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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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Mg2+-dependent activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with dialyzed HDP at 37°C for 30 min in the presence (CaCl2 = 10 mM, MgCl2 = 50 mM) or absence of divalent cations. Microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown. A cross-reacting protein with 68 kDa against the anti-TtC2/Bf-2-SP antibody by Western blotting is shown by (*).
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pone-0036783-g004: Mg2+-dependent activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with dialyzed HDP at 37°C for 30 min in the presence (CaCl2 = 10 mM, MgCl2 = 50 mM) or absence of divalent cations. Microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown. A cross-reacting protein with 68 kDa against the anti-TtC2/Bf-2-SP antibody by Western blotting is shown by (*).

Mentions: The proteolytic activation of factor B by factor D in the mammalian complement system requires Mg2+ ions that bind to factor B to induce a conformational change, leading to the complex formation with C3 [4]. We prepared cation-free HDP by dialysis. As expected, the activation of TtC2/Bf-1 and TtC2/Bf-2 by microbes was absolutely Mg2+-dependent (Figure 4). In these experiments, the anti-TtC2/Bf-2-SP antibody, but not the two types of anti-TtC2/Bf-1 antibodies, cross-reacted with an unknown protein with apparent molecular mass of 68 kDa, and the cross-reacting protein was not a derivative from TtC2/Bf-2, since the 68-kDa protein was observed in the absence of Mg2+ ions (Figure 4, right panels). The mammalian complement system is also activated by PAMPs, such as LPS of Gram-negative bacteria, and by zymosan of fungi [2], [3]. HDP in the absence or presence of cations was incubated with PAMPs including LPS, lipoteichoic acid, peptidoglycan, zymosan, and laminarin (Figure 5). Zymosan, laminarin, and LPS effectively activated TtC2/Bf-1 in the presence of Mg2+, but none of the PAMPs could activate TtC2/Bf-2. In order to know whether TtC2/Bf-1 or TtC2/Bf-2 is present as a complex with TtC3 in hemolymph plasma, HDP was mixed with the anti-TtC3 antibody and immunoprecipitated by protein G Sepharose. TtC2/Bf-1 and TtC2/Bf-2 were detected in the resulting immunoprecipitates by Western blotting, revealing that TtC2/Bf-1 and TtC2/Bf-2 are present in the complex form with TtC3 (Figure 6).


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)

Mg2+-dependent activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with dialyzed HDP at 37°C for 30 min in the presence (CaCl2 = 10 mM, MgCl2 = 50 mM) or absence of divalent cations. Microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown. A cross-reacting protein with 68 kDa against the anti-TtC2/Bf-2-SP antibody by Western blotting is shown by (*).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036783-g004: Mg2+-dependent activation of TtC2/Bf-1 and TtC2/Bf-2.Microbes were incubated with dialyzed HDP at 37°C for 30 min in the presence (CaCl2 = 10 mM, MgCl2 = 50 mM) or absence of divalent cations. Microbes were removed by centrifugation, and 20 µl of the supernatants were subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown. A cross-reacting protein with 68 kDa against the anti-TtC2/Bf-2-SP antibody by Western blotting is shown by (*).
Mentions: The proteolytic activation of factor B by factor D in the mammalian complement system requires Mg2+ ions that bind to factor B to induce a conformational change, leading to the complex formation with C3 [4]. We prepared cation-free HDP by dialysis. As expected, the activation of TtC2/Bf-1 and TtC2/Bf-2 by microbes was absolutely Mg2+-dependent (Figure 4). In these experiments, the anti-TtC2/Bf-2-SP antibody, but not the two types of anti-TtC2/Bf-1 antibodies, cross-reacted with an unknown protein with apparent molecular mass of 68 kDa, and the cross-reacting protein was not a derivative from TtC2/Bf-2, since the 68-kDa protein was observed in the absence of Mg2+ ions (Figure 4, right panels). The mammalian complement system is also activated by PAMPs, such as LPS of Gram-negative bacteria, and by zymosan of fungi [2], [3]. HDP in the absence or presence of cations was incubated with PAMPs including LPS, lipoteichoic acid, peptidoglycan, zymosan, and laminarin (Figure 5). Zymosan, laminarin, and LPS effectively activated TtC2/Bf-1 in the presence of Mg2+, but none of the PAMPs could activate TtC2/Bf-2. In order to know whether TtC2/Bf-1 or TtC2/Bf-2 is present as a complex with TtC3 in hemolymph plasma, HDP was mixed with the anti-TtC3 antibody and immunoprecipitated by protein G Sepharose. TtC2/Bf-1 and TtC2/Bf-2 were detected in the resulting immunoprecipitates by Western blotting, revealing that TtC2/Bf-1 and TtC2/Bf-2 are present in the complex form with TtC3 (Figure 6).

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