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Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

Tagawa K, Yoshihara T, Shibata T, Kitazaki K, Endo Y, Fujita T, Koshiba T, Kawabata S - PLoS ONE (2012)

Bottom Line: Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown.TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent.We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

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Microbe-induced activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with HDP at 37°C for 30 min. The microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting as described in Figure 2A. Lane 1, HDP; lane 2, HDP+E. coli; lane 3, HDP+S. aureus; lane 4, HDP+P. pastoris. Each experiment was performed at least three times. Representative blots are shown.
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pone-0036783-g003: Microbe-induced activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with HDP at 37°C for 30 min. The microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting as described in Figure 2A. Lane 1, HDP; lane 2, HDP+E. coli; lane 3, HDP+S. aureus; lane 4, HDP+P. pastoris. Each experiment was performed at least three times. Representative blots are shown.

Mentions: To evaluate whether or not microbes trigger the activation of TtC2/Bf-1 and TtC2/Bf-2, hemocyanin-depleted hemolymph plasma (HDP) was incubated with Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Staphylococcus aureus), and fungi (Pichia pastoris). The conversion of TtC2/Bf-1 and TtC2/Bf-2 to their active forms was analyzed by Western blotting. HDP was used for these experiments because a large amount of hemocyanin subunits with 70 kDa interrupted the detection of the activation fragments derived from TtC2/Bf-1 and TtC2/Bf-2. TtC2/Bf-1 and TtC2/Bf-2 were converted to the active forms by mixing with microbes (Figure 3). The anti-TtC2/Bf-1-CCP antibody recognized the N-terminal part of TtC2/Bf-1 with 63 kDa (Figure 3, left panel), and the C-terminal proteolytic fragment of TtC2/Bf-1 with 74 kDa was detected with another anti-TtC2/Bf-1-SP antibody (Figure S5). On the other hand, the anti-TtC2/Bf-2-SP antibody recognized the C-terminal proteolytic fragment of TtC2/Bf-2 with 70 kDa (Figure 3, right panel).


Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner.

Tagawa K, Yoshihara T, Shibata T, Kitazaki K, Endo Y, Fujita T, Koshiba T, Kawabata S - PLoS ONE (2012)

Microbe-induced activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with HDP at 37°C for 30 min. The microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting as described in Figure 2A. Lane 1, HDP; lane 2, HDP+E. coli; lane 3, HDP+S. aureus; lane 4, HDP+P. pastoris. Each experiment was performed at least three times. Representative blots are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351276&req=5

pone-0036783-g003: Microbe-induced activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with HDP at 37°C for 30 min. The microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting as described in Figure 2A. Lane 1, HDP; lane 2, HDP+E. coli; lane 3, HDP+S. aureus; lane 4, HDP+P. pastoris. Each experiment was performed at least three times. Representative blots are shown.
Mentions: To evaluate whether or not microbes trigger the activation of TtC2/Bf-1 and TtC2/Bf-2, hemocyanin-depleted hemolymph plasma (HDP) was incubated with Gram-negative bacteria (Escherichia coli), Gram-positive bacteria (Staphylococcus aureus), and fungi (Pichia pastoris). The conversion of TtC2/Bf-1 and TtC2/Bf-2 to their active forms was analyzed by Western blotting. HDP was used for these experiments because a large amount of hemocyanin subunits with 70 kDa interrupted the detection of the activation fragments derived from TtC2/Bf-1 and TtC2/Bf-2. TtC2/Bf-1 and TtC2/Bf-2 were converted to the active forms by mixing with microbes (Figure 3). The anti-TtC2/Bf-1-CCP antibody recognized the N-terminal part of TtC2/Bf-1 with 63 kDa (Figure 3, left panel), and the C-terminal proteolytic fragment of TtC2/Bf-1 with 74 kDa was detected with another anti-TtC2/Bf-1-SP antibody (Figure S5). On the other hand, the anti-TtC2/Bf-2-SP antibody recognized the C-terminal proteolytic fragment of TtC2/Bf-2 with 70 kDa (Figure 3, right panel).

Bottom Line: Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown.TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent.We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.

Show MeSH
Related in: MedlinePlus