Shun Ichiro Kawabata

Tachylectin-2: crystal structure of a specific GlcNAc/GalNAc-binding lectin involved in the innate immunity host defense of the Japanese horseshoe crab Tachypleus tridentatus.

Tachylectin‐2, isolated from large granules of the hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), is a 236 amino acid protein belonging to the lectins. It binds specifically to N‐acetylglucosamine and N‐acetylgalactosamine and is a part of the innate immunity host defense system of the horseshoe crab. The X‐ray structure of tachylectin‐2 was solved at 2.0 Å resolution by the multiple isomorphous replacement method and this molecular model was employed to solve the X‐ray structure of the complex with N‐acetylglucosamine. Tachylectin‐2 is the first protein displaying a five‐bladed β‐propeller structure. Five four‐stranded antiparallel β‐sheets of W‐like topology are arranged around a central water‐filled tunnel, with the water molecules arranged as a pentagonal dodecahedron. Tachylectin‐2 exhibits five virtually identical binding sites, one in each β‐sheet. The binding sites are located between adjacent β‐sheets and are made by a large loop between the outermost strands of the β‐sheets and the connecting segment from the previous β‐sheet. The high number of five binding sites within the single polypeptide chain strongly suggests the recognition of carbohydrate surface structures of pathogens with a fairly high ligand density. Thus, tachylectin‐2 employs strict specificity for certain N‐acetyl sugars as well as the surface ligand density for self/non‐self recognition.

Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity

Two antifungal peptides (Pn-AMP1 and Pn-AMP2) have been purified to homogeneity from seeds of Pharbitis nil. The amino acid sequences of Pn-AMP1 (41 amino acid0 residues) and Pn-AMP2 (40 amino acid residues) were identical except that Pn-AMP1 has an additional serine residue at the carboxyl-terminus. The molecular masses of Pn-AMP1 and Pn-AMP2 were confirmed as 4299.7 and 4213.2 Da, respectively. Both the Pn-AMPs were highly basic (pI 12.02) and had characteristics of cysteine/glycine rich chitin-binding domain. Pn-AMPs exhibited potent antifungal activity against both chitin-containing and non-chitin-containing fungi in the cell wall. Concentrations required for 50% inhibition of fungal growth were ranged from 3 to 26 micrograms/ml for Pn-AMP1 and from 0.6 to 75 micrograms/ml for Pn-AMP2. The Pn-AMPs penetrated very rapidly into fungal hyphae and localized at septum and hyphal tips of fungi, which caused burst of hyphal tips. Burst of hyphae resulted in disruption of the fungal membrane and leakage of the cytoplasmic materials. To our knowledge, Pn-AMPs are the first hevein-like proteins that show similar fungicidal effects as thionins do.

A Newly Identified Horseshoe Crab Lectin with Binding Specificity to O-antigen of Bacterial Lipopolysaccharides

We identified a novel horseshoe crab hemocyte-derived lectin, which we named tachylectin-4. It has more potent hemagglutinating activity against human A-type erythrocytes than a previously identified hemocyte lectin with an affinity toN-acetylglucosamine, tachylectin-2. The purified tachylectin-4 is an oligomeric glycoprotein of 470 kDa, composed of subunits of 30 and 31.5 kDa. Ca2+ at 10 mmenhanced the hemagglutinating activity 4-fold, and the activity was inhibited by EDTA and o-phenanthroline.l-Fucose and N-acetylneuraminic acid at 100 mm completely inhibited the activity of tachylectin-4. The activity was also inhibited more strongly by bacterial S-type lipopolysaccharides (LPS) but not by R-type LPS lacking O-antigen. The most effective S-type LPS was from Escherichia coliO111:B4, and the minimum concentration required for inhibiting agglutination against human A-type erythrocytes (0.1 μg/ml) was 160-fold lower than those of S-type LPS from Salmonella minnesota. Therefore, colitose (3-deoxy-l-fucose), a unique sugar present in the O-antigen of E. coli O111:B4 with structural similarity to l-fucose, is the most probable candidate for a specific ligand of tachylectin-4. A cDNA coding for tachylectin-4 was isolated from a hemocyte cDNA library. The open reading frame of the 1344-base pair cDNA coded for the mature protein with 232 amino acids. There is no significant sequence similarity to any other known LPS-binding lectins, whereas tachylectin-4 is homologous to the NH2-terminal domain with unknown functions of Xenopus laevis pentraxin 1.

Role of lectins in the innate immunity of horseshoe crab

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.

Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crabTachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to ω-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescence-labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity

A glycoprotein (M r = 43 000) from horseshoe crab hemocytes with antimicrobial activity against Gram‐negative bacteria was purified. The internal peptide sequences coincided exactly with the deduced amino acid sequence of a cDNA clone, designated limulus factor D, which was isolated by screening a hemocyte cDNA library with an anti‐human plasminogen antibody. The open reading frame codes for a precursor of factor D of 394 amino acid residues, including an NH2‐terminal signal sequence. The COOH‐terminal domain of factor D has significant sequence homology with the catalytic domain of mammalian serine proteases, in particular with human tissue plasminogen activator (32% identity), except for the substitution of Ser of the active site triad to Gly. Factor D has a unique NH2‐terminal domain with weak sequence homology with part of the mammalian interleukin‐6 receptor α‐chain. Factor D is likely to have an important role in host defense mechanisms.

