The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

Abstract

Background: Factor C is autocatalytically activated on lipopolysaccharides. Results: The N-terminal Arg of factor C is essential for the autocatalytic activation. Conclusion:The N-terminal Arg of factor C is required to be in sufficiently close vicinity for the autocatalytic activation. Significance: The recombinant factor C with restricted and homogeneous N-glycans may be useful for further structurefunction studies.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade

“…An Inhibitory Effect of Triton X-100 on Factor B Activation by ␣-Factor C in the Presence of LPS-Autocatalytic activation of factor C on LPS is inhibited by adding the synthetic surfactant Triton X-100, especially above the critical micelle concentration of Triton X-100 (0.24 mM) (5,24). To determine whether Triton X-100 inhibits the activation of factor B by ␣-factor C, wild-type factor B was incubated with ␣-factor C in the presence of LPS (0.44 nM) and various concentrations of Triton X-100.…”

“…Expression of Recombinant Proteins-Recombinant proteins were expressed as described previously (5). In brief, recombinant proteins were expressed in HEK293S GnTI Ϫ cells and secreted into the culture medium.…”

“…The amidase activity of activated factor B was assayed with 0.4 mM Boc-Leu-Thr-Arg-MCA, as described previously (5). Molar concentrations of pNA and 7-amino-4-methylcoumarin released were measured, respectively, by absorbance at 405 nm and fluorometrically with excitation at 380 nm and emission at 440 nm.…”

“…Microbalance-Surface plasmon resonance analysis was performed as described previously (5). Biotinylated LPS (2.3 M in 10 mM HEPES-NaOH, pH 7.4, containing 150 mM NaCl) was immobilized on a sensor chip SA of a BIAcore X system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's specifications.…”

“…1). In addition, the N-terminal Arg residue of factor C and the distance between the N terminus and the tripeptide are absolutely required for specific proteinprotein interactions to form multiple oligomers of factor C on the surface of LPS, leading to autocatalytic activation (5).…”