The Amino Acid Sequence of Coagulogen Isolated from Southeast Asian Horseshoe Crab, Tachypleus gigas12

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Abstract
The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology, in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a closer phylogenetic relationship with each other than with American horseshoe crab, based on amino acid sequence data. Moreover, the sequence comparison indicated that the A and B chain regions, which participate in the gel formation, have extremely high homology in the three coagulogens, whereas the peptide C region (released during the gelation) is relatively variable.