Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (M_r= 123 000) composed of a heavy chain (M_r= 80 000) and a light chain (M_r= 43 000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511–521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (M_r= 123 000) in the hemocyte lysate. The heavy chain had the NH₂-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH₂-terminal part of the molecule. The light chain had an NH₂-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the ligth chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human β₂-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH₂-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-.

These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.