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Shun-ichiro Kawabata - One of the best experts on this subject based on the ideXlab platform.
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Factor C Acts as a Lipopolysaccharide-Responsive C3 Convertase in Horseshoe Crab Complement Activation
Journal of Immunology, 2008Co-Authors: Shigeru Ariki, Teizo Fujita, Shusaku Takahara, Toshio Shibata, Takaaki Fukuoka, Aya Ozaki, Yuichi Endo, Takumi Koshiba, Shun-ichiro KawabataAbstract:The complement system in vertebrates plays an important role in host defense against and clearance of invading microbes, in which complement component C3 plays an essential role in the opsonization of pathogens, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. In an effort to understand the molecular activation mechanism of invertebrate C3, we isolated and characterized an ortholog of C3 (designated TtC3) from the Horseshoe Crab Tachypleus tridentatus. Flow cytometric analysis using an Ab against TtC3 revealed that the Horseshoe Crab complement system opsonizes both Gram-negative and Gram-positive bacteria. Evaluation of the ability of various pathogen-associated molecular patterns to promote the proteolytic conversion of TtC3 to TtC3b in hemocyanin-depleted plasma indicated that LPS, but not zymosan, peptidoglycan, or laminarin, strongly induces this conversion, highlighting the selective response of the complement system to LPS stimulation. Although originally characterized as an LPS-sensitive initiator of hemolymph coagulation stored within hemocytes, we identified factor C in hemolymph plasma. An anti-factor C Ab inhibited various LPS-induced phenomena, including plasma amidase activity, the proteolytic activation of TtC3, and the deposition of TtC3b on the surface of Gram-negative bacteria. Moreover, activated factor C present on the surface of Gram-negative bacteria directly catalyzed the proteolytic conversion of the purified TtC3, thereby promoting TtC3b deposition. We conclude that factor C acts as an LPS-responsive C3 convertase on the surface of invading Gram-negative bacteria in the initial phase of Horseshoe Crab complement activation.
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recognition of pathogens and activation of immune responses in drosophila and Horseshoe Crab innate immunity
Immunobiology, 2006Co-Authors: Shoichiro Kurata, Shigeru Ariki, Shun-ichiro KawabataAbstract:In innate immunity, pattern recognition receptors discriminate between self- and infectious non-self-matter. Mammalian homologs of the Drosophila Toll protein, which are collectively referred to as Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS) and lipoproteins, whereas the Drosophila Toll protein does not act as a PAMP receptor, but rather binds to Spatzle, an endogenous peptide. In Drosophila, innate immune surveillance is mediated by members of the peptidoglycan recognition protein (PGRP) family, which recognize diverse bacteria-derived peptidoglycans and initiate appropriate immune reactions including the release of antimicrobial peptides and the activation of the prophenoloxidase cascade, the latter effecting localized wound healing, melanization, and microbial phagocytosis. In the Horseshoe Crab, LPS induces hemocyte exocytotic degranulation, resulting in the secretion of various defense molecules, such as coagulation factors, antimicrobial peptides, and lectins. Recent studies have demonstrated that the zymogen form of the serine protease factor C, a major granular component of hemocyte, also exists on the hemocyte surface and functions as a biosensor for LPS. The proteolytic activity of activated factor C initiates hemocyte exocytosis via a G protein mediated signal transduction pathway. Furthermore, it has become clear that an endogenous mechanism for the feedback amplification of the innate immune response exists and is dependent upon a granular component of the Horseshoe Crab hemocyte.
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proline rich cell surface antigens of Horseshoe Crab hemocytes are substrates for protein cross linking with a clotting protein coagulin
Journal of Biological Chemistry, 2002Co-Authors: Tsukasa Osaki, Fuminori Tokunaga, Sadaaki Iwanaga, Nozomu Okino, Shun-ichiro KawabataAbstract:Monoclonal antibodies were raised against hemocytes of the Horseshoe Crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the Horseshoe Crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.
