes and, ultimately, in its high antibacterial activity and potent anti-inflammatory activity. In addition to elucidating the antimicrobial action of coprisin, this work may provide insight into the mechanism of action of insect defense systems.
... Many invertebrate (11) and fungal (12) defensins have a cysteine-stabilized ␣-helix/␤-sheet structure similar to the structure of ␤-defensins from vertebrates. Among others, structures have been solved for Phormia defensin (13), Sarcophaga defensin (14), drosomycin (15), heliomycin (16), terramycin (17), MGD-1 (18), lucifensin (19), and plectasin (20). They all form an ␣-helix and two anti-parallel ␤-strands (␣␤␤scaffold) with the ␣-helix stabilized by two disulfide bridges to one strand of the ␤-sheet. ...
... The cDNA clone encoded a putative defensin-like peptide that was named eurocin. The gene encodes 90 amino acids (signal peptide (amino acids (aa) [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], propeptide (aa , and defensin peptide (aa 49 -90)). The mature peptide consists of 42 amino acids (Fig. 1) and has a molecular mass of 4.3 kDa. ...
Article
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Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.
... The three-dimensional (3D) structures of Phormia defensin A 17,18 and Sarcophaga sapecin 19 have been derived from two-dimensional nmr, which yielded comparable architectures for both molecules ( Figure 2). Phormia defensin consists of three distinct domains: an amino-terminal loop, a single ␣-helix, and a two-stranded antiparallel ␤-sheet. ...
... Moreover, insect defensins and mammalian defensins possess six cysteine residues engaged in three intramolecular disulfide bridges. However, subsequent elucidation of the global fold of mammalian defensins 29,30 and insect defensins [17][18][19] has established that these two classes of molecules are structurally unrelated. The structure of mammalian defensins is dominated by a three-stranded antiparallel ␤-sheet stabilized by three disulfide bridges, but contains no ␣-helix. ...
Article
Full-text available
Antimicrobial peptides are pivotal elements of the innate immune defense against bacterial and fungal infections. Within the impressive list of antimicrobial peptides available at present, more than half have been characterized in arthropods. Cysteine-rich antimicrobial peptides represent the most diverse and widely distributed family among arthropods and, to a larger extent, among invertebrates. Proeminent groups of cysteine-rich peptides are peptides with the CSαβ motif and peptides forming an hairpin-like β-sheet structure. Although these substances exhibit a large structural diversity and a wide spectrum of activity, they have in common the ability to permeabilize microbial cytoplasmic membranes. Drosophila has proved a remarkable system for the analysis of the regulation of expression of gene encoding antimirobial cysteine-rich peptides. These studies have unraveled the striking parallels that exist between insect immunity and innate immunity in mammals that point to a common ancestry of essential aspects of innate immunity. © 1999 John Wiley & Sons, Inc. Biopoly 47: 465–477, 1998
... Sapecin from Sarcophaga peregrina (Hanzawa et al. 1990, PDB entry: 1L4V) and insect defensin A from Phormia terranova (Cornet et al. 1995, PDB entry: 1ICA) are two well studied insect defensins, for which the 3D structure has already been determined. Many of the studies have focused on elucidating the defensin-membrane interactions as these are believed to cause antimicrobial activity, which for insect defensins primarily is observed against Gram-positive bacteria. ...
... Lucifensin has a high sequence similarity of 90.0 and 87.5% with the two insect defensins sapecin (Hanzawa et al. 1990) and insect defensin A (Cornet et al. 1995), respectively. Also the tertiary structure of lucifensin is very similar to the structures of the two insect defensins. ...
... This effect reßects a prominent impact of the Val-to-Ile substitution on the difference between the hydrophobicity of these defensins, indicating signiÞcant hydrophobic interaction of the side chain of Ile with lipidic C-18 stationary phase groups of the RP-HPLC column. The tertiary structure of lucifensin recently determined (Nygaard et al. 2012) using nuclear magnetic resonance (NMR) showed a high degree of similarity to the structure of other insect defensins: sapecin (Hanzawa et al. 1990) and insect defensin A (Cornet et al. 1995). Lucifensin adopts a characteristic insect defensin structure that includes an N-terminal loop (residues 1Ð12), followed by an ␣-helix (residues 13Ð23) that is linked by a turn to a pair of ␤-strands (residues 28 Ð31 and 34-38) folded into an antiparallel ␤-sheet (Nygaard et al. 2012). ...
... The Cys3ÐCys30 disulÞde bridge connects the N-terminal loop with the Þrst ␤-strand, and the other two bridges (Cys16 ÐCys36, Cys20 ÐCys38) link the ␣-helix and second ␤-strand. The ␣-helix and ␤-structure connected by two disulÞde bridges form a common structural element typical for insect defensins, known as the cysteine-stabilized ␣␤ (CS␣␤) motif, and is essential for their antimicrobial activity (Cornet et al. 1995, Hanzawa et al. 1990). The altered residue (ValÐtoÐIle) is located in the N-terminal loop following the Ser-Gly-Thr-Gly motif conserved in both lucifensin (L. ...
Article
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A novel homolog of insect defensin, designated lucifensin II (Lucilia cuprina Wiedemann [Diptera: Calliphoridae] defensin), was purified from hemolymph extract from larvae of the blowfly L. cuprina. The full-length primary sequence of this peptide of 40 amino acid residues and three intramolecular disulfide bridges was determined by electrospray ionization-orbitrap mass spectrometry and Edman degradation and is almost identical to the previously identified sequence of lucifensin (Lucilia sericata Meigen defensin). Lucifensin II sequence differs from that of lucifensin by only one amino acid residue, that is, by isoleucine instead of valine at position 11. The presence of lucifensin II also was detected in the extracts of other larval tissues, such as gut, salivary glands, fat body, and whole body extract.
... The PFP indicated that this protein is involved in antimicrobial humoral response (Gene Ontology: 0019730). According to the definition, this type of immune response against microbes is seen in the antimicrobial humoral response of Drosophila melanogaster and Mus musculus, being similar to the function of several defensins [1,8,12,44,45,47,48]. ...
... Overall, they have been known to be particularly resistant to bacterial infection [49]. Insects are a promising source of antimicrobial agents, and several studies have suggested that insect defensins are a potential alternative in the control of pathogenic bacteria [1,8,45]. This paper reports the identification of a defensin from the Hessian fly, MdesDEF-2. ...
Article
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The CSαβ defensins are one of the most ancient antimicrobial peptide classes and are distributed in plants, invertebrates, and fungi. In the insect immunity, the defensins play a crucial role in protection against pathogens. The discovery of novel insect defensins could be a vital tool in developing novel antimicrobial agents, which are urgently needed because of growing resistance in pathogenic bacteria and the resulting reduction in the effectiveness of conventional antibiotics over the years. In this context, novel insect defensins could be identified from the potential resource of model insects. Here, a novel defensin, MdesDEF-2, was identified from the model insect Mayetiola destructor, the most destructive insect pest of wheat worldwide. The in silico identification of MdesDEF-2 was done through searching by regular expression in M. destructor's protein sequences available at NCBI. MdesDEF-2 has 36 amino acid residues and its model was composed of two β-strands and one α-helix showing three disulfide bridges. According to the classification of CSαβ defensins, MdesDEF-2 belongs to the group of ancient insect-type defensins. The molecular dynamics simulation revealed that MdesDEF-2 has a very flexible N-terminal loop. Moreover, phylogenetic analysis together with functional predictions indicated that MdesDEF-2 could have antibacterial activity without causing membrane disruption. However, while the actual activity of MdesDEF-2 is still unclear, it is evident that its role in the biology of M. destructor is similar to that of its paralogue, MdesDEF-1, protecting the insect against microbial invasion. Figure From Mayetiola destructor protein sequences to in silico structural and evolutionary data of MdesDEF-2, a novel defensin from the Hessian fly
... Comparisons purely based on primary sequences are error-prone (Grishin, 2001) and conservation within secondary and tertiary structure is higher as compared to the primary sequences (Shafee et al., 2016). Currently only nine peptides have been characterized using solution NMR spectroscopy, including four antifungal, i.e., heliomicin (Lamberty et al., 2001a), drosomycin (Landon et al., 1997), termicin (Da Silva et al., 2003), and ARD1 (Landon et al., 2004) as well as five anti-gram-positive peptides, i.e., phormicin (Cornet et al., 1995), sapecin (Hanzawa et al., 1990), Def-AAA (Landon et al., 2008), lucifensin (Nygaard et al., 2012), and coprisin (Lee et al., 2013). Nevertheless, these studies provide valuable information about structure-activity relations for both antibacterial as well as antifungal insect defensins and shed light on structural determinants underlying biological activity. ...
