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

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

Horseshoe Crab Aquaculture as a Sustainable Endotoxin Testing Source

“…One protein band, identified as the L-chain of Factor C, migrated faster in the commercial LAL (∼43 kDa) than in the aquaculture LAL (∼52 kDa), which was confirmed via Western blotting with rabbit polyclonal antibody (COCH, ABclonal, Inc., Woburn, MA, United States; data not shown). Factor C typically generates a 43 kDa subunit (L-chain) under denaturing conditions (Shibata et al, 2018), and thus, the aquaculture-derived Factor C L-chain subunit could migrate at a higher molecular weight due to posttranslational modifications. As lysates prepared from freshly harvested wild HSCs revealed, this higher molecular weight form of Factor C does not appear to be a consequence of aquaculture but rather an effect of commercial LAL preparation ( Figure 6C).…”

Immune Responses to Gram-Negative Bacteria in Hemolymph of the Chinese Horseshoe Crab, Tachypleus tridentatus

“…Obviously, the innate immune mechanism of horseshoe crabs is unique and complex, especially against Gram-negative bacteria containing LPS in the cell wall membrane. In previous studies, many innate immunity-related genes have been reported in Chinese horseshoe crab (13,14,17,20), but the immune response mechanism based on highthroughput analysis of pathogen challenge has been rarely reported.…”

Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

“…Takayama et al [21] observed LAL activation with their monomeric LPS preparations, and Mueller et al [14] did not. As discussed above, a recent study revealed that monomeric LPS cannot activate Factor C [17]. Although there was a proposed mechanism that Factor C can be activated by monomeric LPS before [22], no direct evidence was provided.…”

“…LAL activation is triggered by intermolecular activation of Factor C molecules on LPS aggregates [17]. The Factor C activation occurs on the surface of LPS aggregates after active transition state Factor C forms the dimer or its multimers on the LPS aggregates.…”