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Functional Conversion of Hemocyanin to Phenoloxidase by Horseshoe Crab Antimicrobial Peptides
Journal of Biological Chemistry, 2001Co-Authors: Taku Nagai, Tsukasa Osaki, Shun-ichiro KawabataAbstract:Abstract Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the Horseshoe Crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297–35301). Here we show that chitin-binding antimicrobial peptides of the Horseshoe Crab induce the intrinsic phenoloxidase activity of hemocyanin. Tachyplesin, a major Tachypleus antimicrobial peptide with an amphiphilic structure, converted the hemocyanin to phenoloxidase. Surface plasmon resonance analysis revealed the specific interaction of tachyplesin with hemocyanin at K d = 3.4 × 10− 6 m. The chemical modification of Trp or Tyr in tachyplesin, but not Lys or Arg, dramatically reduced the affinity to hemocyanin, suggesting that the binding site is located in the hydrophobic face of tachyplesin. Hemocyanin has no affinity with chitin, but it significantly binds to tachyplesin-coated chitin, leading to the expression of phenoloxidase activity. The chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing.
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Horseshoe Crab acetyl group recognizing lectins involved in innate immunity are structurally related to fibrinogen
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Soutaro Gokudan, Sadaaki Iwanaga, Tatsushi Muta, Ryoko Tsuda, Kumiko Koori, Takeshi Kawahara, Noriaki Seki, Yoshimitsu Mizunoe, Sun Nyunt Wai, Shun-ichiro KawabataAbstract:We have characterized and cloned newly isolated lectins from hemolymph plasma of the Horseshoe Crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a Horseshoe Crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of “bouquet arrangement” of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The Horseshoe Crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the Horseshoe Crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.
Mark L Botton - One of the best experts on this subject based on the ideXlab platform.
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Effects of a Beach Nourishment Project in Jamaica Bay, New York, on Horseshoe Crab (Limulus polyphemus) Spawning Activity and Egg Deposition
Estuaries and Coasts, 2018Co-Authors: Mark L Botton, Matthew Sclafani, Christina P. Colón, John Rowden, Susan Elbin, Debra Kriensky, Kim Mckown, Robert MaddenAbstract:Restoration of Horseshoe Crab spawning habitats through beach nourishment may be considered as a potential strategy to enhance reproductive success in areas where estuarine beaches have been lost to coastal erosion and development. The US Army Corps of Engineers performed a beach nourishment project at Plumb Beach (Jamaica Bay, Brooklyn, NY) in 2012 to stabilize the shoreline. While the addition of sand was done to protect infrastructure, it created an opportunity to examine the responses of American Horseshoe Crabs ( Limulus polyphemus ) to beach nourishment using a BACI (before-after control impact) design. During Spring 2012, before beach nourishment, Horseshoe Crabs made minimal use of the highly degraded western section of Plumb Beach in comparison to a nearby reference site, as quantified by numbers of spawning adults at high tide and densities of Horseshoe Crab eggs in core samples. In the first post-nourishment field season (Spring 2013), there was no detectable increase in Horseshoe Crab spawning activity on the newly restored beach. In 2014 and 2015, the density of spawning females began to increase at the nourished beach, although their numbers and especially the density of Horseshoe Crab eggs remained much lower than at the reference site. Three years after beach nourishment, differences in sediments texture (mean grain diameter, percent gravel, sorting, skewness, and hardness) were still evident between the nourishment and reference sites. Our results suggest that (1) at this site, beach nourishment appeared to bring about only slow increases in Horseshoe Crab spawning density after several seasons and (2) subtle differences in beach geomorphology over relatively short distances can be detected by Horseshoe Crabs and may underlie their selection of specific nesting sites.