... For the primarily antibacterial defensins only five available structures represent a very limited number given the large number of different peptide sequences ( Table 1). Additionally it is worth to mention that phormicin, sapecin, and lucifensin only differ by individual amino acids and thus it is not surprising that their three-dimensional topologies are highly similar (Hanzawa et al., 1990;Cornet et al., 1995;Nygaard et al., 2012). In an attempt to increase activity against Staphylococcus aureus a detailed study was reported using the Anopheles defensin as well as an alignment of 40 insect defensin sequences as basis for the design of 45 peptide mutants (Landon et al., 2008). ...
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Insects make up the largest and most diverse group of organisms on earth with several million species to exist in total. Considering the sheer number of insect species and the vast variety of ways they interact with their environment through chemistry, it is clear that they have significant potential as a source of new lead molecules. They have adapted to a range of ecological habitats and exhibit a symbiotic lifestyle with various microbes such as bacteria and fungi. Accordingly, numerous antimicrobial compounds have been identified including for example defensin peptides. Insect defensins were found to have broad-spectrum activity against various gram-positive/negative bacteria as well as fungi. They exhibit a unique structural topology involving the complex arrangement of three disulfide bonds as well as an alpha helix and beta sheets, which is known as cysteine-stabilized αβ motif. Their stability and amenability to peptide engineering make them promising candidates for the development of novel antibiotics lead molecules. This review highlights the current knowledge regarding the structure-activity relationships of insect defensin peptides and provides basis for future studies focusing on the rational design of novel cysteine-rich antimicrobial peptides.
... The majority of invertebrate defensins are mainly active against Gram-positive bacteria, with a few exceptions being active against some Gram-negative bacteria and fungi [8]. The structures of some invertebrate defensins and plectasin, a defensin from a fungus, but structurally also belonging to the invertebrate defensins, have been identified, and are all comprised of an a-helix and two antiparallel b-strands stabilized by three or four disulfide bridges, constituting the common CSab motive [9][10][11][12]. ...
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Defensins are a major family of antimicrobial peptides found throughout the phylogenetic tree. From the spider species: Cupiennius salei, Phoneutria reidyi, Polybetes pythagoricus, Tegenaria atrica, and Meta menardi, defensins belonging to the 'ancestral' class of invertebrate defensins were cloned and sequenced. The deduced amino acid sequences contain the characteristic six cysteines of this class of defensins and reveal precursors of 60 or 61 amino acid residues. The mature peptides consist of 37 amino acid residues, showing up to 70% identities with tick and scorpion defensins. In C. salei, defensin mRNA was found to be constitutively expressed in hemocytes, ovaries, subesophageal nerve mass, hepatopancreas, and muscle tissue. This is the first report presenting and comparing antimicrobial peptides belonging to the family of defensins from spiders.
... Specifically, the second intron of defensin 1 out of two, which is not represented in defensin 2, is the first intron found in the coding part of the defensin gene in arthropods. The short aminated C-terminal elongations are revealed only in the hymenopteran de- fensins [20,21]. These elongations from 11 amino acids seem possibly acquiring the defensin molecule form of alpha-helix that stabilizes amino Cends [14]. ...
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Specific conditions of life of the honeybee require effective mechanisms of anti-infectious protection, one of the most important components of which are dephenyl Sina - a family of antimicrobial peptides. In honeybee defensins are present as two different peptides -defensina 1 and 2 having a similarity between only 55.8%. Defensin 1, synthesized in the salivary glands, plays an important role in social immunity, whereas defensin 2, synthesized by cells of the fat body and hemolymph, is an important factor in the individual immunity of the honey bee. Defensins inducible, controlled interaction Toll and Imd signaling pathways and have a broad spectrum of antimicrobial action. Специфические условия жизни медоносной пчелы требуют наличия эффективных механизмов противоинфекционной защиты, одним из важнейших компонентов которой являются дефен-сины — семейство антимикробных пептидов. У медоносной пчелы дефенсины присутствуют в виде двух разных пептидов —дефенсина 1 и 2, обладающих сходством между собой лишь на 55.8 %. Дефенсин 1, синтезируемый в слюнных железах, выполняет важную роль в социальном иммунитете, тогда как дефенсин 2, синтезируемый клетками жирового тела и гемолимфы, является важным фактором в системе индивидуального иммунитета медоносной пчелы. Дефенсины индуцибельны, контролируются взаимодействием Toll и Imd сигнальных путей и обладают широким спектром антимикробного действия. Ilyasov R.A., Gaifullina L.R., Saltykov E.S., Poskryakov A.V., Nikolenko A.G. Antimicrobial protection of defensins in the honeybees. Journal of Evolutionary Biochemistry and Physiology. 2012, v. 48 (5) . P. 425-432.
... A class of such anti-microbial peptides known as insect defensins (Ganz 2003) are induced in the haemolymph of dipterians (Hoffmann andHoffmann 1990, Hara andYamakawa 1995) in response to bacterial invasion or injury Natori 1988, Lambert et al. 1989). One such defensin, sapecin homologue B isolated from Sacrophaga peregrina (flesh fruit fly), was highly active against Gram-positive bacteria and to a lesser extent against Gram-negative bacteria (Matsuyama and Natori 1990). 1 H-NMR studies of sapecin B (Bontems et al. 1991) identified a structural motif with an a-helix and a b-sheet linked by three intra-molecular disulphide bonds (Hanzawa et al. 1990). This motif shares similarities with a scorpion venom, charybdotoxin, that inhibits potassium channels (Yamada and Natori 1993, Miller et al. 1985, Gimenez-Gallego et al. 1988). ...
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Combinatorial search of the antimicrobial peptide R7SLCLLHCRLK from flesh fruit fly yielded a substantially more active peptide of the sequence KLKL5KLK-NH2 that had signal sequence character as revealed by Neural-network survey. Bioinformatics survey of KLKLnKLK revealed a sigmoidal relationship between SSP and the intervening Leu stretch. Synthetic enantiomeric KLKLnKLK peptides inhibited Escherichia coli signal peptidase-I, invitro, in correlation with their SSPs; KLKL6(7)KLK exterted maximum inhibition. Both (l)-and (d)-forms were bactericidal to Gram-positive and Gram-negative bacteria. However, the protease-resistant (d)-KLKL6KLK-NH2 proved more potent than (d)-KLKL6KLK-NH2 at inhibiting the bacterial protein secretion prior to inducing bacterial lysis. Kinetic analyses of the interaction of these peptides with the signal peptidase-I revealed competitive inhibition with Ki of 10μM and 35μM for the (d)- and (l)-forms, respectively. The left and right-handed helicity of the respective peptides assessed by CD concurs with their probable interaction at the active site of signal peptidase-I.
... Three-dimensional (3D) models of navidefensin1-1 and navidefensin2-1 were constructed by comparative modeling with sapecin's structure (PDB: 1L4V) [36] as a template, which confirmed their typical CSαβ folding, as identified by an α-helix and a two-stranded β-sheet. Residues spanning S15-S23 in navidefensin1-1 and S18-A26 in navidefensin2-1 form an α-helix with separate hydrophobic (A16, A17 and L22 in navidefensin1-1 or A19, A21, V22, L25, and A26 in navidefensin2-1) and hydrophilic (S15, N20, and S23 in navidefensin1-1 or S18 and R23 in navidefensin2-1) surfaces. ...
