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A mutant equipped with a regenerated disulfide for the missing his loop of a serine protease zymogen in the horseshoe crab coagulation cascade

Yamashita, K;Takeshita, N;Arita, A;Shibata, T;Kobayashi, Y;Kawabata, SI;

The lipopolysaccharide-triggered coagulation cascade in horseshoe crabs is composed of three zymogens belonging to the trypsinogen family: prochelicerase C, prochelicerase B (proB), and the proclotting enzyme (proCE). Trypsinogen-family members contain three conserved disulfides located around the active site. While it is known that proB evolutionarily lost one of the disulfides, the His-loop disulfide, the roles of the missing His-loop disulfide in proB remain unknown. Here we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulfide. The activation rate by upstream ?-chelicerase C for proB-murasame was indistinguishable from that for wild-type (WT) proB. The resulting protease chelicerase B-murasame exhibited an 8-fold higher kcat value for downstream proCE than WT chelicerase B, whereas the Km value of chelicerase B-murasame was equivalent to that of WT chelicerase B. WT serpins-1, -2, and -3, identified as scavengers for the cascade, had no reactivity against WT chelicerase B, whereas chelicerase B-murasame was inhibited by WT serpin-2, suggesting that WT chelicerae B may trigger as-yet-unsolved phenomena after performing its duty in the cascade. The reconstituted lipopolysaccharide-triggered cascade containing proB-murasame exhibited ?5-fold higher CE production than that containing WT proB. ProB-murasame might be used as a high value-adding reagent for lipopolysaccharide detection.