Mussel Defensin MGD-1

MGD-1 is a 39-residue defensin-like peptide isolated from the edible Mediterranean mussel, Mytilus galloprovincialis. This peptide is characterized by the presence of four disulfide bonds. We report here its solid-phase synthesis and an easy way to improve the yield of the four native disulfide bonds. Synthetic and native MGD-1 display similar antibacterial activity, suggesting that the hydroxylation of Trp28 observed in native MGD-1 is not involved in the antimicrobial effect. The three-dimensional solution structure of MGD-1 has been established using (1)H NMR and mainly consists of a helical part (Asn7-Ser16) and two antiparallel beta-strands (Arg20-Cys25 and Cys33-Arg37), together giving rise to the common cystine-stabilized alpha-beta motif frequently observed in scorpion toxins. In MGD-1, the cystine-stabilized alpha-beta motif is stabilized by four disulfide bonds (Cys4-Cys25, Cys10-Cys33, Cys14-Cys35, and Cys21-Cys38), instead of by the three disulfide bonds commonly found in arthropod defensins. Except for the Cys21-Cys38 disulfide bond which is solvent-exposed, the three others belong to the particularly hydrophobic core of the highly constrained structure. Moreover, the C4-P5 amide bond in the cis conformation characterizes the MGD-1 structure. MGD-1 and insect defensin A possess similar bactericidal anti-Gram-positive activity, suggesting that the fourth disulfide bond of MGD-1 is not essential for the biological activity. In agreement with the general features of antibacterial peptides, the MGD-1 and defensin A structures display a typical distribution of positively charged and hydrophobic side chains. The positively charged residues of MGD-1 are located in three clusters. For these two defensin peptides isolated from insects and mollusks, it appears that the rather well conserved location of certain positively charged residues and of the large hydrophobic cluster are enough to generate the bactericidal potency and the Gram-positive specificity.

Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II. Hydrophilic and hydrophobic areas of mytilin account for 63% and 37%, respectively, a ratio very close to that of MGD-1 (64% and 36%). One linear and three cyclic fragments were designed from the interstrand loop sequence known to retain the biological activities in MGD-1. Only the fragment of 10 amino acids (C10C) constrained by two disulfide bonds in a stable beta-hairpin structure was able to inhibit the mortality of Palaemon serratus shrimp injected with white spot syndrome virus (WSSV). Fifty percent inhibition was obtained by in vitro pre-incubation of WSSV with 45 microM of C10C compared with 7 microM for mytilin. Interaction between the fragment and the virus occurred very rapidly as 40% survival was recorded after only 1 min of pre-incubation. In addition, C10C was capable of inhibiting in vitro growth of Vibrio splendidus LGP32 (MIC 125 microM), Vibrio anguillarum (MIC 2mM), Micrococcus lysodeikticus and Escherichia coli (MIC 1mM). Destroying the cysteine-stabilized alphabeta structure or shortening the C10C fragment to the C6C fragment with only one disulfide bond resulted in loss of both antiviral and antibacterial activities. Increasing the positive net charge did not enforce the antibacterial activity and completely suppressed the antiviral one. The C10C-designed peptide from mytilin appeared comparable in composition and structure with protegrin, tachyplesin and polyphemusin.

To elucidate the structural features of the mussel defensin MGD1 required for antimicrobial activity, we synthesized a series of peptides corresponding to the main known secondary structures of the molecule and evaluated their activity towards Gram-positive and Gram-negative bacteria, and filamentous fungi. We found that the nonapeptide corresponding to residues 25-33 of MGD1 (CGGWHRLRC) exhibited bacteriostatic activity once it was cyclized by a non-naturally occurring disulfide bridge. Longer peptides corresponding to the amino acid sequences of the alpha-helical part or to the beta-strands of MGD1 had no detectable activity. The bacteriostatic activity of the sequence 25-33 was strictly dependent on the bridging of Cys25 and Cys33 and was proportional to the theoretical isoelectric point of the peptide, as deduced from the variation of activity in a set of peptide analogues of the 25-33 sequence with different numbers of cationic charges. By using confocal fluorescence microscopy, we found that the cyclic peptides bound to Gram-positive bacteria without apparent lysis. However, by using a fluorescent dye, we observed that dead bacteria had been permeated by the cyclic peptide 25-33. Sequence comparisons in the family of arthopod defensins indicate that MGD1 belongs to a subfamily of the insect defensins, characterized by the constant occurrence of both positively charged and hydrophobic amino acids in the loop. Modelling studies showed that in the MGD1 structure, positively charged and hydrophobic residues are organized in two layered clusters, which might have a functional significance in the docking of MGD1 to the bacterial membrane.