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Antimicrobial peptides (AMPs) are an essential component of innate immunity which can rapidly respond to diverse microbial pathogens. Insects, as a rich source of AMPs, attract great attention of scientists in both understanding of the basic biology of the immune system and searching molecular templates for anti-infective drug design. Despite a large number of AMPs have been identified from different insect species, little information in terms of these peptides is available from parasitic insects. By using integrated computational approaches to systemically mining the Hymenopteran parasitic wasp Nasonia vitripennis genome, we establish the first AMP repertoire whose members exhibit extensive sequence and structural diversity and can be distinguished into multiple molecular types, including insect and fungal defensin-like peptides (DLPs) with the cysteine-stabilized alpha-helical and beta-sheet (CSalphabeta) fold; Pro- or Gly-rich abaecins and hymenoptaecins; horseshoe crab tachystatin-type AMPs with the inhibitor cystine knot (ICK) fold; and a linear alpha-helical peptide. Inducible expression pattern of seven N. vitripennis AMP genes were verified, and two representative peptides were synthesized and functionally identified to be antibacterial. In comparison with Apis mellifera (Hymenoptera) and several non-Hymenopteran model insects, N. vitripennis has evolved a complex antimicrobial immune system with more genes and larger protein precursors. Three classical strategies that are likely responsible for the complexity increase have been recognized: 1) Gene duplication; 2) Exon duplication; and 3) Exon-shuffling. The present study established the N. vitripennis peptidome associated with antimicrobial immunity by using a combined computational and experimental strategy. As the first AMP repertoire of a parasitic wasp, our results offer a basic platform for further studying the immunological and evolutionary significances of these newly discovered AMP-like genes in this class of insects.
... Using N -terminal sequencing, we determined the entire amino acid sequence (40 amino acids) of peptide in fraction M15, but we succeeded in determining only ten N -terminal amino acids in fraction M14. Provided that cysteines are in positions X (Table 4), the sequence of peptide M15 almost completely matches with S. peregrina sapecin [59]. The only difference is in the position 34 (G for S. bullata and A for S. peregrina). ...
Article
We chose the larvae of fleshfly Sarcophaga bullata to map the peptide and protein immune response. The hemolymph of the third-instar larvae of S. bullata was used for isolation. The larvae were injected with bacterial suspension to induce an antimicrobial response. The hemolymph was separated into crude fractions, which were subdivided by RP-HPLC, gel electrophoresis, and free-flow electrophoresis. In several fractions, we determined significant antimicrobial activities against the pathogenic bacteria Escherichia coli, Staphylococcus aureus, or Pseudomonas aeruginosa. Among antimicrobially active compounds we identified dipeptide β-alanyl-L-tyrosine, protein transferrin, and two variants of peptide sapecin. We also partially characterized two novel antimicrobially active polypeptides; odorant-binding protein 99b, and a peptide which remains unidentified.
... They report that these components have features in common with insect antimicrobial peptides belonging to the defensin group [1,15] for which the generic term insect defensins was proposed some time ago [12]. It is surprising that structural characterisation of Lucilia defensin has not yet been reported, since the first insect defensins isolated from related dipteran species were entirely characterised two decades ago [11,12,[17][18][19][20]. In this paper, we report for the first time the primary sequence of L. sericata defensin, for which we coined the name lucifensin. ...
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A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.
... Insect defensins are among the best-characterized antimicrobial peptides. A study of the threedimensional structures of Phormia defensin A (Bonmatin et al., 1992;Cornet et al., 1995) and Sarcophaga sapecin (Hanzawa et al., 1990) revealed that insect defensins consist of an aminoterminal loop, a single a-helix, and a two-stranded anti-parallel b-sheet folded into the cysteine-stabilized ab (Csab) motif (Cornet et al., 1995). Since three disulfide bridges support this motif, insect defensin sequences require six highly conserved cysteine residues. ...
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We purified an antibacterial peptide from the larval hemolymph of a coleopteran beetle, Acalolepta luxuriosa. Using structure analysis and cDNA cloning, this peptide was identified as a novel member of the insect defensin family and named A. luxuriosa defensin 1. A. luxuriosa defensin 1 shares high sequence similarity with other defensins, especially coleopteran defensins. A dendrogram of coleopteran insect defensins based on sequence homology revealed that A. luxuriosa defensin is closer to Tenebrionoidea defensin than to Scarabaeoidea defensin, which parallels the evolutionary relationship of these coleopteran insects. Although A. luxuriosa defensin 1 was most homologous with Tenebrio molitor tenecin 1, its antibacterial spectrum was broader, affecting the growth of Gram-positive and Gram-negative bacteria, suggesting that the variability of the antibacterial spectrum results from small sequence differences.
... In contrast to cecropins, defensins mainly attack Gram positive bacteria (Boman, 1991). They are 4 kDa cationic peptides with a characteristic six cysteine/three disulfide bridge pattern and three domains, a flexible amino-terminal loop, a central αhelix and a carboxy-terminal anti-parallel β-sheet (Hanzawa et al., 1990;Bonmatin et al., 1992;Bulet et al., 1999). Several reports have described the presence of defensins in different insect species (Bulet et al., 1999), but not in Lepidoptera. ...
Article
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The mechanisms of the innate immunity in the insects have been reviewed. In particular, thecellular component (phagocytosis, encapsulation, melanization, nodule formation, wound healing,hemolymph clotting and transplantation) and the humoral component (lectins, cytokine-like moleculesand anti-microbial peptides) of the hemolymph have been investigated.
... The structure of insect defensins is composed of an Nterminal loop, an α-helix, followed by an antiparallel βsheet ( Fig. 1a) (Bonmatin et al. 1992;Cornet et al. 1995;Hanzawa et al. 1990). The α-helix and β-sheet are linked by two intramolecular disulfide bonds, forming a "cysteine-stabilized alpha beta (CSαβ)" or "loop-helix-beta-sheet" structure (Cornet et al. 1995). ...
Article
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Insects are one of the major sources of antimicrobial peptides/proteins (AMPs). Since observation of antimicrobial activity in the hemolymph of pupae from the giant silk moths Samia Cynthia and Hyalophora cecropia in 1974 and purification of first insect AMP (cecropin) from H. cecropia pupae in 1980, over 150 insect AMPs have been purified or identified. Most insect AMPs are small and cationic, and they show activities against bacteria and/or fungi, as well as some parasites and viruses. Insect AMPs can be classified into four families based on their structures or unique sequences: the α-helical peptides (cecropin and moricin), cysteine-rich peptides (insect defensin and drosomycin), proline-rich peptides (apidaecin, drosocin, and lebocin), and glycine-rich peptides/proteins (attacin and gloverin). Among insect AMPs, defensins, cecropins, proline-rich peptides, and attacins are common, while gloverins and moricins have been identified only in Lepidoptera. Most active AMPs are small peptides of 20-50 residues, which are generated from larger inactive precursor proteins or pro-proteins, but gloverins (~14 kDa) and attacins (~20 kDa) are large antimicrobial proteins. In this mini-review, we will discuss current knowledge and recent progress in several classes of insect AMPs, including insect defensins, cecropins, attacins, lebocins and other proline-rich peptides, gloverins, and moricins, with a focus on structural-functional relationships and their potential applications.
... These protein species play an important role in the innate immune systems of those organisms and mainly affect Gram-positive bacteria and appear to be the abundant group throughout the inducible AMP [10]. Recently, a novel insect defensin-like peptide DLP4 is isolated and identified in immunized hemolymph and various tissues of BSF; it shares high sequence similarity (60-67.5%) to other insect defensins, such as sapecin, and lucifensin [11][12][13]. Structurally, defensin and defensin-like AMPs have a common conserved three intermolecular disulfide bridges motif with a molecular size of 3-4 kDa [14]. Ubiquitin was first isolated from bovine thymus as an adenylate cyclase stimulating polypeptide [15].Ubiquitin is a highly conserved polypeptide consisting of 74-76 amino acid residues with a molecular weight of 8. 4-8.5 kDa. ...
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Black soldier fly (BSF) larvae are often exposed to organic waste which harbors abundant zoonotic pathogens. We investigated the ability of BSF larvae to inhibit the zoonotic pathogens naturally found in pig manure. The zoonotic pathogens populations were detected by using selective medium during the conversion. Results showed that the viability of the zoonotic pathogens in pig manure was significantly affected. After eight days of conversion, the Coliform populations were undetected, and Staphylococcus aureus and Salmonella spp. decreased significantly on the eighth day. Antimicrobial assays of the purified recombinant defensin-like peptide 4 (DLP4) showed that this peptide exhibits inhibitory activity against S. aureus, Salmonella enterica serovar typhimurium, and Escherichia coli in vitro. Bacteria BSF-CL and BSF-F were isolated from the larvae gut, and both inhibited the growth of S. aureus and E. coli, but Salmonella spp. was sensitive to the BSF-CL strain (but not to the BSF-F strain). The results from our experiments indicate that BSF larvae are capable of functionally inhibiting potential zoonotic pathogens in pig manure through a variety of mechanisms including antimicrobial peptides expression and the gut associate microorganisms. This study provides a theoretical basis for further study on the combined mechanism of BSF larvae immunity and its gut microbes against the zoonotic pathogens in pig manure.
