Purified horseshoe crab factor G. Reconstitution and characterization of the (1-->3)-beta-D-glucan-sensitive serine protease cascade.
Horseshoe crab hemocyte lysate responds to (1-->3)-beta-D-glucans, initiating an enzymatic cascade, which culuminates in clot formation. We have purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1-->3)-beta-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently associated subunits alpha (72 kDa) and beta (37 kDa). In the presence of (1-->3)-beta-D-glucans such as curdlan and paramylon, factor G is autocatalytically activated to an active serine protease named factor G. This activation is accompanied by limited proteolysis of both subunits: the 72-kDa subunit alpha is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is shortened to 34 kDa. Longer incubations with (1-->3)-beta-D-glucans result in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa fragment to 32 kDa, with concomitant loss of amidase activity. Reconstitution experiments using purified proteins participating in the hemolymph clotting cascade demonstrate that factor G is capable of activating proclotting enzyme directly, resulting in the conversion of coagulogen to coagulin gel. Thus, purified factor G is shown to be the primary initiator of the (1-->3)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.
Muta
T
T
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Seki
N
N
Takaki
Y
Y
Hashimoto
R
R
Oda
T
T
Iwanaga
A
A
Tokunaga
F
F
Iwanaga
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001779:Blood Coagulation Factors / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D005936:Glucans / Q000378:metabolism*; D006737:Horseshoe Crabs; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D047071:beta-Glucans
D001779:Blood Coagulation Factors; D005936:Glucans; D047071:beta-Glucans; C033363:beta-1,3-glucan; C085383:coagulation factor G, horseshoe crab; D012697:Serine Endopeptidases