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Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.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.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

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.

No MeSH data available.


Related in: MedlinePlus

Domain structures of limulus clotting factors. The arrowheads indicate cleavage sites for zymogen activation.10) 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.42), 43)
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f3-83_110: Domain structures of limulus clotting factors. The arrowheads indicate cleavage sites for zymogen activation.10) 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.42), 43)

Mentions: Fig. 3 summarizes the gross structures of these new clotting factors. All the factors except for coagulogen are typical glycoproteins and differ from each other in molecular mass. The initiation factor, factor C sensitive to LPS, comprises one EGF-like domain, five short consensus repeats (SCR, also called CCP or the sushi domain) found mainly in mammalian complements, one C-type lectin domain, and a serine protease domain, the latter of which is located in the COOH-terminal portion.13) The finding of SCR in factor C makes it the first protein in invertebrates that has been discovered to contain this type of domain.b) The fact that this initiator of the clotting cascade contains SCR led us to speculate that coagulation and complement systems may have evolved from a common origin.8), 13) Recently, Koshiba et al.48) reported that the LPS-binding site is present in the NH2-terminal cysteine-rich region of the zymogen factor C molecule (Fig. 3), and that it contains a tripeptide sequence, such as RWR and KYK, that is conserved in the hemocyte-drived anti-LPS factor13) and other mammarian LPS-recognizing proteins.


Biochemical principle of Limulus test for detecting bacterial endotoxins.

Iwanaga S - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2007)

Domain structures of limulus clotting factors. The arrowheads indicate cleavage sites for zymogen activation.10) 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.42), 43)
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756735&req=5

f3-83_110: Domain structures of limulus clotting factors. The arrowheads indicate cleavage sites for zymogen activation.10) 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.42), 43)
Mentions: Fig. 3 summarizes the gross structures of these new clotting factors. All the factors except for coagulogen are typical glycoproteins and differ from each other in molecular mass. The initiation factor, factor C sensitive to LPS, comprises one EGF-like domain, five short consensus repeats (SCR, also called CCP or the sushi domain) found mainly in mammalian complements, one C-type lectin domain, and a serine protease domain, the latter of which is located in the COOH-terminal portion.13) The finding of SCR in factor C makes it the first protein in invertebrates that has been discovered to contain this type of domain.b) The fact that this initiator of the clotting cascade contains SCR led us to speculate that coagulation and complement systems may have evolved from a common origin.8), 13) Recently, Koshiba et al.48) reported that the LPS-binding site is present in the NH2-terminal cysteine-rich region of the zymogen factor C molecule (Fig. 3), and that it contains a tripeptide sequence, such as RWR and KYK, that is conserved in the hemocyte-drived anti-LPS factor13) and other mammarian LPS-recognizing proteins.

Bottom Line: This gelation reaction of the lysate, so-called Limulus test, has been widely employed as a simple and very sensitive assay method for endotoxins.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.

View Article: PubMed Central - PubMed

Affiliation: The Chemo-Sero-Therapeutic Research Institute, 1-6-1 Okubo, Kumamoto 812-8581, Japan . ; Research adviser.

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.

No MeSH data available.


Related in: MedlinePlus