Purification, Characterization, and cDNA Cloning of a 27-kDa Lectin (L10) from Horseshoe Crab Hemocytes

Okino, Nozomu; Kawabata, Shun Ichiro; Saito, Tetsu; Hirata, Michimasa; Takagi, Toshio; Iwanaga, Sadaaki

doi:10.1074/jbc.270.52.31008

Cited by 69 publications

(50 citation statements)

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“…Tachylectin-2, one of the hemocyte-derived lectins (14), exhibits specificity against blood group A antigen, and it has high affinity for monosaccharides, such as GlcNAc and GalNAc (14). Such being the case, the inhibitory effects of LPSs and LTAs on the hemagglutinating activity of tachylectin-2 were also examined and compared with those of tachylectin-3 (Table II).…”

Section: Resultsmentioning

confidence: 99%

“…Biotinylation of Polyclonal Antibodies against Tachylectins-An antiserum against tachylectin-3 was raised in rabbits, as described (13,14). Polyclonal antibodies against tachylectin-1 (13), tachylectin-2 (14) and tachylectin-3 were purified from rabbit antisera with the Ampure PA kit (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

“…Hemagglutinating Activity and Inhibition Assay-Hemagglutinating activity and the inhibition assay were done as described (14,17).…”

Section: Methodsmentioning

confidence: 99%

“…Polyclonal antibodies against tachylectin-1 (13), tachylectin-2 (14) and tachylectin-3 were purified from rabbit antisera with the Ampure PA kit (Amersham Pharmacia Biotech). The IgG fractions obtained were dialyzed against 0.1 M NaHCO 3 and biotinylated with EZ-Link™ NHS-LC-Biotin, according to a protocol provided by the manufacturer (Pierce).…”

Section: Methodsmentioning

confidence: 99%

“…Tachylectin-1, identical to an L-granule-derived protein L6 (12), binds to both S-and R-types of LPSs and inhibits the growth of Gram-negative bacteria (13). Tachylectin-2, identical to another L-granulederived protein, L10 (12), exhibits binding specificity to DGlcNAc or D-GalNAc and agglutinates a certain strain of Staphylococcus (14). Tachylectins-4 has affinity for L-fucose and binds to S-type LPSs from several Gram-negative bacteria * This work was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Science, Sports and Culture of Japan (to S.-i.…”

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confidence: 99%

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A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

Inamori

Saito

Iwaki

et al. 1999

Journal of Biological Chemistry

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A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B-and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gramnegative bacteria but not by R-type LPSs lacking Oantigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.Immunity to infectious agents is mediated by two general systems, innate and acquired. Acquired immunity, found only in vertebrates, is one function of B and T lymphocytes, which produce an infinite multitude of specific antigen receptors and antibodies through somatic gene rearrangement. On the other hand, innate immunity is phylogenetically older than acquired immunity, and a certain form of innate immunity is present in all multicellular organisms. Non-self-recognizing proteins involved in innate immunity seem to recognize mainly carbohydrate moieties on pathogens; for instance, a receptor CD14 (1) on macrophages for bacterial lipopolysaccharides (LPSs), 1 Creactive protein (2), and the mannose-binding lectin of the collectin family in mammalian plasma (3). Furthermore, the innate immunity in invertebrates is also triggered by polysaccharides, such as LPSs and ␤-1,3-glucans, as seen in the hemolymph coagulation system in horseshoe crab (4 -9) and the phenoloxidase-mediated melanization system in crustaceans and insects (10).In the Japanese horseshoe crab, Tachypleus tridentatus, one of the major defense systems is carried by hemolymph that contains granular hemocytes comprising 99% of the total hemocytes (11). These granular hemocytes are fil...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Hemagglutinating Activity and Inhibition Assay-Hemagglutinating activity and the inhibition assay were done as described (14,17).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

Inamori

Saito

Iwaki

et al. 1999

Journal of Biological Chemistry

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Two novel ficolin‐like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus

Söderhäll

2011

Proteomics

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To isolate pathogen-associated molecular patterns (PAMPs)-binding molecules, the bacterium, Staphylococcus aureus was used as an affinity matrix to find bacteria-binding proteins in the plasma of the freshwater crayfish, Pacifastacus leniusculus. Two new bacteria-binding ficolin-like proteins (FLPs) were identified by 2-DE and MS analysis. The FLPs have a fibrinogen-related domain (FReD) in their C-terminal and a repeat region in their N-terminal regions with putative structural similarities to the collagen-like domain of vertebrate ficolins and mannose binding lectins (MBLs). Phylogenetic analysis shows that the newly isolated crayfish FLP1 and FLP2 cluster separately from other FReD-containing proteins. A tissue distribution study showed that the mRNA expression of FLP occurred mainly in the hematopoietic tissue (Hpt) and in the hepatopancreas. Recombinant FLPs exhibited agglutination activity of Gram-negative bacteria Escherichia coli and Aeromonas hydrophila in the presence of Ca(2+) . The FLPs could bind to A. hydrophila, E. coli as well as S. aureus as judged by bacteria adsorption. Moreover, the FLPs may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria which had been injected into the hemolymph. When Gram-negative bacteria coated with FLPs were incubated with Hpt cells, a lower death rate of the cells was found compared with control treatment. Our results suggest that FLPs function as pattern recognition receptors in the immune response of crayfish.

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Purification and Assays of Tachylectin-2

Kawabata

Shibata

2020

Methods in Molecular Biology

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Purification, Characterization, and cDNA Cloning of a 27-kDa Lectin (L10) from Horseshoe Crab Hemocytes

Abstract: We separated granular components of horseshoe crab hemocytes by a combination of centrifugation on sucrose density gradient and high performance liquid chromatography, and a 27-kDa protein termed L10 was found to be a major component in the large granules (Shigenaga, T., Takayenoki

Cited by 69 publications

References 35 publications

A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

Two novel ficolin‐like proteins act as pattern recognition receptors for invading pathogens in the freshwater crayfish Pacifastacus leniusculus

Purification and Assays of Tachylectin-2

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