A newly identified horseshoe crab lectin with specificity for blood group A antigen recognizes specific O-antigens of bacterial lipopolysaccharides

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 forN-acetyl-d-glucosamine orN-acetyl-d-galactosamine. However, the activity of tachylectin-3 was not inhibited by these twoN-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 Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was fromEscherichia 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.

Limulus Intracellular Coagulation Inhibitor Type 3 PURIFICATION, CHARACTERIZATION, cDNA CLONING, AND TISSUE LOCALIZATION

We reported that limulus intracellular coagulation inhibitor type-1 (LICI-1) (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547) and LICI type-2 (LICI-2) (Miura, Y., Kawabata, S., Wakamiya, Y., Nakamura, T., and Iwanaga, S. (1995) J. Biol. Chem. 270, 558-565) found in the hemocyte lysate belong to the serpin family. The LICI-1 specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C (k1 = 2.5 × 106 M−1 s−1), whereas LICI-2 inhibits preferentially limulus clotting enzyme (k1 = 4.3 × 105 M−1 s−1). In our ongoing studies on limulus serpin, we found another inhibitor, named LICI type-3 (LICI-3), which strongly inhibits (1,3)-β-D-glucan-sensitive serine protease, factor (k1 = 3.9 × 105 M−1 s−1). Thus, the limulus hemolymph coagulation cascade is effectively regulated by at least the three endogenous serpins. LICI-3, newly identified in hemocytes, is a single chain glycoprotein with an apparent Mr = 53,000, the largest one among known limulus serpins. A cDNA sequence for LICI-3 coded a mature protein of 392 amino acids, of which 68 residues were confirmed by peptide sequencing. LICI-3 showed significant sequence similarity to LICI-1 (45.8% identity) and LICI-2 (33.7% identity). LICI-3 contained a putative reactive site, -Arg-Ser-, distinct from that of LICI-2 (-Lys-Ser-) but the same as that of LICI-1. Expression of LICI-3 mRNA was detected only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. Immunoblotting of the hemocyte-derived large and small granules with antiserum against LICI-3 suggested that it is stored specifically in large granules, as in the case of LICI-1 and LICI-2, and is released in response to external stimuli.

Biochemical and physicochemical characterization of normal and variant forms of human MTH1 protein with antimutagenic activity

8-Oxo-7,8-dihydro-2-deoxyguanosine 5-triphosphate (8-oxo-dGTP) is produced during cellular metabolism, and its misincorporation into DNA causes mutation. Human cells possess an enzyme that hydrolyzes 8-oxo-dGTP to the corresponding nucleoside monophosphate, thereby preventing misincorporation of 8-oxo-7,8-dihydroguanine into DNA. Sequence analyses of the MTH1 gene, encoding the 8-oxo-7,8-dihydro-2-deoxyguanosine 5-triphosphatase (8-oxo-dGTPase) protein in human cell lines revealed that a G to A base substitution frequently occurs at codon 83, which causes a change of valine to methionine in the MTH1 protein [Wu, C. et al., Biochem. Biophys. Res. Commun. 214 (1995) 1239-1245]. Here we isolated cDNAs for the two types of MTH1 protein and expressed them in Escherichia coli mutT-. cells, devoid of their own 8-oxo-dGTPase activity. The two forms of proteins were purified to physical homogeneity, and amino acid analyses confirmed that the variant protein, Met83-MTH1, indeed carries the corresponding amino acid substitution. Met83-MTH1, but not normal type Val83-MTH1, was separated into two peaks in hydrophobic interacting chromatography. 8-Oxo-dGTPase activity of Met83-MTH1 is more thermolabile than that of Val83-MTH1. Circular dichroism (CD) and fluorescence spectroscopic analyses confirmed this conclusion. CD further indicated that Met83-MTH1 has a higher alpha-helix content.

Purification, Characterization, and Amino Acid Sequence of an Embryonic Lectin in Perivitelline Fluid of the Horseshoe Crab

Hemagglutinating activity in perivitelline fluid of the horseshoe crab embryo dramatically increases during the third and fourth molt of the embryo. A 27-kDa lectin, which we named tachylectin-P (TL-P), was newly identified in perivitelline fluid of the horseshoe crab Tachypleus tridentatus. TL-P preferentially agglutinated human A-type erythrocytes, and the activity was inhibited by N-acetyl group-containing monosaccharides. The amino acid sequence analysis indicated that TL-P is almost structurally the same as a hemocyte-derived lectin with no hemagglutinating activity, tachylectin-1 (TL-1), and that 218 out of 221 amino acid residues in total were conserved between the two lectins. Despite the high sequence similarity, biological and biochemical characteristics of TL-P differed from those of TL-1: (i) unlike TL-1, TL-P agglutinates several animal-derived erythrocytes; (ii) unlike TL-1, TL-P has no significant affinity for bacterial lipopolysaccharides or antibacterial activity; (iii) Based on apparent molecular masses determined by gel filtration, TL-P forms a dimer in solution, while TL-1 is present as a monomer; (iv) and TL-P interacts with endogenous proteins of 13 and 14 kDa present in the perivitelline fluid; however, neither has any affinity for TL-1. We propose that TL-P may have an important role in completing embryonic development by interacting with endogenous glycoproteins orN-acetylhexosamines.