...  ÷àñòíîñòè, âòîðîé èíòðîí defensin 1 èç èìåþùèõñÿ äâóõ, êîòîðûé íå ïðåäñòàâëåí ó defensin 2, ÿâëÿåòñÿ ïåðâûì èíòðîíîì, íàéäåííûì â êîäèðóþùåé ÷àñòè ãåíà äåôåíñèíà ÷ëåíèñòîíîãèõ. Êîðîòêèå àìèíèðîâàííûå Ñ-êîíöåâûå óäëèíåíèÿ íàéäåíû òîëüêî â äåôåíñèíàõ ïåðåïîí÷àòîêðûëûõ [20,21]. Ýòè óäëèíåíèÿ â 11 àìèíîêèñëîò, âîçìîaeíî, ïîçâîëÿþò ïðèíèìàòü ìîëåêóëå äåôåíñèíà ñòðóêòóðó àëüôà-ñïèðàëè, êîòîðàÿ ñòàáèëèçèðóåò àìèíèðîâàííûå Ñ-êîíöû [14]. ...
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Specific conditions of life of the honeybee require effective mechanisms of anti-infectious protection, one of the most important components of which are dephenyl Sina - a family of antimicrobial peptides. In honeybee defensins are present as two different peptides -defensina 1 and 2 having a similarity between only 55.8%. Defensin 1, synthesized in the salivary glands, plays an important role in social immunity, whereas defensin 2, synthesized by cells of the fat body and hemolymph, is an important factor in the individual immunity of the honey bee. Defensins inducible, controlled interaction Toll and Imd signaling pathways and have a broad spectrum of antimicrobial action. Специфические условия жизни медоносной пчелы требуют наличия эффективных механизмов противоинфекционной защиты, одним из важнейших компонентов которой являются дефен-сины — семейство антимикробных пептидов. У медоносной пчелы дефенсины присутствуют в виде двух разных пептидов —дефенсина 1 и 2, обладающих сходством между собой лишь на 55.8 %. Дефенсин 1, синтезируемый в слюнных железах, выполняет важную роль в социальном иммунитете, тогда как дефенсин 2, синтезируемый клетками жирового тела и гемолимфы, является важным фактором в системе индивидуального иммунитета медоносной пчелы. Дефенсины индуцибельны, контролируются взаимодействием Toll и Imd сигнальных путей и обладают широким спектром антимикробного действия. Ilyasov R.A., Gaifullina L.R., Saltykova E.S., Poskryakov A.V., Nikolaenko A.G. Defensins in the Honeybee Antiinfectious Protection. Journal of Evolutionary Biochemistry and Physiology, 2013, V. 49 (1), P. 1-9.
... Code PDB Nom Auteurs Défensines de plantes (13) (Fant et al., 1999) - (Fant et al., 1998) - (Bruix et al., 1993a) - (Bruix et al., 1993b) Silva et al., 2003) - (Lamberty et al., 2001) - (Landon et al., 1997) - (Cornet et al., 1995) - (Hanzawa et al., 1990) Défensines de champignons (4) (Blanc et al., 1996) - (Landon et al., 1996) - (Housset et al., 1994) - (Bontems et al., 1992) Tableau 1 -Tableau regroupant toutes les protéines comportant le motif CSαβ répertoriées dans la Protein Data Bank (PDB) ...
Article
My thesis focuses on the AhPDF1 plant defensin from Arabidopsis halleri. AhPDF1 is a 51-residue, cysteine-rich protein involved in the plant defense, and playing an antifungal role. AhPDF1 defensin has eight cysteins involved in its four disulfide bridges, and presents a folding in CSαβ. Recent work on AhPDF1 allowed to identify a new function : the tolerance to heavy metals, especially zinc tolerance. The overall objective of the project that fits my thesis is to understand, at the atomic level and in relation to the oxidation state of cysteins, the mechanism by which the plant defensins confer zinc tolerance. In a major part of my thesis I worked on the production of AhPDF1 defensin first in Escherichia coli and in Pichia pastoris. Then, I developed the chemical synthesis of AhPDF1 and optimized the most delicate step of the oxidative folding. After producing AhPDF1 defensin in sufficient quantity and quality, I realized its structural study by NMR. Furthermore, this structure was used as starting structure, for modeling by homology all other defensins currently identified from Arabidopsis halleri and Arabidopsis thaliana. Finally, I learnt how to master the conditions that maintain the protein in a partially reduced state in order to achieve the first assay of zinc chelation with defensin.
... Conservative analysis and sequence alignment showed that both DLP2 and DLP4 have high sequence similarity (60-65%) to insect defensins: sapecin 16 , insect defensin A 12 , and lucifensin 8 , which are composed of 40 amino acid residues (Fig. 1A). DLP2 and DLP4 were predicted to adopt the characteristic structure of insect defensins including a loop (residues 1-12), three pairs of disulfide bonds, α-helix (residues [13][14][15][16][17][18][19][20][21][22][23] and an antiparallel β-sheet (residues 28-31 and 34-38) (Fig. 1B), which folding into a typical CSαβ conformation. Additionally, an amphipathic surface can be observed in DLP2, DLP4, sapecin, and lucifensin ( Fig. 1C), indicating that these peptides maybe interact with anionic membranes of bacteria. ...
Article
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Methicillin-resistant Staphylococcus aureus (MRSA), are the most frequent cause of sepsis, which urgently demanding new drugs for treating infection. Two homologous insect CSαβ peptides-DLP2 and DLP4 from Hermetia illucens were firstly expressed in Pichia pastoris, with the yields of 873.5 and 801.3 mg/l, respectively. DLP2 and DLP4 displayed potent antimicrobial activity against Gram-positive bacteria especially MRSA and had greater potency, faster killing, and a longer postantibiotic effect than vancomycin. A 30-d serial passage of MRSA in the presence of DLP2/DLP4 failed to produce resistant mutants. Macromolecular synthesis showed that DLP2/DLP4 inhibited multi-macromolecular synthesis especially for RNA. Flow cytometry and electron microscopy results showed that the cell cycle was arrested at R-phase; the cytoplasmic membrane and cell wall were broken by DLP2/DLP4; mesosome-like structures were observed in MRSA. At the doses of 3 7.5 mg/kg DLP2 or DLP4, the survival of mice challenged with MRSA were 80 100%. DLP2 and DLP4 reduced the bacterial translocation burden over 95% in spleen and kidneys; reduced serum pro-inflammatory cytokines levels; promoted anti-inflammatory cytokines levels; and ameliorated lung and spleen injury. These data suggest that DLP2 and DLP4 may be excellent candidates for novel antimicrobial peptides against staphylococcal infections.
... Defensins are 4-6 kDa cyclic peptides and are the most widespread insect's AMPs (Čeřovský and Bém, 2014). S. peregrine sapecin possesses an N-terminal flexible loop, a central α-helix and a C-terminal anti-parallel β-sheet (Hanzawa et al., 1990). L. sericata defensin (lucifensin) differs from sapecin by five amino acid residues (Čeřovský and Bém, 2014). ...
... They report that these components have features in common with insect antimicrobial peptides belonging to the defensin group [1,15] for which the generic term insect defensins was proposed some time ago [12]. It is surprising that structural characterisation of Lucilia defensin has not yet been reported, since the first insect defensins isolated from related dipteran species were entirely characterised two decades ago [11,12,[17][18][19][20]. In this paper, we report for the first time the primary sequence of L. sericata defensin, for which we coined the name lucifensin. ...
... On the basis of different structural features, AMPs can roughly be divided into the following groups: Peptides rich in cysteines, such as defensins and protegrins, adopt a relatively rigid antiparallel ␤-sheet or ␣-helix/␤-sheet architecture stabilized by 2 to 4 intramolecular disulfide bridges (13,14). Molecules in this group virtually all contain a unifying structural signature, named the ␥-core motif (15). ...
