Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade |
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Authors: | Yuki Kobayashi Toshiaki Takahashi Toshio Shibata Shunsuke Ikeda Takumi Koshiba Hikaru Mizumura Toshio Oda Shun-ichiro Kawabata |
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Institution: | From the ‡Department of Biology, Faculty of Sciences.;the §Graduate School of Systems Life Sciences, and ;the ¶Institute for Advanced Study, Kyushu University, Fukuoka 812-8581, Japan and ;the ‖LAL Research and Development Group, Seikagaku Corporation, Higashiyamato, Tokyo 207-0021, Japan |
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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. |
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Keywords: | endotoxin enzyme processing lipopolysaccharide (LPS) recombinant protein expression serine protease |
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