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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 plasma lectins on the deposition of TtC3b on microbes.Microbes were incubated with HDP, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry, as described in Figure 7A (blue line). The dotted line indicates autonomous fluorescence. The red line indicates flow-cytometric data obtained by pretreatment of HDP with the anti-TtCRP-1 (Tachypleus tridentatus C-reactive protein-1) antibody (A), anti-TL-5A (Tachylectin-5A) antibody (B), or anti-TL-1 (Tachylectin-1) antibody (C) at 10 µg/ml for 1 hour at 4°C. Mean fluorescence intensity of E. coli: (A) AF, 7.2; HDP, 266; HDP+antibody, 276; (B) AF, 6.83; HDP, 110; HDP+antibody, 155; (C) AF, 8.96; HDP, 201; HDP+antibody, 79. MFI of S. aureus: (A) AF, 7.2; HDP, 66; HDP+antibody, 26; (B) AF, 7.2; HDP, 66; HDP+antibody, 18; (C) AF, 3.6; HDP, 42; HDP+antibody, 25. Mean fluorescence intensity of P. pastoris: (A) AF, 5.8; HDP, 96; HDP+antibody, 160; (B) AF, 5.8; HDP, 96; HDP+antibody, 150; (C) AF, 6.04; HDP, 50; HDP+antibody, 16. In (A), (B), and (C), one representative experiment out of three repeats is shown.
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pone-0036783-g008: Effects of plasma lectins on the deposition of TtC3b on microbes.Microbes were incubated with HDP, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry, as described in Figure 7A (blue line). The dotted line indicates autonomous fluorescence. The red line indicates flow-cytometric data obtained by pretreatment of HDP with the anti-TtCRP-1 (Tachypleus tridentatus C-reactive protein-1) antibody (A), anti-TL-5A (Tachylectin-5A) antibody (B), or anti-TL-1 (Tachylectin-1) antibody (C) at 10 µg/ml for 1 hour at 4°C. Mean fluorescence intensity of E. coli: (A) AF, 7.2; HDP, 266; HDP+antibody, 276; (B) AF, 6.83; HDP, 110; HDP+antibody, 155; (C) AF, 8.96; HDP, 201; HDP+antibody, 79. MFI of S. aureus: (A) AF, 7.2; HDP, 66; HDP+antibody, 26; (B) AF, 7.2; HDP, 66; HDP+antibody, 18; (C) AF, 3.6; HDP, 42; HDP+antibody, 25. Mean fluorescence intensity of P. pastoris: (A) AF, 5.8; HDP, 96; HDP+antibody, 160; (B) AF, 5.8; HDP, 96; HDP+antibody, 150; (C) AF, 6.04; HDP, 50; HDP+antibody, 16. In (A), (B), and (C), one representative experiment out of three repeats is shown.

Mentions: In T. tridentatus hemolymph plasma, there are several types of Ca2+-dependent lectins, such as C-reactive protein-1 (TtCRP-1, a homolog to CrCRP-1) [9] and tachylectin-5A (TL-5A, a homolog to CL5a) [10]. To evaluate whether or not the Ca2+-dependent lectins are involved in the enhancement of TtC3b deposition, microbes were incubated with HDP pretreated with antibodies against TtCRP-1 and TL-5A. These antibodies effectively inhibited TtC3b deposition on S. aureus but not on E. coli or P. pastoris (Figures 8A and 8B). On the other hand, a Ca2+-independent lectin has been identified in plasma, Tachypleus plasma lectin-1 with 26 kDa (TPL-1, an ortholog of CrGBP) [11]–[13]. A polyclonal antibody previously prepared against a hemocyte-derived tachylectin-1 (TL-1, an isolectin for TPL-1) [14] had cross-reactivity against TPL-1 and its isolectins with 25∼27-kDa in hemolymph plasma (Figure S6). HDP was treated with the anti-TL-1 antibody and was mixed with microbes. TtC3b deposition was inhibited almost completely on P. pastoris and was inhibited partially on E. coli and S. aureus (Figure 8C), suggesting that TPL-1 has an essential role in TtC3b deposition on fungi.


