Purified Horseshoe Crab Factor G

Abstract

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.

Innate immune response in insects: recognition of bacterial peptidoglycan and amplification of its recognition signal

“…These observations imply that excess linearized Lystype PG acts as a competitive inhibitor by sequestering Tm-PGRP-SA molecules, thereby impairing the initial activating complex composed of clustered PGRP-SA molecules bound to one linearized PG molecule. Similar observations were reported in the recognition of β-1,3-glucan by Factor G in horseshoe crab, and in the recognition of LPS by the proPO system of crayfish (45,46).…”

Characterization and Properties of a 1,3-β-d-Glucan Pattern Recognition Protein of Tenebrio molitor Larvae That Is Specifically Degraded by Serine Protease during Prophenoloxidase Activation

“…Interestingly, glucan binding proteins from silkworm, tobacco hornworm, fall webworm, fly, and mealworm are all replaced with other residues, suggesting that these GRPs do not have glucanase enzyme activity. However, other 1,3-␤-Dglucan recognition or binding proteins, such as factor-G of horseshoe crab (33), coelomic cytolytic factor 1 of earthworm (39), LGBP of crayfish (10), and GNBPs of mosquito (38) and silkworm (36), have all conserved Glu residues in the active site as the bacterial glucanase (Fig. 5).…”

“…These proteins contain a similar domain with those of ␤-1,3-and ␤-1,4-bacterial glucanase (48) and ␤-1,3-glucanase of sea urchin Strongylocentrotus purpuratus (49). It was reported that this region also exists in the GNBPs of B. mori (Bm-GNBP) (36), Drosophila melanogaster (Dm-GNBP) (35), and Hyphantria cunea (Hc-GNBP) (37); putative GNBP of Anopheles gambiae (Ag-GNBP) (38); coagulation factor G of the horseshoe crab Tachypleus tridentatus (Factor-G) (33), LGBP of the crayfish Pacifastacud leniuschlus (10), and Coelomic cytolytic factor 1 of an earthworm (CCF-1) (39). But, as shown in Fig.…”

“…Although several pattern recognition proteins, such as PGN recognition proteins (PGRPs) (23-31), 1,3-␤-D-glucan recognition protein (GRP) (11,12,(32)(33)(34), Gram-negative bacteriabinding proteins (GNBPs) (35)(36)(37)(38), and LPS and 1,3-␤-D-glucan-binding proteins (LGBPs) (10,39), have been identified in invertebrates, the molecular mechanism and structural basis of microbial recognition are poorly defined. We were able to reconstitute in vitro the downstream components of the proPO cascade using biochemically purified compounds from the large insect, Holotrichia diomphalia larvae (18).…”

A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides

“…This response is important for the host defense's ability to engulf and kill invading microbes. The granular components include two PAMP-sensitive zymogens, factor C (20) and factor G (21,22). These serine protease zymogens are autocatalytically activated by LPS and ␤-1,3-glucans, respectively.…”