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 Mg2+-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg2+-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca2+-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca2+-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.
Highlights
Metazoans are endowed with a variety of host-defense mechanisms against invading pathogens
We previously demonstrated that C3 (TtC3), identified in the Japanese horseshoe crab Tachypleus tridentatus, is proteolytically activated by a lipopolysaccharide (LPS)-sensitive serine protease factor C in the presence of LPS, leading to C3b deposition on Gram-negative bacteria [6]
We identified two structurally different types of C2/factor B in T. tridentatus (TtC2/Bf-1 and TtC2/Bf-2), and characterized whether the two complement factors participate in C3b deposition on microbes, including Gram-positive and -negative bacteria and fungi, in the presence or absence of plasma lectins
Summary
Metazoans are endowed with a variety of host-defense mechanisms against invading pathogens. Pattern-recognition proteins, such as mammalian Toll-like receptors and Drosophila peptidoglycanrecognition proteins, recognize pathogen-associated molecular patterns (PAMPs), and activate signaling cascades that induce inflammation, phagocytosis, and subsequent adaptive immunity [1,2]. The mammalian complement system is an important immune surveillance system involving more than 30 factors in plasma or on cytoplasmic membrane; complement C3 is activated via three convergent pathways (classical, alternative, and lectin) [3]. The activation of C3 can lead to a variety of host-defense systems, including the promotion of phagocytosis and the formation of a membrane-attack complex. The alternative pathway is triggered by a small fraction of C3, in which the thioester bond is hydrolyzed to C3(H2O), exposing a binding site for complement factor B, a serine protease zymogen [4]. The resulting C3a attracts phagocytes to an inflammation locus, and C3b binds to the surfaces of target cells, leading to C3bBb (a membrane-bound C3 convertase), which amplifies C3b deposition on the surfaces of target cells
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