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

Keisuke Yamashita, Naoki Takeshita, Aina Arita, Toshio Shibata, Yuki Kobayashi, Shun Ichiro Kawabata

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)489-500
Number of pages12
JournalJournal of biochemistry
Volume170
Issue number4
DOIs
Publication statusPublished - Oct 1 2021

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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