The lipopolysaccharide (LPS)-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 disulphides located around the active site. While it is known that proB evolutionarily lost one of the disulphides, the His-loop disulphide, the roles of the missing His-loop disulphide in proB remain unknown. Here, we prepared a proB mutant, named proB-murasame, equipped with a regenerated His-loop disulphide. The activation rate by upstream α-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 LPS-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 LPS detection.

中文翻译：

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一种配备再生二硫化物的突变体，用于马蹄蟹凝血级联中缺失的丝氨酸蛋白酶酶原 His 环

马蹄蟹中脂多糖 (LPS) 触发的凝血级联反应由属于胰蛋白酶原家族的三种酶原组成：前螯合酶 C、前螯合酶 B (proB) 和前凝血酶 (proCE)。胰蛋白酶原家族成员包含三个位于活性位点周围的保守二硫化物。虽然已知 proB 在进化上失去了一种二硫化物，即 His-loop 二硫化物，但缺失的 His-loop 二硫化物在 proB 中的作用仍然未知。在这里，我们准备了一个 proB 突变体，名为 proB-murasame，配备了再生的 His-loop 二硫化物。上游 α-螯合酶 C 对 proB-murasame 的激活率与野生型 (WT) proB 的激活率没有区别。所得蛋白酶螯合酶 B-murasame 对下游 proCE 的 kcat 值比 WT 螯合酶 B 高 8 倍，而螯合酶 B-murasame 的 Km 值与 WT 螯合酶 B 相当。WT serpins-1、-2 和 -3 被确定为级联的清除剂，对 WT 螯合酶 B 没有反应性，而螯合酶 B-murasame 是被 WT serpin-2 抑制，表明 WT chelicerae B 在级联中履行其职责后可能触发尚未解决的现象。重组的含有 proB-murasame 的 LPS 触发级联显示出比含有 WT proB 高 5 倍的 CE 产量。ProB-murasame 可用作 LPS 检测的高附加值试剂。表明 WT chelicerae B 在级联中履行其职责后可能会触发尚未解决的现象。重组的含有 proB-murasame 的 LPS 触发级联显示出比含有 WT proB 高 5 倍的 CE 产量。ProB-murasame 可用作 LPS 检测的高附加值试剂。表明 WT chelicerae B 在级联中履行其职责后可能会触发尚未解决的现象。重组的含有 proB-murasame 的 LPS 触发级联显示出比含有 WT proB 高 5 倍的 CE 产量。ProB-murasame 可用作 LPS 检测的高附加值试剂。