Abstract
The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.
Highlights
The melanization reaction is an essential immune response in arthropods that should be tightly regulated
No significant differences (p Ͼ 0.05) were found between the inhibitory activity in protamine-treated (55.4 Ϯ 1.11 IU/ml; mean Ϯ S.E., n ϭ 5) and untreated (54.0 Ϯ 0.31 IU/ml; mean Ϯ S.E., n ϭ 5) lobster hemocyte lysate (LHL) prepared in 450 mM NaCl, indicating that protamine sulfate did not impair the inhibitory activity under the experimental conditions used
These results might suggest that trypsin inhibitors in the LHL are bound to nucleic acids, probably electrostatically, and that this binding is abrogated by high ionic strength
Summary
The melanization reaction is an essential immune response in arthropods that should be tightly regulated. The addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme. Novel Trypsin Inhibitor Regulates Immune Response in Lobsters this sense, components of the proPO-activating system may associate to form a large noncovalent complex, which localizes the melanization to the surface of invading microorganisms or at the injury site (8 –10) This complex ensures a high local concentration of quinone products where necessary, whereas it avoids their dissemination. Experimental evidence indicated that panulirin is implicated in the regulation of the proPO-activating system in the spiny lobster
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