TY - JOUR
T1 - Roles of the clip domains of two protease zymogens in the coagulation cascade in horseshoe crabs
AU - Yamashita, Keisuke
AU - Shibata, Toshio
AU - Takahashi, Toshiaki
AU - Kobayashi, Yuki
AU - Kawabata, Shun-Ichiro
N1 - Publisher Copyright:
© 2020 Yamashita et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2020/6/26
Y1 - 2020/6/26
N2 - 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 a-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 a-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 a-chelicerase C and LPS, where a-chelicerase C is ready to activate proB, leading to chelicerase B-mediated activation of proCE via its clip domain.
AB - 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 a-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 a-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 a-chelicerase C and LPS, where a-chelicerase C is ready to activate proB, leading to chelicerase B-mediated activation of proCE via its clip domain.
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U2 - 10.1074/jbc.ra119.012452
DO - 10.1074/jbc.ra119.012452
M3 - Article
C2 - 32409575
AN - SCOPUS:85087320823
SN - 0021-9258
VL - 295
SP - 8857
EP - 8866
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -