Abstract
Horseshoe crab hemolymph coagulation is believed to be triggered by the autocatalytic activation of serine protease zymogen factor C to the active form, α-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 α-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.
Highlights
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*
Proteolytic cascades in blood/hemolymph coagulation involve serine protease zymogens and are triggered through the interaction of initiator zymogens with biologic substances derived from host tissues or microbes to amplify and propagate biologic reactions [27,28,29,30]
We used several mutants of factor C to show that the Phe737 site of the S941A mutant was cleaved LPS-dependently by the F737P mutant (Fig. 5), clearly indicating the presence of the active transition state of factor C with an active conformation induced by the interaction with LPS without cleavage of the Phe737 site
Summary
(Phe737 site) (Fig. 1), corresponding to the Arg15–Ile bond in chymotrypsinogen numbering [21]. The resulting -factor C exhibits amidase activity against the synthetic substrates for ␣-thrombin with ϳ70% specific activity compared with that of ␣-factor C but has neither the LPS-binding activity nor the activating activity for factor B [22]. These data suggest that LPS interaction with ␣-factor C is required to maintain its proteolytic activity against factor B. Factor C*, is only speculative, and its proteolytic activity has not been validated It remains unclear whether the autocatalytic cleavage at the Phe737 site occurs intramolecularly within factor C bound to LPS or intermolecularly between the molecules of factor C. We show that the autocatalytic activation of factor C is the unidentified intermolecular event between the factor C* molecules on the LPS surface
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