Intermolecular autocatalytic activation of serine protease zymogen factor C through an active transition state responding to lipopolysaccharide

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 Phe 737 –Ile 738 bond (Phe 737 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 Phe 737 site of a catalytic Ser-deficient mutant of factor C is LPS-dependently hydrolyzed by a Phe 737 site–uncleavable mutant, clearly indicating the existence of the active transition state of factor C without cleavage of the Phe 737 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 Phe 737 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 Phe 737 site to be cleaved by inducing a conformational change around the Phe 737 site or by acting as a scaffold to induce specific protein–protein interactions between factor C* molecules.