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Effects of copper and cadmium on development and superoxide dismutase levels in Horseshoe Crab (Limulus polyphemus) embryos
SpringerPlus, 2015Co-Authors: Mary G. Hamilton, Christopher Esposito, Mia Malin, Lucas Cusumano, Mark L BottonAbstract:Pollution by metals may adversely affect organisms through the generation of reactive oxygen species (ROS). In this study, we examined the sublethal effects of two metals, copper and cadmium, on Horseshoe Crab ( Limulus polyphemus ) embryos. Exposure to copper or cadmium at concentrations of 0.01–10 mg/L for periods of 4, 8, 16 and 24 h had minimal effect on embryo survival except at 100 mg/L Cu. However, metal-exposed embryos took significantly longer to hatch into first instar (“trilobite”) larvae than seawater controls. Levels of superoxide dismutase (SOD), believed to be important in the response to oxidative stress, were determined by Western blotting. Both the Cu/Zn and Mn cofactor forms of SOD tended to be somewhat elevated in metal-exposed embryos, but the increases were neither dose nor time-dependent. Likewise, SOD enzymatic activity showed no significant differences comparing embryos exposed to metals with seawater controls. We conclude that the protective role of SOD’s against ROS produced in response to metal exposure appears to be limited in Horseshoe Crab embryos, at least under our experimental conditions.
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Physiological responses of Horseshoe Crab (Limulus polyphemus) embryos to osmotic stress and a possible role for stress proteins (HSPs)
Marine Biology, 2011Co-Authors: Morgan P. Greene, Mary G. Hamilton, Mark L BottonAbstract:We tested the effects of osmotic stress on survival, developmental rate, and level of HSPs on American Horseshoe Crab ( Limulus polyphemus ) embryos. Animals were maintained in the laboratory at an ambient salinity of 20 ppt and then exposed to 4-h osmotic shocks at salinities of 10, 30, 40, 50, and 60 ppt, with a control group at 20 ppt. Horseshoe Crab embryos had 100% developmental success (defined as individuals reaching the first instar or trilobite larval stage) at all salinities. However, osmotic stresses, especially hyperosmotic conditions, slowed the rate of development. Embryos subjected to osmotic stress showed higher levels of HSP70 and HSP90 than control animals kept at a salinity of 20 ppt. HSPs are of value to Horseshoe Crab embryos in surviving the fluctuating salinities that are typical of estuarine beach habitats.
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developmental ecology of the american Horseshoe Crab limulus polyphemus
Current Zoology, 2010Co-Authors: Mark L Botton, Richard A Tankersley, Robert E LovelandAbstract:During spawning events, Horseshoe Crab eggs are released from the female's oviducts, and fertilized by one or more males. Eggs are shaped by the female into discrete clutches deposited in nests at depths of 10−20 cm on intertidal estuarine beaches. Distinguishing between fresh eggs and the early developmental stages is obfuscated by the large amount of dense, opaque yolk. The first unambiguous confirmation of development is the formation of the rudimentary prosomatic appendages at the "limb bud" stage. Several days thereafter, the outer chorion is shed and the developing embryo expands and undergoes a series of molts within the clear inner egg membrane. The trilobite (first instar) stage thus attained may remain within the beach sedi- ments for several more weeks, until hatching is facilitated by environmental factors such as hydration, agitation, and osmotic shock that accompany the infiltration of seawater into the nests. Trilobites exhibit endogenous circatidal swimming rhythms that are entrained by mechanical agitation, suggesting that peaks in larval swimming are timed to coincide with periods of high water and the inundation of the nests. Larval swimming is limited and does not appear to result in long-distance dispersal. The limited dispersal of the larvae has important implications for the population dynamics of relatively isolated populations. The rate of larval development is highly plastic and is influenced by temperature, salinity, dissolved oxygen, and the presence of pollutants. The broad environmental tolerances of Horseshoe Crab embryos and larvae are important in understanding their current geographic distribution and their evolutionary persistence (Current Zoology 56 (5): 550-562, 2010).
