TY - JOUR
T1 - Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide
AU - Shibata, Toshio
AU - Kobayashi, Yuki
AU - Ikeda, Yuto
AU - Kawabata, Shun ichiro
N1 - Funding Information:
This study was supported by the Joint Research Fund of Seikagaku Corp. The authors declare that they have no conflicts of interest with the contents of this article. We thank Naoki Hirakawa (Kyushu University, Fukuoka, Japan) for preparation of factor C recombinants containing the PA tag and for preliminary experiments regarding the autocatalytic activation of these recombinants. We also thank Prof. Shouichi Higashi (Yokohama City University, Yokohama, Japan) for helpful discussions regarding autocatalytic activation of serine protease zymogens.
Publisher Copyright:
© 2018 Shibata et al.
PY - 2018/7/20
Y1 - 2018/7/20
N2 - Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, -factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversion to -factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site– uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules.
AB - Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, -factor C, belonging to the trypsin family, through an active transition state of factor C responding to bacterial lipopolysaccharide (LPS), designated factor C*. However, the existence of factor C* is only speculative, and its proteolytic activity has not been validated. In addition, it remains unclear whether the proteolytic cleavage of the Phe737–Ile738 bond (Phe737 site) of factor C required for the conversion to -factor C occurs intramolecularly or intermolecularly between the factor C molecules. Here we show that the Phe737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe737 site– uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe737 site. Moreover, we found the following facts using several mutants of factor C: the autocatalytic cleavage of factor C occurs intermolecularly between factor C* molecules on the LPS surface; factor C* does not exhibit intrinsic chymotryptic activity against the Phe737 site, but it may recognize a three-dimensional structure around the cleavage site; and LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* by interacting with the N-terminal region but also to allow the Phe737 site to be cleaved by inducing a conformational change around the Phe737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules.
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U2 - 10.1074/jbc.RA118.002311
DO - 10.1074/jbc.RA118.002311
M3 - Article
C2 - 29866883
AN - SCOPUS:85050394505
VL - 293
SP - 11589
EP - 11599
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -