Citation

The lipopolysaccharide (LPS)-triggered horseshoe crab coagulation cascade is composed of three protease zymogens, prochelicerase C (proC), prochelicerase B (proB) and the proclotting enzyme (proCE). In this study, we found that Ca ²⁺ ions increase the production of the clotting enzyme as a result of a cascade reaction reconstituted by recombinant proteins of wild-type (WT) proC, WT proB and WT proCE. We divided the cascade into three stages: autocatalytic activation of WT proC on the surface of LPS into WT α-chelicerase C (Stage 1); activation of WT proB on the surface of LPS into WT chelicerase B by WT α-chelicerase C (Stage 2) and activation of WT proCE into WT CE by chelicerase B (Stage 3). Ca²⁺ ions enhanced the proteolytic activation in Stage 2, but not those in Stages 1 and 3. Moreover, we performed isothermal titration calorimetry to clarify the interaction of LPS or the recombinant zymogens with Ca²⁺ ions. LPS interacted with Ca²⁺ ions at an association constant of K_a = 4.7 × 10⁴ M⁻¹, but not with any of the recombinant zymogens. We concluded that LPS bound with Ca²⁺ ions facilitates the chain reaction of the cascade as a more efficient scaffold than LPS itself.