A Structural Perspective on the Interaction between Lipopolysaccharide and Factor C, a Receptor Involved in Recognition of Gram-negative Bacteria

Koshiba, Takumi; Hashii, Tomoyuki; Kawabata, Shun Ichiro

doi:10.1074/jbc.m609198200

Cited by 57 publications

(40 citation statements)

References 23 publications

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“…The LAL test depends on the activation of the initiating protease of the blood clotting system by LPS 14 , with a read-out of clot formation or of a colorimetric assay for protease activity. The two elements of immunity that have received the greatest attention have been the proteins of the plasma and the several proteins and peptides of the secretory granules of the blood cells.…”

Section: Discussionmentioning

confidence: 99%

Blood collection from the American Horseshoe Crab, Limulus polyphemus

Armstrong

Conrad

2008

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The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and α2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 -400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 -5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.

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Section: Discussionmentioning

confidence: 99%

Blood collection from the American Horseshoe Crab, Limulus polyphemus

Armstrong

Conrad

2008

JoVE

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“…The granular hemocyte is filled with two distinct types of granules, each of which selectively stores different types of defense molecules, including coagulation factors, protease inhibitors, lectins, and antimicrobial peptides (7,8). The hemocyte is highly sensitive to LPS, and stimulation of the hemocyte by LPS leads to the immediate release of defense molecules by degranulation (9,10). The coagulation cascade of horseshoe crabs is composed of a clottable protein coagulogen and four serine protease zymogens, including factor C, factor G, factor B, and the proclotting enzyme (7).…”

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confidence: 99%

Factor G Utilizes a Carbohydrate-Binding Cleft That Is Conserved between Horseshoe Crab and Bacteria for the Recognition of β-1,3-d-Glucans

Ueda

Ohwada

Abe³

et al. 2009

The Journal of Immunology

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In the horseshoe crab, the recognition of β-1,3-d-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes β-1,3-d-glucans. To gain an insight into the recognition of β-1,3-d-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than ∼30 and 3 μM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble β-1,3-d-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble β-1,3-d-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a β-sheet in a predicted β-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for β-1,3-d-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

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“…In response to the stimulation from bacterial lipopolysaccharides (LPS), the defense molecules stored in granules are secreted by hemocyte exocytosis (8,10,11). The coagulation cascade of these zymogens is triggered by LPS or ␤-1,3-glucans, which finally leads to the conversion of coagulogen into coagulin, resulting in noncovalent coagulin homopolymers through head-to-tail interaction (12,13).…”

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A Cysteine-rich Protein from an Arthropod Stabilizes Clotting Mesh and Immobilizes Bacteria at Injury Sites

Matsuda¹,

Osaki²,

Hashii³

et al. 2007

Journal of Biological Chemistry

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Hemolymph coagulation in arthropods plays key roles in host defense, including sealing wounds to staunch bleeding and immobilizing invading microorganisms. We have previously reported that horseshoe crab transglutaminase (TGase) promotes cross-linking of a clotting protein (coagulin) with hemocyte-derived proteins (proxins), resulting in the formation of stable coagulin fibrils. Here we show that TGase also cross-links proxins to another hemocyte-derived protein named stablin. Stablin is a cysteine-rich protein of 131 residues. Surface plasmon resonance analysis revealed the specific interaction of stablin with proxin-1 at K d ‫؍‬ 4.0 ؋ 10 ؊9 M. Stablin was predominantly localized in the large granules of hemocytes and secreted by lipopolysaccharide-induced exocytosis. Interestingly, stablin bound to chitin at K d ‫؍‬ 1.5 ؋ 10 ؊8 M, as determined by using a quartz-crystal microbalance. Stablin also interacted with lipopolysaccharides and lipoteichoic acids and exhibited bacterial agglutinating activity against Gram-positive and -negative bacteria. Immunostaining showed that stablin is co-localized with coagulin in the clotting fibrils that effectively trap bacteria. Moreover, an anti-stablin antibody strongly inhibited the proper formation of the clotting fibrils. These data suggest that stablin promotes the formation of the clotting mesh and the immobilization of invading microbes at injury sites. In arthropods, the TGase-mediated cross-linking may play an important role in the initial stage of host defense, wound closure, and healing, as in the case of mammals.

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A Structural Perspective on the Interaction between Lipopolysaccharide and Factor C, a Receptor Involved in Recognition of Gram-negative Bacteria

Cited by 57 publications

References 23 publications

Blood collection from the American Horseshoe Crab, Limulus polyphemus

Blood collection from the American Horseshoe Crab, Limulus polyphemus

Factor G Utilizes a Carbohydrate-Binding Cleft That Is Conserved between Horseshoe Crab and Bacteria for the Recognition of β-1,3-d-Glucans

A Cysteine-rich Protein from an Arthropod Stabilizes Clotting Mesh and Immobilizes Bacteria at Injury Sites

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