title: The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.
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.
authors: From the Graduate School of Systems Life Sciences.
authors: Yuki
authors: Y
authors: Kobayashi
authors: From the Graduate School of Systems Life Sciences.
authors: Takafumi
authors: T
authors: Shiga
authors: Department of Biology, Faculty of Sciences, and Institute for Advanced Study, Kyushu University, Fukuoka 812-8581.
authors: Toshio
authors: T
authors: Shibata
authors: the Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, and.
authors: Miyuki
authors: M
authors: Sako
authors: the Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, and.
authors: Katsumi
authors: K
authors: Maenaka
authors: From the Graduate School of Systems Life Sciences, Department of Biology, Faculty of Sciences, and.
authors: Takumi
authors: T
authors: Koshiba
authors: the LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan.
authors: Hikaru
authors: H
authors: Mizumura
authors: the LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan.
authors: Toshio
authors: T
authors: Oda
authors: From the Graduate School of Systems Life Sciences, Department of Biology, Faculty of Sciences, and skawascb@kyudai.jp.
authors: Shun-ichiro
authors: S
authors: Kawabata
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060829:Arthropod Proteins / Q000378:metabolism*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D057809:HEK293 Cells; D006737:Horseshoe Crabs / Q000201:enzymology*; D006801:Humans; D007113:Immunity, Innate / Q000235:genetics*; D008070:Lipopolysaccharides / Q000633:toxicity; D057057:Serine Proteases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
chemical_list: D060829:Arthropod Proteins; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D057057:Serine Proteases
keywords: Chemical Cross-linker; Coagulation Cascade; Glycoprotein; Lipopolysaccharide (LPS); Protein-protein Interaction; Proteolytic Cleavage; Recombinant Protein Expression; Serine Protease