title: The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.
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.
authors.affiliation: From the Biochemistry Department, Center for Pharmaceuticals Research and Development, Ave. 26 No. 1605 e/ Ave. 51 y Boyeros, Plaza, CP 10400, Havana, Cuba.
authors.forename: Rolando
authors.initials: R
authors.lastname: Perdomo-Morales
authors.forename: Vivian
authors.initials: V
authors.lastname: Montero-Alejo
authors.forename: Gerardo
authors.initials: G
authors.lastname: Corzo
authors.forename: Vladimir
authors.initials: V
authors.lastname: Besada
authors.forename: Yamile
authors.initials: Y
authors.lastname: Vega-Hurtado
authors.forename: Yamile
authors.initials: Y
authors.lastname: González-González
authors.forename: Erick
authors.initials: E
authors.lastname: Perera
authors.forename: Marlene
authors.initials: M
authors.lastname: Porto-Verdecia
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004156:Catechol Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006434:Hemocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D033521:Palinuridae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D060829:Arthropod Proteins; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D014361:Trypsin Inhibitors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D014357:Trypsin
keywords: Crustacean; Host Defense; Innate Immunity; Invertebrates; Melanization; Prophenoloxidase-activating System; Protease Inhibitor; Serine Protease
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