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A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa, which was a glycoprotein having an N-linked sugar chain, was deglycosylated by N-glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1–6(Manα1–3)Manβ1–4GlcNAcβ1–4(Fucα1–6)GlcNAc and Manα1–6(GlcNAcβ1–2Manα1–3)Manβ1–4GlcNAcβ1–4(Fucα1–6))GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.

The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

Symbioses between zooxanthellae (Symbiodinium spp.) and marine invertebrates, including corals, are common in shallow marine environments. The zooxanthellae contribute to host nutrition by translocating photosynthetic products and enabling them to effloresce in oligotrophic conditions. Coral mainly acquire Symbiodinium spp. by capturing freeswimming cells from the environment (1). Cultured Symbiodinium cells show a diel growth cycle with alternation between motile and non-motile cell stages once a day (2, 3), and the cell divides only during the latter stage (2). When associated with a host, however, cells are arrested in a non-motile stage while healthy cell division is maintained (4). We deduced that host-directed and chemical-based mechanisms are responsible for this phenomenon since SLL-2, a lectin that binds to carbohydrate chains with a D-galactosyl moiety, produced by the octocoral Sinularia lochmodes, is localized on the cell surface of the Symbiodinium harbored in the host (5). Here we describe SLL-2 as the key chemical factor for arresting Symbiodinium in the cell-dividing, non-motile stage, while some nonsymbiotic microalgae were even destroyed by SLL-2. Symbiotic associations between photosynthetic dinoflagellates (Symbiodinium spp.) and invertebrate hosts such as corals are crucial for the survival of the host animals since Symbiodinium supplies organic compounds to them and enables them to prosper in the oligotrophic environment

Carbohydrate-binding proteins that bind their carbohydrate ligands with high affinity are rare and therefore of interest because they expand our understanding of carbohydrate specificity and the structural requirements that lead to high-affinity interactions. Here, we use NMR and isothermal titration calorimetry techniques to determine carbohydrate specificity and affinities for a novel cyanobacterial protein, MVL, and show that MVL binds oligomannosides such as Man(6)GlcNAc(2) with sub-micromolar affinities. The amino acid sequence of MVL contains two homologous repeats, each comprising 54 amino acid residues. Using multi-dimensional NMR techniques, we show that MVL contains two novel carbohydrate recognition domains composed of four non-contiguous regions comprising approximately 15 amino acid residues each, and that these residues make numerous intermolecular contacts with their carbohydrate ligands. NMR screening of a comprehensive panel of di-, tri-, and high-mannose oligosaccharides establish that high-affinity binding requires at least the presence of a discrete conformation presented by Manbeta(1-->4)GlcNAc in the context of larger oligomannosides. As shown by sedimentation equilibrium and gel-filtration experiments, MVL is a monodisperse dimer in solution, and NMR data establish that the three-dimensional structure must be symmetric. MVL inhibits HIV-1 Envelope-mediated cell fusion with an IC(50) value of approximately 30 nM.

The microstructure and orientation distribution of aragonite crystals in the nacreous layer of cultured Pteria penguin are investigated in this paper. Helical patterns formed by growth forefronts of the nacreous layer for good-quality shells are observed using a laser microscope, whereas no clear pattern is observed on the nacreous layer surfaces of bad-quality shells. The observed aragonite crystals are plate-shaped and hexagonal for both the good and bad-quality shells, in which the top and bottom faces are parallel to the (001) plane and the side faces are parallel to the {110} and (010) planes. The aragonite crystals in the nacreous layer of good-quality shells seem to be harmonically oriented along a crystallographic direction. These orientation distributions basically indicate that the (001) basal planes are parallel to the inner shell surface, and the [100] and [010] axes are oriented in almost the same direction, respectively. Some of the aragonite crystals are rotated about the c axis by approximately � 60 � from the basic orientation distribution. On the other hand, the aragonite crystals of bad-quality shells seem to be randomly oriented in the nacreous layer. These orientation distributions indicate that the (001) basal planes are parallel to the inner shell surface in a similar manner as those of good-quality shells, but their [100] and [010] axes are randomly oriented about the c axis. Therefore, it is considered that such different orientation distributions result in different qualities of pearls that are developed in the shells.

