Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization.
Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.
Muta
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Hashimoto
R
R
Miyata
T
T
Nishimura
H
H
Toh
Y
Y
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000235:genetics; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004220:Disulfides / Q000032:analysis; D010450:Endopeptidases / Q000032:analysis / Q000235:genetics*; D004792:Enzyme Precursors / Q000032:analysis / Q000235:genetics*; D006434:Hemocytes / Q000201:enzymology*; D006737:Horseshoe Crabs / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D007124:Immunoenzyme Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D001779:Blood Coagulation Factors; D004220:Disulfides; D004792:Enzyme Precursors; D010446:Peptide Fragments; D012333:RNA, Messenger; D004247:DNA; D010450:Endopeptidases; C014775:pro-clotting enzyme