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* Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka, Japan, and
Department of Immunology, Fukushima Medical University School of Medicine, Fukushima, Japan
The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the horseshoe crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the horseshoe crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of horseshoe crab complement activation.
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1 This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Priority Area 839 to S.K. and T.F., No. 13143203 to S.K., No. 13143204 to T.F., and No. 18370045 to S.K.) and by the Naito Foundation (to S.K.).
2 The sequences reported in this article have been deposited in GenBank database (accession no. AB353279 for TtC3).
3 Current address: Department of Biochemistry, School of Medicine, Sapporo Medical University, Sapporo, Japan.
4 Address correspondence and reprint requests to Dr. Shun-ichiro Kawabata, Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-Ku, Fukuoka, Japan. E-mail address: skawascb{at}kyudai.jp
5 Abbreviations used in this paper: Df, complement factor D; Bf, complement factor B; PAMP, pathogen-associated molecular pattern; HDP, hemocyanin-depleted plasma; PVDF, polyvinylidene fluoride; MCA, 4-methylcoumaryl-7-amide; AMC, 7-amino-4-methylcoumarin; PPACK, D-Phe-Pro-Arg-chloromethylketone; Boc, N-tert-butoxycarbonyl; Bzl, benzyl.
6 The online version of this article contains supplementary material.
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