Role of Limulus Hemocytes in the Biological Defense System.

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Horseshoe Crab hemocyte lysate responds to (1*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*3) -D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently-asociated subnits alpha (72 kDa) and beta (37 kDa). In the prescence of (1*3) -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 proteolyses of Arg-X bonds in 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. 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 coagulo … More gen to coagulin gel. Analyzes of cDNAs for both subunits indicates that the subunits are derived from separate mRNA species and thus encoded by different genes. Subunit beta is a serine protease zymogen consisting of 278 residues, which is homologous to horseshoe crab factor B.Subunit alpha, on the other hand, is a new type of mozaic protein consisting of 654 residues with intriguing features. The NH_2-terminal portion of this subnit is similar to bacterial beta-1,3-glucanases. Its 126-amino-acid COOH-terminus exhibits a repetitive sequence having partial homology to xylanases. Between these regions there are three repeating units of 47 amino acids, whose similarity to carbohydrate binding proteins. These may be the (1*3) -beta-D-glucan binding domain (s) of factor G.Thus, factor G,the primary initiator of the (1*3) -beta-D-glucan-sensitive coagulation pathway in the horseshoe crab, is a structurally unique heterodimeric serine protease zymogen and as such represents a new class of active defense proteins. Less