Tachystatin A

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to omega-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescence-labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

Tachystatin B is an antimicrobial and a chitin-binding peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus) consisting of two isopeptides called tachystatin B1 and B2. We have determined their solution structures using NMR experiments and distance geometry calculations. The 20 best converged structures of tachystatin B1 and B2 exhibited root mean square deviations of 0.46 and 0.49 A, respectively, for the backbone atoms in Cys(4)-Arg(40). Both structures have identical conformations, and they contain a short antiparallel beta-sheet with an inhibitory cystine-knot (ICK) motif that is distributed widely in the antagonists for voltage-gated ion channels, although tachystatin B does not have neurotoxic activity. The structural homology search provided several peptides with structures similar to that of tachystatin B. However, most of them have the advanced functions such as insecticidal activity, suggesting that tachystatin B may be a kind of ancestor of antimicrobial peptide in the molecular evolutionary history. Tachystatin B also displays a significant structural similarity to tachystatin A, which is member of the tachystatin family. The structural comparison of both tachystatins indicated that Tyr(14) and Arg(17) in the long loop between the first and second strands might be the essential residues for binding to chitin.

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded beta-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist omega-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that omega-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and omega-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and omega-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe(9) of tachystatin A might be an essential residue for binding to chitin.