MUTA Tatsushi Kyushu University, Graduate School of Medical Science, Associate Professor, 大学院・医学系研究科, 助教授 (60222337)
KAWANAO Keiichi Toyama Medical and Pharmaceutical University, Faculty of Pharmaceutical Science, Professor, 薬学部, 教授 (10136492)
|Budget Amount *help
¥9,700,000 (Direct Cost : ¥9,700,000)
Fiscal Year 1999 : ¥3,400,000 (Direct Cost : ¥3,400,000)
Fiscal Year 1998 : ¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1997 : ¥3,700,000 (Direct Cost : ¥3,700,000)
Antimicrobial polypeptides, named tachystatins A, B, and C, were newly identified from hemocytes of the Japanese 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 the most effective antimicrobial polypeptide with ICィイD250ィエD2 values of 0.3-1.2 μg/ml against these microorganisms. Tachystatin A is homologous to tachystatin B with 40% sequence identity, but tachystatin C has no significant sequence similarity to those of tachystatins A and B, but the positions of six cysteines are conserved. Tachystatins A and B showed sequence similarity to ω-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. However, tachystatins A and B exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. On the other hand, tachystatins A and B, and C bound significantly to chitin. The chitin-binding affinities of tachystatins were quantified, and a causal relationship was observed between binding affinity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell-lysis activity. The septum between a mother cell and bud, a chitin-rich region, was strongly stained by fluorescence labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. Furthermore, only tachystatin C had a strong hemolytic activity and contained a unique COOH-terminal sequence that appears to form an amphiphilic β-sheet, suggesting that it has an important role in the hemolytic activity of tachystatin C. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral polypeptides, with adaptive functions.