Article
Both vertebrates and invertebrates employ alpha-helical antimicrobial peptides (AMPs) as an essential component of their innate immune system. However, evolutionary relation of these immune molecules remains unresolved. Venoms, as key weapons of venomous arthropods for prey and defense, receive increasing recognition as an emerging source of such peptides. From a cDNA library prepared from the venom gland of the scorpion Mesobuthus eupeus, clones encoding precursors of two new AMPs, named meucin-13 (IFGAIAGLLKNIF-NH(2)) and meucin-18 (FFGHLFKLATKIIPSLFQ), have been isolated. The precursor of meucins consists of a signal peptide, a mature peptide, and an acidic propeptide, in which dibasic residues as the typical processing signal are located between the mature and propeptide. Meucin-13 is an ortholog of several previously described AMPs from scorpion venom and has also detectable sequence similarity to temporins, a large family of AMPs from frog skin, whereas meucin-18 displays some similarity to AMPs from diverse origin including arthropod venoms, fish mast cells, and frog skins. These two meucin peptides form alpha-helical structure in the presence of 50% trifluoroethanol (TFE), a membrane-mimicking environment, as identified by circular dichroism (CD) spectroscopy. This finding is further verified by their NMR structures that show a typical alpha-helical amphipathic design, a structural prerequisite for cytolytic activity. Meucins exhibit extensive cytolytic effects on both prokaryotic and eukaryotic cells (gram(+) and gram(-) bacteria, fungi, yeasts, rabbit erythrocytes, and rat dorsal root ganglion cells) at micromolar concentrations. It is remarkable that muecin-18 was 2- to >14-fold more potent than meucin-13 against nearly all the cells tested. Structural differences in hydrophilic/hydrophobic balance and cationic amino acid location between two meucins could account for their differential potency. Despite these differences, commonalities at precursor organization, three-dimensional structure, and biological function suggests that meucins are two evolutionarily related AMPs and likely originated from a common ancestor by gene duplication. Our work presented here also provides new insights into an evolutionary link among AMPs from invertebrates and vertebrates and clues for evolutionary convergence between AMPs and virus fusion domains.
... Tridimensional structure and sequence alignment of insect and mollusk defensins, presenting the insect defensins: (a) lucifensin -PDB: 2LLD from Lucilia sericata [71], (b) defensin A -PDB: 1ICA from Phormia terranovae [16], (c) DEF-AAA -PDB: 2NY8 from Anopheles gambiae [53], (d) sapecin -PDB: 1L4V from Sarcophaga peregrina [35], (e) termicin -PDB: 1MM0 from Pseudacanthotermes spiniger [18], and (f) heliomicin -PDB: 1I2U from Heliothis virescens [51]; and the mussel defensins: (g) MGD-1 -PDB: 1FJN from Mytilus galloprovincialis [109] and (h) Cg-Def -PDB: 2B68 from Crassostrea gigas [33] structures and the (i) insect and (j) mussel defensins sequence alignment. The colors in the figure represent the cysteines and disulfide bonds (yellow) and positive (Arg, Lys and His -blue) and negative (Glu and Asp -red) charged residues. ...
Article
Antimicrobial peptides (AMPs) seem to be promising alternatives to common antibiotics, which are facing increasing bacterial resistance. Among them are the cysteine-stabilized αβ defensins. These peptides are small, with a length ranging from 34 to 54 amino acid residues, cysteine-rich and extremely stable, normally composed of an α-helix and three β-strands stabilized by three or four disulfide bonds and commonly found in several organisms. Moreover, animal and plant CSαβ defensins present different specificities, the first being mainly active against bacteria and the second against fungi. The role of the CSαβ-motif remains unknown, but a common antibacterial mechanism of action, based on the inhibition of the cell-wall formation, has already been observed in some fungal and invertebrate defensins. In this context, the present work aims to group the data about CSαβ defensins, highlighting their evolution, conservation, structural characteristics, antibacterial activity and biotechnological perspectives. Copyright © 2015. Published by Elsevier Inc.
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Article
Defensins are a family of small, variably cationic proteins which are highly abundant in the granules of mammalian phagocytes. Three defensins, HNP-1, 2, and 3, comprise 30–50% of total protein in azurophil granules of human neutrophils. Some defensins are broadly antimicrobial, antiviral and cytotoxic, while others are chemotactic, opsonic, or may modulate hormonal responses. The defensin molecule typically consists of 29–34 amino acids with a conserved pattern of disulfide linkage among its 6 cysteines. The three-dimensional fold of defensins forms a highly amphiphilic molecule. Microbicidal and cytotoxic properties of defensins are most likely a consequence of their ability to insert into biological membranes and to generate pores. Defensins are synthesized by phagocytes or their precursors as a 94–95 amino acid charge-neutralized preprodefensin, an arrangement which may avoid cytotoxic injury to the phagocyte. Although defensins were recognized only recently, the existence of homologs in certain invertebrates suggests that they are ancestral components of the host defense system.
Article
Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves—to inhibit competitors—but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter.
Article
Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in E. coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra- disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin. Copyright © 2015. Published by Elsevier Inc.
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Background: "Invertebrate defensins" belong to the cysteine-stabilized alpha-beta (CS-αβ), also known as the scorpion toxin-like, superfamily. Some other peptides belonging to this superfamily of defensive peptides are indistinguishable from "defensins," but have been assigned other names, making it unclear what, if any, criteria must be met to qualify as an "invertebrate defensin." In addition, there are other groups of defensins in invertebrates and vertebrates that are considered to be evolutionarily unrelated to those in the CS-αβ superfamily. This complicates analyses and discussions of this peptide group. This paper investigates the criteria for classifying a peptide as an invertebrate defensin, suggests a reference cysteine array that may be helpful in discussing peptides in this superfamily, and proposes that the superfamily (rather than the name "defensin") is the appropriate context for studying the evolution of invertebrate defensins with the CS-αβ fold. Methods: CS-αβ superfamily sequences were identified from previous literature and BLAST searches of public databases. Sequences were retrieved from databases, and the relevant motifs were identified and used to create a conceptual alignment to a ten-cysteine reference array. Amino acid sequences were aligned in MEGA6 with manual adjustments to ensure accurate alignment of cysteines. Phylogenetic analyses were performed in MEGA6 (maximum likelihood) and MrBayes (Bayesian). Results: Across invertebrate taxa, the term "defensin" is not consistently applied based on number of cysteines, cysteine spacing pattern, spectrum of antimicrobial activity, or phylogenetic relationship. The analyses failed to reveal any criteria that unify "invertebrate defensins" and differentiate them from other defensive peptides in the CS-αβ superfamily. Sequences from various groups within the CS-αβ superfamily of defensive peptides can be described by a ten-cysteine reference array that aligns their defining structural motifs. Conclusions: The proposed ten-cysteine reference array can be used in addition to current nomenclature to compare sequences in the CS-αβ superfamily and clarify their features relative to one another. This will facilitate analysis and discussion of "invertebrate defensins" in an appropriate evolutionary context, rather than relying on nomenclature.
Article
Pricking the body wall of Sarcophaga peregrina (flesh fly) larvae with a needle activated the immune system of this insect and induced various immune molecules, including antibacterial proteins, in the hemolymph. In this review, I summarize and discuss the functions of these immune molecules, with particular emphasis on the dual roles of some of these molecules in defense and development.
Article
Royalisin found in the royal jelly of Apis mellifera is an antimicrobial peptide (AMP). It has a molecular weight of 5.5 kDa, which contains six cysteine residues. In this study, royalisin was overexpressed in Escherichia coli AD494 (DE3) as two oleosin-fusion proteins for preparation of its antibodies and functional purification. The recombinant royalisin, fused with oleosin central hydrophobic domain in both N- and C-termini, was reconstituted with triacylglycerol and phospholipids to form artificial oil bodies (AOBs). The AOBs were then purified to raise the antibodies. These antibodies could recognize both the native and recombinant royalisins, but not oleosin. Another oleosin-intein S-fusion protein was purified by AOBs system, and royalisin was subsequently released from the AOBs through self-splicing of the intein. The recombinant royalisin exhibited high antibacterial activity, which suggested that it was refolded to its functional structure. These results demonstrated that AOBs system is an efficient method to functionally express and purify small AMPs. In addition, it also provides a facile platform for the production of antibodies against small peptides.
Chapter
Scorpions form an order of animals in the phylum of Arthropods. Approximately 1400 species of scorpions, spread into 9 families, are distributed on all major land masses of the world, except Antartica. Scorpions successfully survived over the past 450 million years, having undergone little morphological change since their appearance, despite a number of functional differences regarding their locomotion and respiration apparatus. As a result, scorpions are often considered as “living fossils”, which carry a number of unique biological informations (1). Recent progresses have emerged in the literature regarding the molecular properties of venoms and toxins from scorpions and their ability to act on a wide diversity of prey. In particular, structural studies have shed light on the molecular basis associated with the functional diversity of scorpion toxins.
