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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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Tissue-specific localizations of TtC2/Bf-1 and TtC2/Bf-2 and the presence of their N-glycosylation forms.(A) Tissue extracts (10 µg each, quantified by micro BCA kit, Pierce) were subjected to Western blotting using the anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 or the anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2. Hc, hemocytes; Ms, muscle; Ht, heart; St, stomach; Hp, hepatopancreas; HDP, hemocyanin-depleted plasma. (B) Tissue extracts (2.5 µg each) were incubated with glycopeptidase F (GPF) at 37°C for 17 hours and subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown.
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pone-0036783-g002: Tissue-specific localizations of TtC2/Bf-1 and TtC2/Bf-2 and the presence of their N-glycosylation forms.(A) Tissue extracts (10 µg each, quantified by micro BCA kit, Pierce) were subjected to Western blotting using the anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 or the anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2. Hc, hemocytes; Ms, muscle; Ht, heart; St, stomach; Hp, hepatopancreas; HDP, hemocyanin-depleted plasma. (B) Tissue extracts (2.5 µg each) were incubated with glycopeptidase F (GPF) at 37°C for 17 hours and subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown.

Mentions: The tissue localization of TtC2/Bf-1 and TtC2/Bf-2 was evaluated by Western blotting using specific polyclonal antibodies: an anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 and an anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2 (Figure S3). For TtC2/Bf-1, a 140-kDa antigen was present in hemolymph plasma and hemocytes, and a lower molecular mass antigen of 130 kDa was also detected in hemocytes (Figure 2A). For TtC2/Bf-2, a 140-kDa antigen was detected in hemocytes and hemolymph plasma, and a lower molecular mass antigen of 100 kDa was detected in muscle, heart, and stomach but not in hemocytes. There were six and ten N-glycosylation potential sites in the amino acid sequences of TtC2/Bf-1 and TtC2/Bf-2, respectively (Figure S1), and treatment of the tissue extracts with glycopeptidase F converted the 140-kDa antigen in hemocytes and plasma to a 110-kDa antigen (Figure 2B). This indicated that the N-linked glycosylation was tissue-specific. These results indicate that the major expression sites of TtC2/Bf-1 and TtC2/Bf-2 are hemocytes, and that the glycosylated forms of TtC2/Bf-1 and TtC2/Bf-2 with 140 kDa are secreted from hemocytes to plasma. The mRNAs for TtC2/Bf-1 and TtC2/Bf-2 were detectable in hepatopancreas by reverse–transcription PCR (RT-PCR) (Figure S4), whereas these proteins were not detectable in this tissue by Western blotting (Figure 2A). The concentrations of TtC2/Bf-1 and TtC2/Bf-2 in hemolymph plasma were determined to be 0.24±0.04 mg/ml and 0.18±0.03 mg/ml, respectively, by enzyme-linked immunosorbent assay (ELISA).


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)

Tissue-specific localizations of TtC2/Bf-1 and TtC2/Bf-2 and the presence of their N-glycosylation forms.(A) Tissue extracts (10 µg each, quantified by micro BCA kit, Pierce) were subjected to Western blotting using the anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 or the anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2. Hc, hemocytes; Ms, muscle; Ht, heart; St, stomach; Hp, hepatopancreas; HDP, hemocyanin-depleted plasma. (B) Tissue extracts (2.5 µg each) were incubated with glycopeptidase F (GPF) at 37°C for 17 hours and subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351276&req=5

pone-0036783-g002: Tissue-specific localizations of TtC2/Bf-1 and TtC2/Bf-2 and the presence of their N-glycosylation forms.(A) Tissue extracts (10 µg each, quantified by micro BCA kit, Pierce) were subjected to Western blotting using the anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 or the anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2. Hc, hemocytes; Ms, muscle; Ht, heart; St, stomach; Hp, hepatopancreas; HDP, hemocyanin-depleted plasma. (B) Tissue extracts (2.5 µg each) were incubated with glycopeptidase F (GPF) at 37°C for 17 hours and subjected to Western blotting, as described in Figure 2A. Each experiment was performed at least three times. Representative blots are shown.
Mentions: The tissue localization of TtC2/Bf-1 and TtC2/Bf-2 was evaluated by Western blotting using specific polyclonal antibodies: an anti-TtC2/Bf-1-CCP antibody for TtC2/Bf-1 and an anti-TtC2/Bf-2-SP antibody for TtC2/Bf-2 (Figure S3). For TtC2/Bf-1, a 140-kDa antigen was present in hemolymph plasma and hemocytes, and a lower molecular mass antigen of 130 kDa was also detected in hemocytes (Figure 2A). For TtC2/Bf-2, a 140-kDa antigen was detected in hemocytes and hemolymph plasma, and a lower molecular mass antigen of 100 kDa was detected in muscle, heart, and stomach but not in hemocytes. There were six and ten N-glycosylation potential sites in the amino acid sequences of TtC2/Bf-1 and TtC2/Bf-2, respectively (Figure S1), and treatment of the tissue extracts with glycopeptidase F converted the 140-kDa antigen in hemocytes and plasma to a 110-kDa antigen (Figure 2B). This indicated that the N-linked glycosylation was tissue-specific. These results indicate that the major expression sites of TtC2/Bf-1 and TtC2/Bf-2 are hemocytes, and that the glycosylated forms of TtC2/Bf-1 and TtC2/Bf-2 with 140 kDa are secreted from hemocytes to plasma. The mRNAs for TtC2/Bf-1 and TtC2/Bf-2 were detectable in hepatopancreas by reverse–transcription PCR (RT-PCR) (Figure S4), whereas these proteins were not detectable in this tissue by Western blotting (Figure 2A). The concentrations of TtC2/Bf-1 and TtC2/Bf-2 in hemolymph plasma were determined to be 0.24±0.04 mg/ml and 0.18±0.03 mg/ml, respectively, by enzyme-linked immunosorbent assay (ELISA).

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