Koji Muramoto

Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2

1. Figs. 1B and 2A: Total RNAwas extracted from different cells, and cDNAwas prepared and analyzed for the expression of SLPI and PARs and GAPDH by RT-PCR. However, the patterns of GAPDH in these figures and those in the figures of The Journal of Immunology, 2002, 169: 4594–4603 and in Fig. 2A of Clinical and Diagnostic Laboratory Immunology, 2003, 10: 286–292 are the same. 2. Fig. 2B: Two panels of the expression of PAR-2 with HLE and Cat G are the same.

Isolation and Characterization of Rhamnose-binding Lectins from Eggs of Steelhead Trout (Oncorhynchus mykiss) Homologous to Low Density Lipoprotein Receptor Superfamily

Two l-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 μg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was l-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in l-rhamnose, such asl-arabinose and d-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I orStaphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) ofd-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and β′-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (K a ) of STL2 to lipovitellin and β′ component were 3.44 × 106 and 4.99 × 106, respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

1. Fig. 1E: HSC-2, an oral epithelial cell line, is different from KB cells, but the patterns of their band staining for IL-8, MCP-1, and GAPDH cDNA are the same. 2. Fig. 2D: Bands for MCP-1 and GAPDH cDNA in Fig. 1E and ICAM-1 and GAPDH cDNA in this figure are the same. 3. Fig. 4A: Total RNAwas extracted from KB (lane 2), HSC-2 (lane 3), and PBMCs (lane 4), and cDNAwas prepared and analyzed for the expression of PAR1-4 and GAPDH by RT-PCR. However, three bands of PAR3 and a band of PAR4 are the same. Furthermore, the patterns of GAPDH in this article and those in Fig. 1B and 2A of The Journal of Immunology, 2003, 170: 5690–5696 and in Fig. 2A of Clinical and Diagnostic Laboratory Immunology, 2003, 10: 286–292 are the same. 4. Fig. 4B and 4D: Three panels of the expression of PAR1 with PR3 and PR3 + cytochalasin B (Cyto B) and PAR3 with PR3 + Cyto B are the same. Two panels of the expression of PAR2 with PR3 + cycloheximide (CHX) in Fig. 4B and with trypsin in Fig. 4D are the same.

Galectin containing cells in the skin and mucosal tissues in Japanese conger eel, Conger myriaster: an immunohistochemical study

Congerin is a beta-galactoside binding lectin (galectin) purified from the skin mucus of the Japanese conger, Conger myriaster. To clarify its tissue distribution and productive cells, several tissue samples including skin, buccal cavity wall, tang, pharynx, gills, esophagus, stomach, intestine, liver, kidney, spleen and ovary of conger were stained immunohistochemically using polyclonal rabbit anti-congerin serum. In the epidermis, a number of club cells were strongly stained. Because no agglutinating activity was detected in plasma, it appears evident that congerin is produced and secreted into mucus by those cells. In addition, congerin-positive club cells were distributed in the mucosal epithelium lining the digestive tract preceding the stomach and in the gills. These findings suggest that congerin participates in innate immunity on the intra- and the extra-body surface of the conger. The putative functions of club cells in fish and their contained lectin are discussed.

Rhamnose-binding lectins from steelhead trout (Oncorhynchus mykiss) eggs recognize bacterial lipopolysaccharides and lipoteichoic acid.

The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces.

Diversified Carbohydrate-Binding Lectins from Marine Resources

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.

Functional and structural characterization of multiple galectins from the skin mucus of conger eel, Conger myriaster.

The complete amino acid sequence of an isogalectin, named congerin II, isolated from the skin mucus of conger eel, was determined by sequencing of the protein and its peptides generated by enzymatic and chemical cleavages. Congerin II consisted of 135 amino acids residues containing an acetylated N-terminus. Congerin II was found to be only 46% homologous in sequence to congerin I which was previously determined (Muramoto K., Kamiya H., Biochem. Biophys. Acta, 1992;1116:129-136), suggesting that the galectins with diverse molecular properties are present in the skin mucus of conger eel. However, it was confirmed by analysis of the secondary structures using circular dichroism that both congerins I and II shared similar folds characterized by beta structures. Congerins I and II showed different molecular properties such as thermostability, pH dependency for hemagglutinating activity and for binding specificity against the pyridylamino derivative of lactose. Congerin I showed more strict recognition specificity for lactose than did congerin II. Furthermore, the effects of chemical modification on congerins I and II were investigated in order to identify the type of amino acids involved in their different lectin activities. Modification of tyrosine and lysine residues did not affect the carbohydrate-binding activities of congerins. However, modification of tryptophan, arginine, histidine, glutamic acid and aspartic acid residues led to considerable loss of their activities, and a different mode of binding activity was observed between modified congerins I and II. These results suggest that multiple galectins from conger eel with the same scaffold have different biological functions and properties.

The amino-acid sequence of a lectin from conger eel, Conger myriaster, skin mucus

The amino-acid sequence of a beta-galactoside-binding lectin isolated from the skin mucus of the conger eel Conger myriaster was determined. The lectin (30 kDa) was composed of two identical subunits of 135 amino acid residues with N-acetylserine at the N-terminus and no half-cystinyl residue. It was a 30-34% sequence identical to vertebrate beta-galactoside-binding lectin and proved to be a member of the S-type lectin family.

The application of fluorescein isothiocyanate and high-performance liquid chromatography for the microsequencing of proteins and peptides.

Amino acid derivatives of fluorescein isothiocyanate have been separated by reverse-phase high-performance liquid chromatography in 45 min, using a linear gradient formed from acetone and 10 mM sodium phosphate buffer (pH 7.0) at 60 degrees C. The fluorescence of the derivatives has been used for detection giving a sensitivity of less than 0.5 pmol for a single component. Applications of this method to the sequence analyses of egg white lysozyme and salmon beta-melanotropin are also described.

The amino-acid sequence of a lectin of the acorn barnacle Megabalanus rosa

The complete amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 64 000) is composed of four identical subunits of 138 amino acids. The amino-acid sequence and the location of two interchain and three intrachain disulfide bridges of the subunit were determined by the manual sequencing of peptides derived from the protein by digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase, as well as fragments produced by cleavage with cyanogen bromide. The Cys-Pro-Pro-Cys sequence at the interchain disulfide bridges was the same as that of the hinge region of human immunoglobulin IgG1. The amino acid sequence of M. rosa lectin includes some regions homologous to those in Sarcophaga (flesh fly) lectin.