Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides.
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.
Nagai
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
Osaki
T
T
Kawabata
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D006433:Hemocyanins / Q000378:metabolism*; D006737:Horseshoe Crabs; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010455:Peptides; D017386:Sequence Homology, Amino Acid
D000900:Anti-Bacterial Agents; D010455:Peptides; D006433:Hemocyanins; D014442:Monophenol Monooxygenase