Chapter
An animal’s environment contains smaller entities that may attack it and cause illness or death. The immune system evolved to protect against such threats. It has two branches, one innate and the other adaptive. The former relies on field-tested molecules that have been selected over eons. Since they are gene-encoded, these innate molecules are deployed with little or no delay. The adaptive immune system consists of molecular and cellular machinery that produces custom-tailored molecules. Its handiwork is relatively slow, and many clients in need of its products would be lost if their innate systems did not also exist. This chapter focuses on cysteine-containing antimicrobial peptides that contain one or more internal disulfide bonds. Special emphasis is placed on the evolution of two superfamilies of defensins: small, usually cationic and amphipathic host defense molecules with three or four intramolecular disulfide bonds. The ancient roots of both defensin groups predate the advent of adaptive immunity by hundreds of millions of years. One superfamily includes the α-, β-, γ-, and θ-defensins of vertebrates, and the “big defensins” found in cephalochordates, mollusks, and crustaceans. The other superfamily of defensins is expressed in arthropods, mollusks, and fungi and may have arisen much earlier. Like defensins, the evolution of other families of cysteine-containing AMPs can be traced to the predawn of vertebrate existence. Collectively and individually, antimicrobial peptides provide a broad range of protective effects. Yet, despite their essential contributions to animal existence, and perhaps because specificity ranks higher than efficacy in the view of most immunologists, AMPs have often been undervalued. Ironically, it is precisely because AMPs lack specificity that these broadly efficacious molecules have been conserved and refined for more than one billion years.
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Defensins are widely distributed and abundant 3–4kDa antimicrobial peptides that are variably cationic and contain six disulfide-paired cysteines. Three structurally distinct peptide families have been identified: ‘classical’ defensins, β-defensins and insect defensins. In many animal species, defensin genes are found in clusters with substantial sequence variability outside the core disulfide-linked cysteines. Defensin peptides have been found in the granules of phagocytes and intestinal Paneth cells, on epithelial surfaces of the intestine and the trachea, and in the hemolymph of insects. They are produced from larger precursors by stepwise, tissue-specific, proteolytic processing, a production resembling that of peptide hormones. Microbes in the phagocytic vacuoles of granulocytes and certain macrophages encounter high concentrations of defensins. Increased transcription of defensin genes and stimulus-dependent release of pre-synthesized defensin-containing cytoplasmic granules contribute to the local antimicrobial response.
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A novel super-secondary structure common for many non-homological proteins is considered. This folding pattern, consisting of adjacent along the chain α-helix and β-hairpin, has an aligned packing. It is found that one of the two possible ‘mirror-symmetrical’ topologies is observed in proteins. The α-helix + β-hairpin structures have a similar pattern of hydrophobic residues in their amino acid sequences. The remaining part of a molecule or a domain is almost always located on the same side of the considered folding pattern. These results can be used in the prediction of three-dimensional protein structure and protein design.
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The primary structures of four low molecular mass peptides (Bs 6, 8, 10 and 14) from scorpion Buthus sindicus were elucidated via combination of Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. Bs 8 and 14 are cysteine-rich, thermostable peptides composed of 35–36 residues with molecular weights of 3.7 and 3.4 kDa, respectively. These peptides show close sequence homologies (55–78%) with other scorpion chlorotoxin-like short-chain neurotoxins (SCNs) containing four intramolecular disulfide bridges. Despite the sequence variation between these two peptides (37% heterogeneity) their general structural organization is very similar as shown by their clearly related circular dichroism spectra. Furthermore, Bs6 is a minor component, composed of 38 residues (4.1 kDa) containing six half-cystine residues and having close sequence identities (40–80%) with charybdotoxin-like SCNs containing three disulfide bridges. The non-cysteinic, bacic and thermolabile Bs10 is composed of 34 amino acid residues (3.7 kDa), and belongs to a new class of peptides, with no sequence resemblance to any other so far reported sequence isolated from scorpions. Surprisingly, Bs10 shows some limited sequence analogy with oocyte zinc finger proteins. Results of these studies are discussed with respect to their structural similarities within the scorpion LCNs, SCNs and other biologically active peptides.
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Honey bees defensin have a high level of polymorphism and exist as two peptides - defensin 1 and 2. Defensin 1 is synthesized in the salivary glands and is responsible for social immunity. Defensin 2 is synthesized by cells of the fat body and hemolymph is responsible for individual immunity. Defensins are inducible and controlled by the interaction of Toll and Imd signaling pathways and have a broad spectrum of antimicrobial action. The use of chitosan as an immunomodulator has been shown to lead to an increase in the expression levels of defensin and abaecin in the honey bee organism. Stimulation of the transcriptional activity of the defensin genes will allow for the control of a honey bee colony’s immunity level, and reduce the using of antibiotics and other chemicals. Ilyasov R.A., Gaifullina L.R., Saltykova E.S., Poskryakov A.V., Nikolenko A.G. Review of the Expression of Antimicrobial Peptide Defensin in Honey bees Apis mellifera L. Journal of Apicultural Science. 2012. Vol.56 No.1. P.115-124.
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An active fragment was identified from tenecin 1, an antibacterial protein belonging to the insect defensin family, by synthesizing the peptides corresponding to the three regions of tenecin 1. Only the fragment corresponding to the C-terminal beta-sheet domain showed activity against fungi as well as Gram-positive and Gram-negative bacteria, whereas tenecin 1, the native protein, showed activity only against Gram-positive bacteria. CD spectra indicated that each fragment in a membrane-mimetic environment might adopt a secondary structure corresponding to its region in the protein. The leakage of dye from liposomes induced by this fragment suggested that this fragment acts on the membrane of pathogens as a primary mode of action. A comparison between the structure and the activity of each fragment indicated that a net positive charge was a prerequisite factor for activity. To the best of our knowledge this is the first report in which the fragment corresponding to the beta-sheet region in antibacterial proteins, which consists of alpha-helical and beta-sheet regions, has been identified as a primary active fragment.
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Based on the amino acid sequence of Allomyrina dichotoma defensin, degenerate primers were synthesized, RT-PCR was done to clone a defensin cDNA and a 114-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended cDNA clone by 3′ and 5′ RACE. The deduced amino acid sequence of the mature portion was identical to that of the purified defensin. Tissue-specific gene expression analyzed by Northern blotting showed that the main sites for defensin gene expression were the fat bodies and hemocytes. The time course of defensin gene expression indicated that expression peaked at 8 to 12 h in larvae injected with Escherichia coli. An analysis of defensin gene expression induction by E. coli or Staphylococcus aureus in the fat body and hemocyte by RT-PCR showed that E. coli induces defensin gene expression more effectively than S. aureus in both tissues.
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The three-dimensional structure of the Sorghum bicolor seed protein γ-thionin SIα1 has been determined by 2D 1H nuclear magnetic resonance (NMR) spectroscopy. The secondary structure of this 47-residue antifungal protein with four disulphide bridges consists of a three-stranded antiparallel sheet and one helix. The helix is tethered to the sheet by two disulphide bridges which link two successive turns of the helix to alternate residues i, i + 2 in one strand. Possible binding sites for antifungal activity are discussed. The same fold has been observed previously in several scorpion toxins. Proteins 32:334–349, 1998. © 1998 Wiley-Liss, Inc.
Article
Synopsis The diversity of biological functions that are exerted by toxins from snake and scorpion venoms is associated with a limited number of structural frameworks. At present, one predominant basic fold has been observed among scorpion toxins whereas six folds have been found among snake toxins. Most toxin folds have the capacity to accept multiple insertions, deletions and mutations and to exert various recognition functions. We suggest that such folds may serve as guides to engineer new protein functions.
Article
Objective: To critically analyze the existing randomized controlled trials (RCTs) on the clinical, economic, and psychological implications of maggot debridement therapy (MDT). Data sources: An exhaustive literature search for English-language publications was conducted using MEDLINE, EMBASE, and PubMed. Study selection: Keywords used for the search were based on the PICO (Population, Intervention, Comparison, Outcome) framework. The titles, abstracts, and relevant full-text articles were screened. Seven RCTs were selected after applying the inclusion and exclusion criteria. Data extraction: Data pertaining to the primary and secondary outcomes of each study were extracted. Data synthesis: The data extracted were evaluated and categorized into clinical, economic, and psychological outcomes pertaining to MDT. A judicious evaluation of these outcomes was made, and the following conclusions were drawn. Conclusions: There exists heterogeneity in the extant RCTs, but MDT appears to be effective for a quick early debridement. For diabetic foot ulcers, MDT improves debridement, controls infection, and enhances wound healing. In chronic peripheral vascular disease ulcers, it aids in early debridement, but the final outcome is equivocal. Further robust integrated health economic and parallel qualitative assessment studies are recommended to understand the cost-effectiveness and patient acceptability and experience.
