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

Publisher: Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology Country of Publication: United States NLM ID: 2985121R Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1083-351X (Electronic) Linking ISSN: 00219258 NLM ISO Abbreviation: J Biol Chem Subsets: MEDLINE

Publication: 2021- : [New York, NY] : Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology
Original Publication: Baltimore, MD : American Society for Biochemistry and Molecular Biology

Arthropod Proteins/*metabolism ; Horseshoe Crabs/*physiology ; Peptide Hydrolases/*metabolism; Animals ; Arthropod Proteins/chemistry ; Blood Coagulation ; Endopeptidases/chemistry ; Endopeptidases/metabolism ; Enzyme Activation ; Enzyme Precursors/chemistry ; Enzyme Precursors/metabolism ; Lipopolysaccharides ; Models, Molecular ; Peptide Hydrolases/chemistry ; Protein Domains

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 k cat / K m 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.
Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with respect to the contents of this article.
(© 2020 Yamashita et al.)

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Keywords: clip domain; coagulation cascade; coagulation factor; endotoxin; enzyme mutation; enzyme processing; lipopolysaccharide (LPS); prochelicerase B (proB); proclotting enzyme (proCE); recombinant protein expression; serine protease

0 (Arthropod Proteins)
0 (Enzyme Precursors)
0 (Lipopolysaccharides)
EC 3.4.- (Endopeptidases)
EC 3.4.- (Peptide Hydrolases)
EC 3.4.99.- (pro-clotting enzyme)

Date Created: 20200516 Date Completed: 20210113 Latest Revision: 20210627

20250114

PMC7324516

10.1074/jbc.RA119.012452

32409575