Capturing the Active Transition States of LPS-Sensitive Protease Precursors in Autocatalytic Activation
Keywords:
Horseshoe crab, Lipopolysaccharide (LPS), Serine protease zymogen, Autocatalytic activation, Transition stateAbstract
Hemolymph coagulation in horseshoe crabs is triggered by the autocatalytic activation of lipopolysaccharide (LPS)-sensitive serine protease zymogen factor C through its transition state (factor C*). However, the existence of factor C* is only speculative, and it remains unknown whether the autocatalytic cleavage of the Phe737-Ile738 bond (the F737 site) of factor C* required for the conversion to an active form α-factor C occurs intramolecularly or intermolecularly. We show that the F737 site of a catalytic Ser941-deficient mutant of factor C is cleaved by an F737 site-uncleavable mutant in the presence of LPS. These data clearly indicate the existence of factor C* without cleavage of the F737 site. We also found the following facts. Firstly, the autocatalytic cleavage at the F737 site of factor C* occurs intermolecularly on the LPS surface. Secondly, factor C* does not exhibit intrinsic chymotryptic activity against the F737 site during the autocatalytic activation. Thirdly, LPS is required not only to complete the substrate-binding site and oxyanion hole of factor C* but also to allow the F737 site to be cleaved.
References
Kawabata S, Muta T, Iwanaga S, 2010, Discovery of the Lipopolysaccharide- and β-1,3-D-Glucan-Mediated Proteolytic Cascade and Unique Proteins in Invertebrate Immunity. J Biochem, 2010(147): 611–618.
Koshiba T, Hashii T, Kawabata S, 2007, A Structural Perspective on the Interaction Between Lipopolysaccharide and Factor C, a Receptor Involved in Recognition of Gram-Negative Bacteria. J Biol Chem, 2007(282): 3962–3967.
Ariki S, Koori K, Osaki T, et al., 2004, A Serine Protease Zymogen Functions as a Pattern - Recognition Receptor for Lipopolysaccharides. Proc Natl Acad Sci USA, 2004(101): 953–958.
Wang J, Tan NS, Ho B, et al., 2002, Modular Arrangement and Secretion of a Multidomain Serine Protease. Evidence for Involvement of Proline-Rich Region and N-Glycans in the Secretion Pathway. J Biol Chem, 2002(277): 36363–36372.
Kobayashi Y, Shiga T, Shibata T, et al., 2014, The N-terminal Arg Residue is Essential for Autocatalytic Activation of a Lipopolysaccharide-Responsive Protease Zymogen. J Biol Chem, 2014(289): 25987–25995.
Tokunaga F, Miyata T, Nakamura T, et al., 1987, Lipopolysaccharide-Sensitive Serine-Protease Zymogen (Factor C) of Horseshoe Crab Hemocytes. Identification and Alignment of Proteolytic Fragments Produced During the Activation Show that it is a Novel Type of Serine Protease. Eur J Biochem, 1987(167): 405–416.
Shibata T, Kobayashi Y, Ikeda Y, et al., 2018, Intermolecular Autocatalytic Activation of Serine Protease Zymogen Factor C Through an Active Transition State Responding to Lipopolysaccharide. J Biol Chem, 2018(293): 11589–11599.
Kobayashi Y, Takahashi T, Shibata T, et al., 2015, Factor B is the Second Lipopolysaccharide-Binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade. J Biol Chem, 2015(290): 19379–19386.
Zhu Y, Thangamani S, Ho B, et al., 2005, The Ancient Origin of the Complement System. EMBO J, 2005(24): 382–394.
Tagawa K, Yoshihara T, Shibata T, et al., 2012, Microbe-Specific C3b Deposition in the Horseshoe Crab Complement System in a C2/Factor B-Dependent or -Independent Manner. PLoS One, 2012(7): e36783.
Ariki S, Takahara S, Shibata T, et al., 2008, Factor C Acts as a Lipopolysaccharide-Responsive C3 Convertase in Horseshoe Crab Complement Activation. J Immunol, 2008(181): 7994–8001.
Urbanová V, Hartmann D, Grunclová L, et al., 2014, IrFC-An Ixodes ricinus Injury-Responsive Molecule Related to Limulus Factor C. Dev Comp Immunol, 2014(46): 439–447.