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The thesis for the degree of candidate of biological sciences. Preservation of the gene pool of dark forest honey bee Ammellifera is the base point for the development of beekeeping and bee breeding in Russia which in turn requires solving a number of problems in basic and applied research. Our work touches the three of them. We were able to prove the existence of the Urals several genetic reserves that should be the basis for the preservation of the gene pool Ammellifera. It was expanded set of genetic markers and statistical approaches used for this purpose. We It managed to obtain new data on the structure of intra honeybee A.mellifera. We hope that the results will allow to approach the problem of preserving the gene pool Ammellifera both in Russia and in other Nordic countries. As is known, the use morphometric methods for this purpose little information when using hybrid bees. It is shown that by using molecular methods may determine which subspecies belong to the bees. However, not all genetic loci can become markers. Only strict selection and integrated use of nuclear loci and mitochondrial genomes will be effective in the identification of subtypes of bees. Equally important in the study played a statistical analysis of results. Statistical methods, selection of genetic markers and the character of the collection object is strictly dependent on the goals of the study. Phylogenetic analysis also allowed for the first time to establish close genetic kinship bees Ural and European populations Ammellifera. In parallel, we have been able to show that the evolutionary branch of M consists only of subspecies Ammellifera, whereas previously it was thought that it includes several other A.mellifera subspecies of the North-West Africa and the Iberian Peninsula. The obtained genetic characteristics of populations of bees Urals unique and will serve as a basis for further research, as well as measures for the conservation and restoration of the entire gene pool Ammellifera the territory of the former habitat subspecies. Furthermore, based on these data, we can develop a strategy for the further development and management of beekeeping Ammellifera populations in Russia and Europe. On the basis of polymorphism intergenic locus COI-COII mitochondrial DNA demonstrated the existence of at least four stored locally populations of Apis mellifera mellifera L. in the Urals: Vishera, Kama region of the South, tatyshlinskoy and Burzyan. An analysis of the frequency of occurrence of combinations of PQQ intergenic locus COI-COII mtDNA showed that uinskaya population, previously hosted by morphometric featured for the population Ammellifera, it is a hybrid. Genetic distance Ammellifera between the Ural and the hybrid population can be Iglinsky accepted for further studies at one of the criteria for the safety of aboriginal population A.m.mellifera. A study of polymorphism loci separate nuclear and mitochondrial DNA revealed a close genetic relationship, a small fraction of inbreeding and a lack of heterozygotes in the Ural population Ammellifera. Phylogenetic analysis based on a comparison of the nucleotide ND2 gene fragment sequences of mitochondrial DNA revealed genetic kinship of the Ural and Western European populations Ammellifera. Comparative analysis mitotypes gene fragment of the mitochondrial ND2 DNA shows that subspecies Ammellifera, probably the only Representative intraspecific evolutionary branches of M, which is thus not It should include not only the African subspecies Apis mellifera sahariensis Baldensperger and Apis mellifera intermissa Maa, but Spanish subspecies of Apis mellifera iberica Goetze. It found that species ancestral form Apis mellifera L., could be With bee evolutionary branch, and not, as previously thought. Диссертация на соискание ученой степени кандидата биологических наук. Сохранение генофонда тёмной лесной медоносной пчелы A.m.mellifera являлось базовым моментом для развития пчеловодства и селекции пчел в России, что в свою очередь требует решения целого ряда задач в области фундаментальных и прикладных исследований. Наша работа касалась трёх из них. Нам удалось доказать существование на Урале нескольких генетических резерватов, которые должны стать основой для сохранения генофонда A.m.mellifera. Был расширен комплекс генетических маркёров и статистических подходов, используемых для этого. Нам удалось получить новые данные по внутривидовой структуре медоносной пчелы A.mellifera. Мы надеемся, что полученные результаты позволят приблизиться к решению проблемы сохранения генофонда A.m.mellifera как в России, так и в других странах Северной Европы. Как известно, использование морфометрических методов для этой цели малоинформативно при работе с гибридными пчелами. Показано, что с помощью молекулярных методов возможно определить, к какому подвиду принадлежат пчелы. Тем не менее, не любые локусы могут стать генетическими маркерами. Только строгий подбор и комплексное использование локусов ядерного и митохондриального геномов будет эффективно в идентификации подвидов пчел. Не менее важную роль в исследовании играла статистическая обработка полученных результатов. Методы статистической обработки, подбор генетических маркеров и характер сбора объекта строго зависят от поставленных целей исследования. Также филогенетический анализ позволил впервые установить тесное генетическое родство пчел уральских и европейских популяций A.m.mellifera. Параллельно мы смогли показать, что эволюционная ветвь М состоит только из подвида A.m.mellifera, тогда как раньше считалось, что в нее входят несколько других подвидов A.mellifera из Северо-Западной Африки и Иберийского полуострова. Полученные нами генетические характеристики популяций пчел Урала уникальны и будут служить основой для дальнейших исследований, а также мероприятий по сохранению и восстановлению генофонда A.m.mellifera на всей территории прежнего ареала подвида. Кроме того, на основе этих данных мы можем разработать стратегию дальнейшего развития пчеловодства и управления популяциями A.m.mellifera в России и Европе. На основе полиморфизма межгенного локуса COI-COII митохондриальной ДНК показано существование как минимум четырёх сохранившихся локальных популяций Apis mellifera mellifera L. на Урале: вишерской, южно-прикамской, татышлинской и бурзянской. Анализ частот встречаемости комбинации PQQ межгенного локуса COI-COII мтДНК показал, что уинская популяция, ранее принимаемая по морфометрическим признакам за популяцию A.m.mellifera, являлась гибридной. Генетическое расстояние между уральской A.m.mellifera и гибридной иглинской популяцией может быть принято в дальнейших исследованиях за один из критериев сохранности аборигенной популяции A.m.mellifera. Исследование полиморфизма отдельных локусов ядерной и митохондриальной ДНК выявило тесное генетическое родство, небольшую долю инбридинга и дефицит гетерозигот в уральских популяциях A.m.mellifera. Филогенетический анализ на основе сравнения нуклеотидных последовательностей фрагмента гена ND2 митохондриальной ДНК показал генетическое родство уральских и западноевропейских популяций A.m.mellifera. Сравнительный анализ митотипов фрагмента гена ND2 митохондриальной ДНК свидетельствует, что подвид A.m.mellifera, вероятно, является единственным представителем внутривидовой эволюционной ветви М, в которую, таким образом, не следует включать не только африканские подвиды Apis mellifera sahariensis Baldensperger и Apis mellifera intermissa Maa, но и испанский подвид Apis mellifera iberica Goetze. Обнаружено, что предковой формой вида Apis mellifera L., могли быть пчелы эволюционной ветви С, а не О, как считалось ранее. Ilyasov R.A. Polymorphism of Apis mellifera mellifera L. in Ural. Thesis of Ph.D. 2006. 204 p.
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We have previously identified a defensin-like peptide (BmDp, Genbank accession number DQ118523), in the larvae of a silkworm, Bombyx mori (B. mori) using wdS20658 cDNA in a Bombyx EST database (Silk Base: http://www.ab.a.u-tokyo.ac.jp/silkbase). A full-length of BmDp cDNA was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) and expressed in Escherichia coli cells in order to obtain a functional peptide. Amino acid sequence comparison of cysteine-rich defensin-like peptides with those of the BmDp showed to be identical protein as BmDefensinA designated as BmdefA gene deposited in the DDBJ database under the accession number AB367525. Sequence alignments revealed that the BmDp belongs to the insect defensin family and is close to galiomicin and spodoptericin, a defensin and a defensin-like protein having antimicrobial activities in phylogenetic analysis. Thus, we evaluated the effect of immune challenge and antimicrobial activity to understand the nature of this anionic BmDp. Semiquantitative RT-PCR analysis showed that BmDp gene expression was inducible by bacterial injection and peaked at a highest level at 8h after bacterial injection. This result suggests that BmDp is related with immune response against bacteria. A purified recombinant GSTBmDp fusion protein did not show direct antimicrobial activity against bacteria and fungi, which is differed from cationic antibacterial insect defensins and neutral antifungal defensin-like peptides. Keywords Bombyx mori -Defensin-like protein-Antimicrobial activity
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A new general technique for the investigation of exchange processes in molecular systems is proposed and demonstrated. Applications comprise the study of chemical exchange, of magnetization transfer by inter‐ and intramolecular relaxation in liquids, and of spin diffusion and cross‐relaxation processes in solids.