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abundance and dispersal potential of Horseshoe Crab limulus polyphemus larvae in the delaware estuary
Estuaries, 2003Co-Authors: Mark L Botton, Robert E LovelandAbstract:The distribution, abundance, and dispersal patterns of Horseshoe Crab (Limulus polyphemus) trilobite larvae were determined from 671 plankton tows taken near a spawning beach in lower Delaware Bay, New Jersey, in 1998 and 1999. In both years, peaks in larval abundance occurred during periods of rough surf (>30 cm wave heights). Planktonic larvae were significantly more abundant nocturnally than during the day, but there was no evidence of a lunar component to larval abundance. Larvae were strongly concentrated inshore; trilobites were 10–100 times more abundant in the immediate vicinity of the shoreline than they were 100–200 m offshore. The strong tendency ofLimulus larvae to remain close to the beach suggests that their capability for long-range dispersal between estuaries is extremely limited. We suggest that limited larval dispersal potential may help explain previously observed patterns of genetic variation among the Mid-Atlantic Horseshoe Crab populations.
Margaret Dah-tsyr Chang - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen- Associated Molecular Patterns of Bacteria
2016Co-Authors: Through Rhamnose, Yu-tsyr Huang, Yuan-chuan Lee, Ee-ling Low, Cheng-hsun Chiu, Shiu-ling Chen, Liang-chi Mao, Margaret Dah-tsyr ChangAbstract:Horseshoe Crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of Horseshoe Crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant Horseshoe Crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associate
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a recombinant Horseshoe Crab plasma lectin recognizes specific pathogen associated molecular patterns of bacteria through rhamnose
PLOS ONE, 2014Co-Authors: Yu-tsyr Huang, Yuan-chuan Lee, Ee-ling Low, Cheng-hsun Chiu, Shiu-ling Chen, Liang-chi Mao, Margaret Dah-tsyr ChangAbstract:Horseshoe Crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of Horseshoe Crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant Horseshoe Crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.
Sadaaki Iwanaga - One of the best experts on this subject based on the ideXlab platform.
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proline rich cell surface antigens of Horseshoe Crab hemocytes are substrates for protein cross linking with a clotting protein coagulin
Journal of Biological Chemistry, 2002Co-Authors: Tsukasa Osaki, Fuminori Tokunaga, Sadaaki Iwanaga, Nozomu Okino, Shun-ichiro KawabataAbstract:Monoclonal antibodies were raised against hemocytes of the Horseshoe Crab Tachypleus tridentatus. All of the antibodies obtained reacted with the same protein bands on SDS-PAGE of hemocyte lysate. Flow cytometry and biotinylation of surface substances on the hemocytes indicated that the antigens are major peripheral proteins of hemocytes. The antigens were purified from hemocyte lysate and were good substrates for the Horseshoe Crab hemocyte transglutaminase (HcTGase). Transglutaminases play an important role during the final stage of blood coagulation in mammals and crustaceans. Although HcTGase did not intermolecularly cross-link a clottable protein coagulogen or its proteolytic product coagulin, HcTGase promoted the cross-linking of coagulin with the surface antigens, resulting in the formation of a stable polymer. We determined the nucleotide sequences for two isoproteins of the antigens. The two proteins containing 271 and 284 residues (66% identity) were composed of tandem repeats of proline-rich segments. We named them proxins-1 and -2 after proline-rich proteins for protein cross-linking. Proxins may form a stable physical barrier against invading pathogens in cooperation with hemolymph coagulation at injured sites.
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the molecular basis of innate immunity in the Horseshoe Crab
Current Opinion in Immunology, 2002Co-Authors: Sadaaki IwanagaAbstract:During the past two decades, the molecular structures and functions have been established for various defense molecules, using Horseshoe Crab (limulus) as a model animal. These defense molecules include clotting factors, proteinase inhibitors, lectins, antimicrobial peptides and other humoral factors found mainly in the hemolymph. These components of the cellular and humoral systems, which together comprise innate immunity, defend Horseshoe Crab effectively from invading microbes.
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Horseshoe Crab acetyl group recognizing lectins involved in innate immunity are structurally related to fibrinogen
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Soutaro Gokudan, Sadaaki Iwanaga, Tatsushi Muta, Ryoko Tsuda, Kumiko Koori, Takeshi Kawahara, Noriaki Seki, Yoshimitsu Mizunoe, Sun Nyunt Wai, Shun-ichiro KawabataAbstract:We have characterized and cloned newly isolated lectins from hemolymph plasma of the Horseshoe Crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a Horseshoe Crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of “bouquet arrangement” of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The Horseshoe Crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the Horseshoe Crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.
