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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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Effects of Mg2+ and Ca2+ on the deposition of TtC3b on microbes.(A) Microbes were incubated with HDP. The deposition of TtC3b on the microbial surfaces was analyzed by flow cytometry using the Alexa 488-conjugated anti-TtC3 antibody (blue line). The dotted line indicates autonomous fluorescence (AF) of microbes. (B) Dialyzed HDP was incubated with various microbes, and the deposition of TtC3b on the microbial surfaces was analyzed in the presence or absence of cations, as described in Figure 7A. Blue line, dialyzed HDP; red line, dialyzed HDP+10 mM Ca2+; green line, dialyzed HDP+50 mM Mg2+; orange line, dialyzed HDP+10 mM Ca2+ and 50 mM Mg2+. Mean fluorescence intensity of E. coli: AF, 5.9; HDP, 250; dialyzed HDP, 266; dialyzed HDP+Ca2+, 236; dialyzed HDP+Mg2+, 286; dialyzed HDP+Ca2+ and Mg2+, 376. MFI of S. aureus: AF, 6.8; HDP, 116; dialyzed HDP, 21; dialyzed HDP+Ca2+, 62; dialyzed HDP+Mg2+, 220; dialyzed HDP+Ca2+ and Mg2+, 231. MFI of P. pastoris: AF, 6.1; HDP, 57; dialyzed HDP, 20; dialyzed HDP+Ca2+, 28; dialyzed HDP+Mg2+, 60; dialyzed HDP+Ca2+ and Mg2+, 139. In (A) and (B), one representative experiment out of three repeats is shown.
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pone-0036783-g007: Effects of Mg2+ and Ca2+ on the deposition of TtC3b on microbes.(A) Microbes were incubated with HDP. The deposition of TtC3b on the microbial surfaces was analyzed by flow cytometry using the Alexa 488-conjugated anti-TtC3 antibody (blue line). The dotted line indicates autonomous fluorescence (AF) of microbes. (B) Dialyzed HDP was incubated with various microbes, and the deposition of TtC3b on the microbial surfaces was analyzed in the presence or absence of cations, as described in Figure 7A. Blue line, dialyzed HDP; red line, dialyzed HDP+10 mM Ca2+; green line, dialyzed HDP+50 mM Mg2+; orange line, dialyzed HDP+10 mM Ca2+ and 50 mM Mg2+. Mean fluorescence intensity of E. coli: AF, 5.9; HDP, 250; dialyzed HDP, 266; dialyzed HDP+Ca2+, 236; dialyzed HDP+Mg2+, 286; dialyzed HDP+Ca2+ and Mg2+, 376. MFI of S. aureus: AF, 6.8; HDP, 116; dialyzed HDP, 21; dialyzed HDP+Ca2+, 62; dialyzed HDP+Mg2+, 220; dialyzed HDP+Ca2+ and Mg2+, 231. MFI of P. pastoris: AF, 6.1; HDP, 57; dialyzed HDP, 20; dialyzed HDP+Ca2+, 28; dialyzed HDP+Mg2+, 60; dialyzed HDP+Ca2+ and Mg2+, 139. In (A) and (B), one representative experiment out of three repeats is shown.

Mentions: Opsonization by C3b is one of the most important responses in the mammalian complement system [2], [3]. To evaluate the opsonization ability of HDP, E. coli, S. aureus, and P. pastoris were mixed with HDP, and TtC3b deposition on microbes was quantified by flow cytometric analysis. The three types of microbes were opsonized with TtC3b (Figure 7A). TtC3b deposition was relatively homogeneous on the surface of E. coli and S. aureus, whereas a bimodal distribution of TtC3b was observed on the surface of P. pastoris. At present, the reason for this discrepancy is unclear.


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)

Effects of Mg2+ and Ca2+ on the deposition of TtC3b on microbes.(A) Microbes were incubated with HDP. The deposition of TtC3b on the microbial surfaces was analyzed by flow cytometry using the Alexa 488-conjugated anti-TtC3 antibody (blue line). The dotted line indicates autonomous fluorescence (AF) of microbes. (B) Dialyzed HDP was incubated with various microbes, and the deposition of TtC3b on the microbial surfaces was analyzed in the presence or absence of cations, as described in Figure 7A. Blue line, dialyzed HDP; red line, dialyzed HDP+10 mM Ca2+; green line, dialyzed HDP+50 mM Mg2+; orange line, dialyzed HDP+10 mM Ca2+ and 50 mM Mg2+. Mean fluorescence intensity of E. coli: AF, 5.9; HDP, 250; dialyzed HDP, 266; dialyzed HDP+Ca2+, 236; dialyzed HDP+Mg2+, 286; dialyzed HDP+Ca2+ and Mg2+, 376. MFI of S. aureus: AF, 6.8; HDP, 116; dialyzed HDP, 21; dialyzed HDP+Ca2+, 62; dialyzed HDP+Mg2+, 220; dialyzed HDP+Ca2+ and Mg2+, 231. MFI of P. pastoris: AF, 6.1; HDP, 57; dialyzed HDP, 20; dialyzed HDP+Ca2+, 28; dialyzed HDP+Mg2+, 60; dialyzed HDP+Ca2+ and Mg2+, 139. In (A) and (B), one representative experiment out of three repeats is shown.
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pone-0036783-g007: Effects of Mg2+ and Ca2+ on the deposition of TtC3b on microbes.(A) Microbes were incubated with HDP. The deposition of TtC3b on the microbial surfaces was analyzed by flow cytometry using the Alexa 488-conjugated anti-TtC3 antibody (blue line). The dotted line indicates autonomous fluorescence (AF) of microbes. (B) Dialyzed HDP was incubated with various microbes, and the deposition of TtC3b on the microbial surfaces was analyzed in the presence or absence of cations, as described in Figure 7A. Blue line, dialyzed HDP; red line, dialyzed HDP+10 mM Ca2+; green line, dialyzed HDP+50 mM Mg2+; orange line, dialyzed HDP+10 mM Ca2+ and 50 mM Mg2+. Mean fluorescence intensity of E. coli: AF, 5.9; HDP, 250; dialyzed HDP, 266; dialyzed HDP+Ca2+, 236; dialyzed HDP+Mg2+, 286; dialyzed HDP+Ca2+ and Mg2+, 376. MFI of S. aureus: AF, 6.8; HDP, 116; dialyzed HDP, 21; dialyzed HDP+Ca2+, 62; dialyzed HDP+Mg2+, 220; dialyzed HDP+Ca2+ and Mg2+, 231. MFI of P. pastoris: AF, 6.1; HDP, 57; dialyzed HDP, 20; dialyzed HDP+Ca2+, 28; dialyzed HDP+Mg2+, 60; dialyzed HDP+Ca2+ and Mg2+, 139. In (A) and (B), one representative experiment out of three repeats is shown.
Mentions: Opsonization by C3b is one of the most important responses in the mammalian complement system [2], [3]. To evaluate the opsonization ability of HDP, E. coli, S. aureus, and P. pastoris were mixed with HDP, and TtC3b deposition on microbes was quantified by flow cytometric analysis. The three types of microbes were opsonized with TtC3b (Figure 7A). TtC3b deposition was relatively homogeneous on the surface of E. coli and S. aureus, whereas a bimodal distribution of TtC3b was observed on the surface of P. pastoris. At present, the reason for this discrepancy is unclear.

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