| Co-Investigator(Kenkyū-buntansha) |
TAWADA Katsuhisa Kyusyu Univ., Faculty of Sci., Associate Professor, 理学部, 助教授 (20029507)
TOKUNAGA Fuminori Kyusyu Univ., Faculty of Sci., Postdoctoral fellow (JSPS), 理学部, 日本学術振興会特別研 (00212069)
MIYATA Toshiyuki Kyusyu Univ., Faculty of Sci., Research Associate, 理学部, 助手 (90183970)
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| Research Abstract |
Limulus proclotting enzyme and factor b associated with endotoxin-sensitive coagulation cascade : novel serine protease zymogens with a new type of "disulfide-knotted domain"
Horseshoe crab (Limulus) hemolymph responds to bacterial endotoxin and results in rapid coagulation. This reaction is composed of a cascade consisting of three serine protease zymogens (factor C, factor B, and proclotting enzyme) and a clottable protein (coagulogen), all of which are released by degranulation of the hemocytes on the stimutation of endotoxin. In the present paper, we describe the structures of proclotting enzyme and factor B. CDNA clonings of all the factors associated with this cascade were completed by this study. Proclotting enzyme is a single-chain glycoprotein with molecular mass of 54 kDa. Upon activation by factor B, it is converted to a two-chain active form clotting enzyme composed of an L (25 kDa) and a H (31 kDa) chains. A CDNA for proclotting enzyme encodes a sequence comprising 29 amino
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acid residues of prepro-sequence and 346 residues of the mature protein. Factor B (64 kDa) is also a glycoprotein that exists as a single-chain form and a two-chain zymogen form with an L (25 kDa) and a H (40 kDa) chains. Factor B zymogen is activated to B by active factor C, which is derived from the zymogen factor C that is sensitive to endotoxin. Factor B has 375 amino acid residues in the mature form, in addition to 25 residues of signal sequence. The entire amino acid sequences of the two proteins are similar, suggeting that these proteins were arisen from a gene duplication. Particularly, the sequence identity of serine protease domains at their C-terminus is calculated to be 43.9%. The N-terminal regions up to 60th residue of both proteins share a similar structure with six cysteines that has not been found in any other proteins. The disulfide locations determined on proclotting enzyme indicate that this region consists of a compact "disulfide-knotted domain". The sequence accompanied with the activation of proclotting enzyme was proven to be an Arg-Ile bond, whereas that of factor B, deduced from sequence alignments with other serine proteases, contained a unique-Arg-Gly-Ile-sequence. Less
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