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.
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 light 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 beta 2-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.
Tokunaga
F
F
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Miyata
T
T
Nakamura
T
T
Morita
T
T
Kuma
K
K
Miyata
T
T
Iwanaga
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003488:Cyanogen Bromide; D004789:Enzyme Activation; D006458:Hemolymph / Q000166:cytology / Q000201:enzymology; D006737:Horseshoe Crabs / Q000201:enzymology*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D008070:Lipopolysaccharides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010447:Peptide Hydrolases; D012697:Serine Endopeptidases; D003488:Cyanogen Bromide