Nagai T, Osaki T, Kawabata S
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
J Biol Chem. 2001 Jul 20;276(29):27166-70. doi: 10.1074/jbc.M102596200. Epub 2001 May 24.
Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the horseshoe crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297-35301). Here we show that chitin-binding antimicrobial peptides of the horseshoe crab induce the intrinsic phenoloxidase activity of hemocyanin. Tachyplesin, a major Tachypleus antimicrobial peptide with an amphiphilic structure, converted the hemocyanin to phenoloxidase. Surface plasmon resonance analysis revealed the specific interaction of tachyplesin with hemocyanin at K(d) = 3.4 x 10(-)6 m. The chemical modification of Trp or Tyr in tachyplesin, but not Lys or Arg, dramatically reduced the affinity to hemocyanin, suggesting that the binding site is located in the hydrophobic face of tachyplesin. Hemocyanin has no affinity with chitin, but it significantly binds to tachyplesin-coated chitin, leading to the expression of phenoloxidase activity. The chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing.
节肢动物血蓝蛋白和酚氧化酶分别作为氧气转运蛋白和参与防御反应的酶发挥不同的生理功能。然而，它们具有结构相似的氧结合中心。我们已经表明，鲎（Tachypleus tridentatus）的凝血酶通过形成复合物而无需蛋白水解切割就能将血蓝蛋白功能性地转化为酚氧化酶（Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297 - 35301）。在此我们表明，鲎的几丁质结合抗菌肽可诱导血蓝蛋白的固有酚氧化酶活性。鲎素（Tachyplesin）是一种具有两亲结构的主要鲎抗菌肽，它能将血蓝蛋白转化为酚氧化酶。表面等离子体共振分析显示鲎素与血蓝蛋白在K(d) = 3.4 x 10(-)6 m时有特异性相互作用。对鲎素中色氨酸或酪氨酸而非赖氨酸或精氨酸的化学修饰显著降低了其与血蓝蛋白的亲和力，这表明结合位点位于鲎素的疏水面上。血蓝蛋白与几丁质没有亲和力，但它能与包被有鲎素的几丁质显著结合，从而导致酚氧化酶活性的表达。包被有抗菌肽的几丁质可能作为血蓝蛋白结合的支架，并且由此产生的酚氧化酶活性似乎作为外骨骼伤口愈合的触发因素发挥作用。
