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

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Abstract
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 (Mr= 123 000) composed of a heavy chain (Mr= 80 000) and a light chain (Mr= 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 (Mr= 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.