A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae.
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Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S. , Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615-621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of Ca2+, the 45-kDa protein was cleaved to a 35-kDa protein. RNA blot hybridization revealed that expression of the 45-kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge. (+info)
Developmental and environmental regulation of antifreeze proteins in the mealworm beetle Tenebrio molitor.
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The yellow mealworm beetle, Tenebrio molitor, contains a family of small Cys-rich and Thr-rich thermal hysteresis proteins that depress the hemolymph freezing point below the melting point by as much as 5. 5 degrees C (DeltaT = thermal hysteresis). Thermal hysteresis protein expression was evaluated throughout development and after exposure to altered environmental conditions. Under favorable growth conditions, small larvae (11-13 mg) had only low levels of thermal hysteresis proteins or thermal hysteresis protein message, but these levels increased 10-fold and 18-fold, respectively, by the final larval instar (> 190 mg), resulting in thermal hysteresis > 3 degrees C. Exposure of small larvae (11-13 mg) to 4 weeks of cold (4 degrees C) caused an approximately 20-fold increase in thermal hysteresis protein concentration, well in excess of the less than threefold developmental increase seen after 4 weeks at 22 degrees C. Exposure of large larvae (100-120 mg) to cold caused 12-fold and sixfold increases in thermal hysteresis protein message and protein levels, respectively, approximately double the maximum levels they would have attained in the final larval instar at 22 degrees C. Thus, thermal hysteresis increased to similar levels (> 4 degrees C) in the cold, irrespective of the size of the larvae (the overwintering stage). At pupation, thermal hysteresis protein message levels decreased > 20-fold and remained low thereafter, but thermal hysteresis activity decreased much more slowly. Exposure to cold did not reverse this decline. Desiccation or starvation of larvae had comparable effects to cold exposure, but surprisingly, short daylength photoperiod or total darkness had no effect on either thermal hysteresis or message levels. As all environmental conditions that caused increased thermal hysteresis also inhibited growth, we postulate that developmental arrest is a primary factor in the regulation of T. molitor thermal hysteresis proteins. (+info)
The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system.
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We have previously suggested that a GroEL homolog produced by the whitefly Bemisia tabaci endosymbiotic bacteria interacts in the insect hemolymph with particles of Tomato yellow leaf curl virus from Israel (TYLCV-Is), ensuring the safe circulative transmission of the virus. We have now addressed the question of whether the nontransmissibility of Abutilon mosaic virus from Israel (AbMV-Is) is related to a lack of association between GroEL and the virus coat protein (CP). Translocation analysis has shown that, whereas TYLCV-Is DNA is conspicuous in the digestive tract, hemolymph, and salivary glands of B. tabaci 8 h after acquisition feeding started, AbMV-Is DNA was detected only in the insect digestive tract, even after 96 h. To determine whether AbMV-Is particles were rapidly degraded in the hemolymph as a result of their inability to interact with GroEL, we have isolated a GroEL gene from B. tabaci and used a yeast two-hybrid assay to compare binding of the CP of TYLCV-Is and AbMV-Is to the insect GroEL. The yeast assay showed that the CPs of the two viruses are able to bind efficiently to GroEL. We therefore suggest that, although GroEL-CP interaction in the hemolymph is a necessary condition for circulative transmission, the nontransmissibility of AbMV-Is is not the result of lack of binding to GroEL in the B. tabaci hemolymph, but most likely results from an inability to cross the gut/hemolymph barrier. (+info)
Purification and characterization of biliverdin-binding protein from larval hemolymph of the swallowtail butterfly, Papilio xuthus L.
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The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The Namino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42% of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type. (+info)
Survival for immunity: the price of immune system activation for bumblebee workers.
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Parasites do not always harm their hosts because the immune system keeps an infection at bay. Ironically, the cost of using immune defenses could itself reduce host fitness. This indirect cost of parasitism is often not visible because of compensatory resource intake. Here, workers of the bumblebee, Bombus terrestris, were challenged with lipopolysaccharides and micro-latex beads to induce their immune system under starvation (i.e., not allowing compensatory intake). Compared with controls, survival of induced workers was significantly reduced (by 50 to 70%). (+info)
A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors.
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Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory. (+info)
Cuticular pro-phenoloxidase of the silkworm, Bombyx mori. Purification and demonstration of its transport from hemolymph.
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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). (+info)
Original involvement of antimicrobial peptides in mussel innate immunity.
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Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects. (+info)