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 plasma lectins on the deposition of TtC3b on microbes.Microbes were incubated with HDP, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry, as described in Figure 7A (blue line). The dotted line indicates autonomous fluorescence. The red line indicates flow-cytometric data obtained by pretreatment of HDP with the anti-TtCRP-1 (Tachypleus tridentatus C-reactive protein-1) antibody (A), anti-TL-5A (Tachylectin-5A) antibody (B), or anti-TL-1 (Tachylectin-1) antibody (C) at 10 µg/ml for 1 hour at 4°C. Mean fluorescence intensity of E. coli: (A) AF, 7.2; HDP, 266; HDP+antibody, 276; (B) AF, 6.83; HDP, 110; HDP+antibody, 155; (C) AF, 8.96; HDP, 201; HDP+antibody, 79. MFI of S. aureus: (A) AF, 7.2; HDP, 66; HDP+antibody, 26; (B) AF, 7.2; HDP, 66; HDP+antibody, 18; (C) AF, 3.6; HDP, 42; HDP+antibody, 25. Mean fluorescence intensity of P. pastoris: (A) AF, 5.8; HDP, 96; HDP+antibody, 160; (B) AF, 5.8; HDP, 96; HDP+antibody, 150; (C) AF, 6.04; HDP, 50; HDP+antibody, 16. In (A), (B), and (C), one representative experiment out of three repeats is shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351276&req=5

pone-0036783-g008: Effects of plasma lectins on the deposition of TtC3b on microbes.Microbes were incubated with HDP, and the deposition of TtC3b on the microbial surface was analyzed by flow cytometry, as described in Figure 7A (blue line). The dotted line indicates autonomous fluorescence. The red line indicates flow-cytometric data obtained by pretreatment of HDP with the anti-TtCRP-1 (Tachypleus tridentatus C-reactive protein-1) antibody (A), anti-TL-5A (Tachylectin-5A) antibody (B), or anti-TL-1 (Tachylectin-1) antibody (C) at 10 µg/ml for 1 hour at 4°C. Mean fluorescence intensity of E. coli: (A) AF, 7.2; HDP, 266; HDP+antibody, 276; (B) AF, 6.83; HDP, 110; HDP+antibody, 155; (C) AF, 8.96; HDP, 201; HDP+antibody, 79. MFI of S. aureus: (A) AF, 7.2; HDP, 66; HDP+antibody, 26; (B) AF, 7.2; HDP, 66; HDP+antibody, 18; (C) AF, 3.6; HDP, 42; HDP+antibody, 25. Mean fluorescence intensity of P. pastoris: (A) AF, 5.8; HDP, 96; HDP+antibody, 160; (B) AF, 5.8; HDP, 96; HDP+antibody, 150; (C) AF, 6.04; HDP, 50; HDP+antibody, 16. In (A), (B), and (C), one representative experiment out of three repeats is shown.
Mentions: In T. tridentatus hemolymph plasma, there are several types of Ca2+-dependent lectins, such as C-reactive protein-1 (TtCRP-1, a homolog to CrCRP-1) [9] and tachylectin-5A (TL-5A, a homolog to CL5a) [10]. To evaluate whether or not the Ca2+-dependent lectins are involved in the enhancement of TtC3b deposition, microbes were incubated with HDP pretreated with antibodies against TtCRP-1 and TL-5A. These antibodies effectively inhibited TtC3b deposition on S. aureus but not on E. coli or P. pastoris (Figures 8A and 8B). On the other hand, a Ca2+-independent lectin has been identified in plasma, Tachypleus plasma lectin-1 with 26 kDa (TPL-1, an ortholog of CrGBP) [11]–[13]. A polyclonal antibody previously prepared against a hemocyte-derived tachylectin-1 (TL-1, an isolectin for TPL-1) [14] had cross-reactivity against TPL-1 and its isolectins with 25∼27-kDa in hemolymph plasma (Figure S6). HDP was treated with the anti-TL-1 antibody and was mixed with microbes. TtC3b deposition was inhibited almost completely on P. pastoris and was inhibited partially on E. coli and S. aureus (Figure 8C), suggesting that TPL-1 has an essential role in TtC3b deposition on fungi.

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