The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen |
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Authors: | Yuki Kobayashi Takafumi Shiga Toshio Shibata Miyuki Sako Katsumi Maenaka Takumi Koshiba Hikaru Mizumura Toshio Oda Shun-ichiro Kawabata |
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Affiliation: | From the ‡Graduate School of Systems Life Sciences.;§Department of Biology, Faculty of Sciences, and ;¶Institute for Advanced Study, Kyushu University, Fukuoka 812-8581.;the ‖Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, and ;the **LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan |
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Abstract: | Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. |
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Keywords: | Glycoprotein Lipopolysaccharide (LPS) Protein-protein Interaction Recombinant Protein Expression Serine Protease Chemical Cross-linker Coagulation Cascade Proteolytic Cleavage |
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