title: Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.
abstract: Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to β-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by β-factor C, in an LPS-dependent manner and that β-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.
authors.affiliation: From the Department of Biology, Faculty of Sciences.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kobayashi
authors.affiliation: the Graduate School of Systems Life Sciences, and.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: From the Department of Biology, Faculty of Sciences, the Institute for Advanced Study, Kyushu University, Fukuoka 812-8581, Japan and.
authors.forename: Toshio
authors.initials: T
authors.lastname: Shibata
authors.affiliation: the Graduate School of Systems Life Sciences, and.
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Ikeda
authors.affiliation: From the Department of Biology, Faculty of Sciences, the Graduate School of Systems Life Sciences, and.
authors.forename: Takumi
authors.initials: T
authors.lastname: Koshiba
authors.affiliation: the LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Mizumura
authors.affiliation: the LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Oda
authors.affiliation: From the Department of Biology, Faculty of Sciences, the Graduate School of Systems Life Sciences, and skawascb@kyudai.jp.
authors.forename: Shun-ichiro
authors.initials: S
authors.lastname: Kawabata
mesh_terms: D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry*; D001665:Binding Sites; D011415:Complement Factor B / Q000737:chemistry*; D004792:Enzyme Precursors / Q000737:chemistry*; D057809:HEK293 Cells; D006737:Horseshoe Crabs / Q000201:enzymology*; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry*; D011485:Protein Binding; D059748:Proteolysis
chemical_list: D060829:Arthropod Proteins; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D011415:Complement Factor B
keywords: endotoxin; enzyme processing; lipopolysaccharide (LPS); recombinant protein expression; serine protease