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A cDNA clone for sapecin, an antibacterial protein produced by an embryonic cell line of Sarcophaga peregrina, was isolated and characterized. This clone was found to encode a precursor of sapecin consisting of 94 residues, with sapecin (40 residues) constituting its carboxyl-terminal half. RNA blot hybridization revealed that the gene for the sapecin precursor is activated in the hemocytes of the third instar larvae of Sarcophaga in response to body injury. Thus, sapecin is probably a defense protein synthesized by Sarcophaga to prevent bacterial infection through the damaged body wall. This gene was also found to be activated in the embryonic and early pupal stages, suggesting that sapecin also plays a role in the ontogenetic processes of Sarcophaga.
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Three antibacterial proteins were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Sequencing studies showed that two of these proteins belong to the sarcotoxin I family, potent antibacterial proteins purified from the hemolymph of Sarcophaga larvae, whereas the other protein, named sapecin, is a new protein consisting of 40 amino acid residues including 6 cysteine residues. Unlike sarcotoxin I, sapecin preferentially represses the growth of various Gram-positive bacteria. The proteins of the sarcotoxin I family produced by this cell line were found to have carboxyl-terminal glycine, whereas sarcotoxin I in the hemolymph has amidated amino acids. This suggests that the embryonic cells lack an enzyme that cleaves off carboxyl-terminal glycine to form a new amidated carboxyl terminus.
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Six microbicidal peptides, NP-1, NP-2, NP-3a, NP-3b, NP-4, and NP-5, from rabbit peritoneal neutrophils were characterized. As a family, the peptides were 32-34 residues in length, were cystine- and arginine-rich, and each contained three intramolecular disulfide bonds. Within their sequences, the six peptides shared 11-residue positions, which included the six half-cystines contained in each peptide. NP-1 and NP-2 differed by a single residue and were identical in their respective sequences to MCP-1 and MCP-2, the peptide analogs from rabbit alveolar macrophages. NP-4 and NP-5 were homologous in 27 of their residues, but NP-3a and NP-3b shared little more than the 11-residue backbone common to all members of this peptide family.
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The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.
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The microbicidal peptides, MCP-1 and MCP-2, of rabbit alveolar macrophages were purified by an improved procedure that employed preparative gel electrophoresis and high performance liquid chromatography. The peptides were arginine- and cystine-rich and lacked free sulfhydryl groups and detectable levels of carbohydrate. Complete sequence determinations revealed that MCP-1 differed from MCP-2 only by the substitution of arginine for leucine at residue 13 from the NH2 terminus and that the molecules were each single chain polypeptides of 33 amino acid residues containing three intramolecular disulfide bonds. The complete amino acid sequences of MCP-1 and MCP-2 are: (sequence in text)
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A method is described for obtaining pure absorption phase spectra in four quadrants in a two-dimensional nuclear magnetic resonance spin exchange experiment. It is shown that phase correction results in a substantial increase in resolution and discrimination while maintaining a signal-to-noise ratio comparable to that of the usual magnitude spectrum. Experimental results are presented for the application of the method to a biological macromolecule, the bovine pancreatic trypsin inhibitor.
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A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.
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Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.
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Solution structures of the rabbit neutrophil defensin NP-5 have been determined by 1H nuclear magnetic resonance (n.m.r.) spectroscopy and distance geometry techniques. This 33 amino acid peptide is part of the oxygen-independent mammalian defense system against microbial infection. The structures were generated from 107 n.m.r. derived inter-residue proton-proton distance constraints. A distance geometry algorithm was then used to determine the range of structures consistent with these distance constraints. These distance geometry calculations employed an improved algorithm that allowed the chirality constraints to be relaxed on prochiral centers when it was not possible to make stereo-specific assignments of protons on these centers. This procedure gave superior results compared with standard distance geometry methods and also produced structures that were more consistent with the original n.m.r. data. Analysis of the NP-5 structures shows that the overall folding of the peptide backbone is well defined by the n.m.r. distance information but that the side-chain group conformations are generally less well defined.
Article
An automated method, based on the principle of simulated annealing, is presented for determining the three-dimensional structures of proteins on the basis of short (less than 5 A) interproton distance data derived from nuclear Overhauser enhancement (NOE) measurements. The method makes use of Newton's equations of motion to increase temporarily the temperature of the system in order to search for the global minimum region of a target function comprising purely geometric restraints. These consist of interproton distances supplemented by bond lengths, bond angles, planes and soft van der Waals repulsion terms. The latter replace the dihedral, van der Waals, electrostatic and hydrogen-bonding potentials of the empirical energy function used in molecular dynamics simulations. The method presented involves the implementation of a number of innovations over our previous restrained molecular dynamics approach [Clore, G.M., Brünger, A.T., Karplus, M. and Gronenborn, A.M. (1986) J. Mol. Biol., 191, 523-551]. These include the development of a new effective potential for the interproton distance restraints whose functional form is dependent on the magnitude of the difference between calculated and target values, and the design and implementation of robust and fully automatic protocol. The method is tested on three systems: the model system crambin (46 residues) using X-ray structure derived interproton distance restraints, and potato carboxypeptidase inhibitor (CPI; 39 residues) and barley serine proteinase inhibitor 2 (BSPI-2; 64 residues) using experimentally derived interproton distance restraints. Calculations were carried out starting from the extended strands which had atomic r.m.s. differences of 57, 38 and 33 A with respect to the crystal structures of BSPI-2, crambin and CPI respectively. Unbiased sampling of the conformational space consistent with the restraints was achieved by varying the random number seed used to assign the initial velocities. This ensures that the different trajectories diverge during the early stages of the simulations and only converge later as more and more interproton distance restraints are satisfied. The average backbone atomic r.m.s. difference between the converged structures is 2.2 +/- 0.3 A for crambin (nine structures), 2.4 +/- 0.3 A for CPI (eight structures) and 2.5 +/- 0.2 A for BSPI-2 (five structures). The backbone atomic r.m.s. difference between the mean structures derived by averaging the coordinates of the converged structures and the corresponding X-ray structures is 1.2 A for crambin, 1.6 A for CPI and 1.7 A for BSPI-2.
Article
We have isolated from the hemolymph of immunized larvae of the dipteran insect Phormia terranovae two peptides that are selectively active against Gram-positive bacteria. They are positively charged peptides of 40 residues containing three intramolecular disulfide bridges and differ from one another by only a single amino acid. These peptides are neither functionally nor structurally related to any known insect immune response peptides but show significant homology to microbicidal cationic peptides from mammalian granulocytes (defensins). We propose the name "insect defensins" for these insect antibiotic peptides.
Article
A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.
Article
"Pseudo-structures" of the 20 common amino acid residues are introduced for use in protein spatial structure determinations, which rely on the use of intramolecular proton-proton distance constraints determined by nuclear Overhauser effects as input for distance geometry calculations. The proposed structures satisfy requirements for the initial structural interpretation of the nuclear magnetic resonance data that arise from the absence of stereospecific assignments and/or limited spectral resolution for certain resonance lines. The pseudo-atoms used as reference points for the experimental distance constraints can be used in conjunction with the real amino acid structures representing the van der Waals' constraints on the spatial molecular structure, or with simplified models in order to reduce the computing time for the distance geometry calculations.
Article
A double quantum filter is inserted into a two-dimensional correlated (COSY) 1H NMR experiment to obtain phase-sensitive spectra in which both cross peak and diagonal peak multiplets have anti-phase fine structure, and in which the cross peaks and the major contribution to the diagonal peaks have absorption lineshapes in both dimensions. The elimination of the dispersive character of the diagonal peaks in phase-sensitive, double quantum-filtered COSY spectra allows identification of cross peaks lying immediately adjacent to the diagonal, which represents a significant improvement over the conventional COSY experiment.
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