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role of lectins in the innate immunity of Horseshoe Crab
Developmental and Comparative Immunology, 1999Co-Authors: Shun-ichiro Kawabata, Sadaaki IwanagaAbstract:We have purified five types of lectins, named tachylectins, from circulating hemocytes and hemolymph plasma of the Japanese Horseshoe Crab, Tachypleus tridentatus. Tachylectin-1 interacts with Gram-negative bacteria probably through 2-keto-3-deoxyoctonate, one of the constituents of lipopolysaccharides (LPS). Tachylectin-1 also binds to polysaccharides such as agarose and dextran with broad specificity. Tachylectin-2 binds to d-GlcNAc or d-GalNAc and recognizes staphylococcal lipoteichoic acids and LPS from several Gram-negative bacteria. In contrast, tachylectins-3 and -4 specifically bind to S-type LPS from several Gram-negative bacteria through a certain sugar moiety on the O-specific polysaccharides (O-antigens). Tachylectin-5 identified in hemolymph plasma has the strongest bacterial agglutinating activity in the five types of tachylectins, and exhibits broad specificity against acetyl group-containing substances. Thus, the innate immune system of Horseshoe Crab may recognize invading pathogens through a combinatorial method using lectins with different specificities against carbohydrates exposed on pathogens. An encounter of these lectins derived from hemocytes and hemolymph plasma at injured sites, in response to the stimulation of LPS, suggests that they serve synergistically to accomplish an effective host defense against invading microbes and foreign substances.
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evolution and phylogeny of defense molecules associated with innate immunity in Horseshoe Crab
Frontiers in Bioscience, 1998Co-Authors: Sadaaki Iwanaga, Shun-ichiro KawabataAbstract:This short review describes the molecular evolution and phylogeny of various defense molecules participating in the host defense of Horseshoe Crab. It is well known that invertebrate animals, which lack adaptive immune systems, have developed various defense systems, so called innate immunity, that respond to common antigens on the surface of potential pathogens. The systems include hemolymph coagulation, melanization, cell agglutination, antimicrobial action, active oxygen formation, and phagocytic action. Among them, hemolymph coagulation and phenoloxidase-mediated melanization, in addition to cell agglutination, are directly induced by foreign substances, that result in the engulfment of invading microbes. The immobilized invaders are finally killed by antimicrobial substances released mainly from many kinds of hemocytes. In the past two decades, we have investigated biochemically various defense molecules, using Horseshoe Crab as a model animal, and established extensively their molecular structures. These results now make it possible to discuss evolution and phylogeny of the defense molecules at a molecular level, in comparison with those derived from vertebrate animals. Here, the authors will describe the present state of our knowledge concerning molecules mainly associated with innate immunity.
Yuan-chuan Lee - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE A Recombinant Horseshoe Crab Plasma Lectin Recognizes Specific Pathogen- Associated Molecular Patterns of Bacteria
2016Co-Authors: Through Rhamnose, Yu-tsyr Huang, Yuan-chuan Lee, Ee-ling Low, Cheng-hsun Chiu, Shiu-ling Chen, Liang-chi Mao, Margaret Dah-tsyr ChangAbstract:Horseshoe Crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of Horseshoe Crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant Horseshoe Crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associate
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a recombinant Horseshoe Crab plasma lectin recognizes specific pathogen associated molecular patterns of bacteria through rhamnose
PLOS ONE, 2014Co-Authors: Yu-tsyr Huang, Yuan-chuan Lee, Ee-ling Low, Cheng-hsun Chiu, Shiu-ling Chen, Liang-chi Mao, Margaret Dah-tsyr ChangAbstract:Horseshoe Crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of Horseshoe Crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant Horseshoe Crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.