Biochemical principle of Limulus test for detecting bacterial endotoxins

Abstract

A hemocyte lysate from horseshoe crab (Limulus) produced a gel, when exposed to Gram-negative bacterial endotoxins, lipopolysaccharides (LPS). This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins. Recent biochemical studies on the principle of Limulus test indicate that the hemocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel. Up to now, six protein components, designated coagulogen, proclotting enzyme, factor B, factor C, and factor G, all of which are closely associated with the endotoxin-mediated coagulation pathway, have been purified and biochemically characterized. The molecular structures of these proteins have also been elucidated. Moreover, the reconstitution experiments using the isolated clotting factors, factor C, factor B, proclotting enzyme and coagulogen in the presence of endotoxin, leads to the formation of coagulin gel. Here, I will focus on the biochemical principle of Limulus test for detecting bacterial endotoxins, and its activation and regulation mechanism on the LPS-mediated coagulation cascade.

Keywords: Limulus test; bacterial endotoxins; clotting factors; horseshoe crab (Tachypleus tridentatus); innate immunity; lipopolysacchalides (LPS).

Fig. 1

A light (A and B) and electron (C) micrographs of horseshoe crab (T. tridentatus) hemocytes/amebocytes, and major defense molecules (C) that have been identified in large and small cell granules.

Fig. 2

LPS- and (1→3)-β-D-glucan mediated clotting cascades found in horseshoe crab (T. tridentatus) hemocytes. Recently, Nagai and Kawabata found that the clotting cascade is linked to prophenoloxidase activation with the hemocyanin functioning as a prophenoloxidase substitude.^, LICI, limulus intracellular coagulation inhibitor. This figure depicts the biochemical principle of the so called limulus test, which is used for detecting bacterial endotoxins. The limulus test is dependent on the protease cascade reaction, and is being used extensively in combination with new technology. Three kinds of LICI shown in the figure inhibit or regulate, respectively, the activation reactions of clotting factors (see text).

Fig. 3

Domain structures of limulus clotting factors. The arrowheads indicate cleavage sites for zymogen activation. The potential oligosaccharide attachment sites are indicated by closed diamonds. Cys-rich: a domain containing a number of cysteine residues, EGF: epidermal growth factor domain, Sushi: this domain corresponds to short consensus repeat (SCR) and also to complement control protein (CCP) often found in mammalian complements, Lectin: structural domain which recognize carbohydrate moiety. Pro-rich: a domain containing many proline residues, Clip: a secondary structure of this domain is similar to feature of clip used as stationary in business work. Recently, a number of the clip domains have been identified in serine-protease originated from invertebrate animals.^,

Fig. 4

Stereo view of a coagulogen monomer showing the A chain, peptide C and B chain, and the secondary structure. The structure is dominated by the β-strands (blue, labeled sequentially B1 to B6), and multiple coils and turns (green) of the B chain. The main α-helical peptide C (red), which is released upon cleavage, covers a reasonable part of the surface at the top. The NH₂-terminal A chain is connected to the B chain by two disulfide bridges (yellow). The whole cysteine-rich structure possesses eight disulfide bridges.

Fig. 5

Hypothetical mechanism of coagulogen gel formation. Upon gelation of coagulogen (pink) by a horseshoe crab clotting enzyme, peptide C (green) is released from the inner portion of the parent molecules. The resulting coagulin (yellow) monomer may selfassemble to form the dimer, trimer, and multimers. The background of this figure shows a fiber-like coagulin gel.