Muta T, Seki N, Takaki Y, Hashimoto R, Oda T, Iwanaga A, Tokunaga F, Iwanaga S
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
J Biol Chem. 1995 Jan 13;270(2):892-7.
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.
鲎血细胞裂解物对（1→3）-β-D-葡聚糖有反应，引发酶促级联反应，最终形成凝块。我们已将丝氨酸蛋白酶原因子G纯化至同质，它可被（1→3）-β-D-葡聚糖直接激活，并启动血淋巴凝血级联反应。因子G是一种异二聚体蛋白，由两个非共价结合的亚基α（72 kDa）和β（37 kDa）组成。在诸如可德胶和副淀粉等（1→3）-β-D-葡聚糖存在的情况下，因子G会自动催化激活为一种名为因子G的活性丝氨酸蛋白酶。这种激活伴随着两个亚基的有限蛋白水解：72 kDa的亚基α被切割成55 kDa和17 kDa的片段，37 kDa的亚基β缩短至34 kDa。与（1→3）-β-D-葡聚糖长时间孵育会导致55 kDa的片段被切割成46 kDa，34 kDa的片段被切割成32 kDa，同时酰胺酶活性丧失。使用参与血淋巴凝血级联反应的纯化蛋白进行的重组实验表明，因子G能够直接激活凝血前酶，导致凝固原转化为凝固蛋白凝胶。因此，纯化的因子G被证明是鲎血细胞裂解物中（1→3）-β-D-葡聚糖敏感凝血途径的主要启动因子。
