Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins

Tsukasa Osaki,Miyuki Omotezako,Ranko Nagayama,Michimasa Hirata,Sadaaki Iwanaga,Jiro Kasahara,Junji Hattori,Isao Ito,Hiroyuki Sugiyama,Shun-Ichiro Kawabata

doi:10.1074/jbc.274.37.26172

Tsukasa Osaki, Miyuki Omotezako + Show 8 more

Open Access

https://doi.org/10.1074/jbc.274.37.26172

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Abstract

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.

Highlights

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus
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
Antimicrobial peptides named tachystatins A, B, and C were newly identified from hemocytes of the Japanese horseshoe crab T. tridentatus

Summary

EXPERIMENTAL PROCEDURES

Materials—Hemocyte debris from the Japanese horseshoe crab Tachypleus tridentatus was prepared as described [22]. Chitin binding Assay—Chitin (0.5 mg) was mixed with antimicrobial substances in 100 ␮l of 20 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl and 2 mM CaCl2, incubated at room temperature for 15 min and centrifuged at 15,000 rpm for 2 min. Hemolytic Activity—Antimicrobial substances dissolved in 0.5 ml of 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl were mixed with the same volume of sheep erythrocytes in the same buffer (final 1%, v/v) and incubated at 37 °C. Tachystatin A-specific DNA Probe and Screening of cDNA Library— The degenerate nucleotide sequences of the primers used for polymerase chain reaction were based on the amino acid sequences of QGFNCV (residues 7–12) and YFPGST (residues 32–37) of tachystatin A. The compositions of external and patch internal solutions were as follows: external solution, 10 mM Hepes-NaOH, pH 7.35, containing 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM glucose, 0.003 mM tetrodotoxin, 10 mM tetraethylammonium chloride, and 1 mM 4-aminopyridine; patch internal solution, 10 mM HepesCsOH, pH 7.35, containing 140 mM CsCl, 1 mM EGTA, 2 mM MgATP, and 0.4 mM NaGTP. ␻-Agatoxin IVA and tachystatins A and B were dissolved in deionized water and diluted with external solution just before use

RESULTS

Big defensin

Wheat germ agglutinina

Tachystatin C Analysis Sequence

DISCUSSION