Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs

Keisuke Yamashita, Toshio Shibata, Toshiaki Takahashi, Yuki Kobayashi, Shun Ichiro Kawabata

Research output: Contribution to journalArticle

Abstract

The lipopolysaccharide (LPS)-triggered coagulation cascade in horseshoe crabs comprises three protease zymogens: prochelicerase C (proC), prochelicerase B (proB), and the proclotting enzyme (proCE). The presence of LPS results in autocatalytic activation of proC to α-chelicerase C, which, in turn, activates proB to chelicerase B, converting proCE to the clotting enzyme (CE). ProB and proCE contain an N-terminal clip domain, but the roles of these domains in this coagulation cascade remain unknown. Here, using recombinant proteins and kinetics and binding assays, we found that five basic residues in the clip domain of proB are required to maintain its LPS-binding activity and activation by α-chelicerase C. Moreover, an amino acid substitution at a potential hydrophobic cavity in proB's clip domain (V55A-proB) reduced both its LPS-binding activity and activation rate. WT proCE exhibited no LPS-binding activity, and the WT chelicerase B-mediated activation of a proCE variant with a substitution at a potential hydrophobic cavity (V53A-proCE) was ∼4-fold slower than that of WT proCE. The kcat/Km value of the interaction of WT chelicerase B with V53A-proCE was 7-fold lower than that of the WT chelicerase B-WT proCE interaction. The enzymatic activities of V55A-chelicerase B and V53A-CE against specific peptide substrates were indistinguishable from those of the corresponding WT proteases. In conclusion, the clip domain of proB recruits it to a reaction center composed of α-chelicerase C and LPS, where α-chelicerase C is ready to activate proB, leading to chelicerase B-mediated activation of proCE via its clip domain.

Original languageEnglish
Pages (from-to)8857-8866
Number of pages10
JournalThe Journal of biological chemistry
Volume295
Issue number26
DOIs
Publication statusPublished - Jun 26 2020

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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