Abstract
Pro-phenoloxidase (proPO) in insects is implicated in the defense against microbes and wounding. The presence of proPO in the cuticle was suggested more than 30 years ago, but it has not been purified. The extract of cuticles of the silkworm, Bombyx mori, was shown to contain two proPO isoforms (F-type and S-type proPOs, which have slightly different mobilities in polyacrylamide gel electrophoresis under nondenaturing conditions). The two isoforms were purified to homogeneity. From hemolymph of the same insect, two types of proPO with the same electrophoretic mobilities as those of cuticular isoforms were separated and were shown to be different at five amino acid residues in one of their subunits. The isoforms in the hemolymph and cuticle were activated by a specific activating enzyme. The resulting active phenoloxidases exhibited almost the same substrate specificities and specific activities toward o-diphenols. The substrate specificities and the susceptibilities to inhibitors, including carbon monoxide, indicated that the purified proPO isoforms were not zymogens of laccase-type phenoloxidase. The proPO in hemolymph was shown to be transported to the cuticle. This demonstration was corroborated by the failure to detect proPO transcripts by Northern analysis of total RNA from epidermal cells. In reversed-phase column chromatography, cuticular and hemolymph proPOs gave distinct elution profiles, indicating that some yet to be identified modification occurs in hemolymph proPO and results in the formation of cuticular proPO. There was little transportation of cuticular proPO to the cuticle when it was injected into the hemocoel. The nature of the modification is described in the accompanying paper (Asano, T., and Ashida, M. (2001) J. Biol. Chem. 276, 11113-11125).
Highlights
Pro-phenoloxidase in insects is implicated in the defense against microbes and wounding
The secretion is directed apically to the cuticular matrix or basolaterally to the hemolymph or bidirectionally to the cuticle and hemolymph [8]. In addition to this secretion, the epithelial cells have been shown to be capable of transcytosis of proteins, a process in which the epithelial cells take up proteins from the hemolymph and secrete them apically to the cuticle or vice versa
One of the authors of this paper reported that immunogold staining of proPO displays an orderly arrayed pattern in the cuticular matrix of the silkworm, B. mori, and that proPO may be transported from the hemolymph to the cuticle through epithelial cell layer [4]
Summary
The cuticle has been shown to have a certain mechanism to sense the presence of bacteria and fungi in an injured part and to relay signals to the underlying epithelial cells to direct the synthesis of a bacteriocidal peptide and its secretion to the cuticular matrix [3]. In the 1980s, this cascade has been shown to be present in the hemolymph of this insect and to be composed of recognition proteins with specific affinity to microbial cell wall components, serine protease zymogens and proPO [5]. One of the authors of this paper reported that immunogold staining of proPO displays an orderly arrayed pattern in the cuticular matrix of the silkworm, B. mori, and that proPO may be transported from the hemolymph to the cuticle through epithelial cell layer [4]. We found that cuticular proPO was not transported when it was injected into the hemocoel, indicating that the mechanism for recognition of subtle differences between cuticular and hemolymph proPOs operates in the process for the transport
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