Section 22
Chapter 21,624

Purified horseshoe crab factor G. Reconstitution and characterization of the (1-3) --D-glucan-sensitive serine protease cascade

Muta, T.; Seki, N.; Takaki, Y.; Hashimoto, R.; Oda, T.; Iwanaga, A.; Tokunaga, F.; Iwanaga, S.

Journal of Biological Chemistry 270(2): 892-897


ISSN/ISBN: 0021-9258
DOI: 10.1074/jbc.270.2.892
Accession: 021623953

Horseshoe crab hemocyte lysate responds to (1 fwdarw 3)-beta-D-glucans, initiating an enzymatic cascade, which culminates in clot formation. We have purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1 fwdarw 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 fwdarw 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 fwdarw 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 fwdarw 3)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.

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