PURIFIED HORSESHOE-CRAB FACTOR-G - RECONSTITUTION AND CHARACTERIZATONOF THE (1-]3)-BETA-D-GLUCAN-SENSITIVE SERINE-PROTEASE CASCADE

Horseshoe crab hemocyte lysate responds to (1-->3)-beta-D-glucans, ini tiating an enzymatic cascade, which culminates in clot formation,We ha ve purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1-->3)-beta-D-glucans and which initiates t he hemolymph clotting cascade, Factor G is a heterodimeric protein com posed of two noncovalently associated subunits alpha (72 kDa) and beta (37 kDa), In the presence of (1-->3)-beta-D-glucans such as curdlan a nd paramylon, factor G is autocatalytically activated to an active ser ine protease named factor ($) over bar G. This activation is accompani ed by limited proteolysis of both subunits: the 72-kDa subunit a is cl eaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is s hortened to 34 kDa. Longer incubations with (1-->3)-beta-D-glucans res ult in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa fragme nt to 32 kDa, with concomitant loss of amidase activity. Reconstitutio n experiments using purified proteins participating in the hemolymph c lotting cascade demonstrate that factor ($) over bar is capable of act ivating proclotting enzyme directly, resulting in the conversion of co agulogen to coagulin gel. Thus, purified factor G is shown to be the p rimary initiator of the (1-->3)-beta-D-glucan-sensitive coagulation pa thway in the horseshoe crab hemocyte lysate.