L-amino acid oxidase (LAAO) activity, as well as mechanisms of antimicrobial action of aplysianin A, a sea hare-derived 340 kDa homotetrameric protein, was determined. Spectrophotometric and High-performance liquid chromatography analyses of aplysianin A indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. Aplysianin A can specifically catalyze oxidation of basic amino acids (L-arginine and L-lysine), and is the first protein from marine invertebrate animals with LAAO activity. Substrate specificity of aplysianin A is markedly different from that of commonly known LAAO, such as snake venom LAAO, which prefer hydrophobic amino acids. Km value of aplysianin A was the smallest of those for all known LAAO reported. In the presence of catalase, the antibacterial activity of aplysianin A was inhibited as expected, indicating that the antibacterial action of aplysianin A results from hydrogen peroxide production during the reaction with substrates. Interestingly, aplysianin A acted as an antibacterial agent even in the presence of excess catalase. Antibacterial assays in various media suggested that this phenomenon was simply attributed to the consumption of amino acids required for bacterial growth in the media by aplysianin A.

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 μg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

Three L-rhamnose-binding lectins, named CSL1, CSL2, and CSL3, were isolated from eggs of chum salmon (Oncorhynchus keta) by affinity chromatography and reversed-phase high-performance liquid chromatography. The yields of CSL1, CSL2, and CSL3 from 2 kg eggs were 2.7, 1.7 and 29.6 mg, respectively. The subunit of CSL1 was composed of 286 amino acid residues with three tandemly repeated domains, while the subunits of both CSL2 and CSL3 were composed of 195 amino acid residues with two tandemly repeated domains. The amino acid sequence homologies among CSL showed 42–52% identities. CSL showed 94–97% sequence identities to three corresponding L-rhamnose-binding lectins, named STL1, STL2 and STL3, from steelhead trout (Oncorhynchus mykiss) eggs. Each CSL showed different hemagglutinating activities against rabbit and human erythrocytes. The lectins also showed different inhibition patterns in the hemagglutination towards rabbit erythrocytes by lipopolysaccharides from Gram-negative bacteria including a fish pathogen, Aeromonas salmonicida. CSL agglutinated Escherichia coli and Bacillus subtilis. These results indicate that CSL could be useful biochemical tools for recognizing specific molecules either in soluble forms or on cell surfaces.

Peptidic fractions which inhibit angiotensin I-converting enzyme (ACE) were separated from peptic digests of soybean by ion exchange chromatography and gel filtration. Further separation of the peptidic fractions by ODS HPLC afforded active peptides, the amino acid sequences of which were identified by Edman 's procedure as: Ile-Ala (inhibitory against ACE with an IC 50 of 153 μM), Tyr-Leu-Ala-Gly-Asn-GIn (14 μM), Phe-Phe-Leu (37 μM), Ile-Tyr-Leu-Leu (42 μM), and Val-Met-Asp-Lys-Pro-Gln-Gly (39 μM). The antihypertensive activity of the soybean peptides was also investigated. Peptide fractions (2.0 g/kg body weight, oral administration) markedly lowered the blood pressure of spontaneously hypertensive rats (SHRs).

Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.

Seven kinds of angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from the hydrolysates of wakame (Undaria pinnatifida) by Protease S "Amano" (from Bacillus stearothermophilus) by using three-step high-performance liquid chromatography (HPLC) on a reverse-phase column. These peptides were identified by amino acid composition analysis, sequence analysis, and liquid chromatography-mass spectrometry (LC-MS), as Val-Tyr (IC(50) = 35.2 microM), Ile-Tyr (6.1 microM), Ala-Trp (18.8 microM), Phe-Tyr (42.3 microM), Val-Trp (3.3 microM), Ile-Trp (1.5 microM), and Leu-Trp (23.6 microM). These peptides have resistance against gastrointestinal proteases in vitro. Each peptide was determined to have an antihypertensive effect after a single oral administration in spontaneously hypertensive rats (SHR). Among them, the blood pressure significantly decreased by Val-Tyr, Ile-Tyr, Phe-Tyr, and Ile-Trp in a dose of 1 mg/kg of body weight (BW). The present study showed that antihypertensive effect in the hydrolysates of wakame by Protease S "Amano" was attributed to these peptides.

The epidermis of premetamorphic leptocephali of Japanese conger Conger myriaster consisted of only a few cell layers including congerin- (a galectin) containing club cells. The epidermis increased in thickness after metamorphosis, mainly due to a preponderant development of club cells. This suggested that the biological significance of congerins increased at this stage, which may be related to a change in life style. Epidermal mucous cells showed only limited distribution. Immuno-positive cells in the mucosal epithelia of the buccal cavity wall and oesophagus were not found in the youngest individuals. Though these cells were observed at the commencement of metamorphosis, the development of the epithelia and club cells was apparently retarded compared to that of the epidermis. No immuno-staining was found in the gills at any stages.

The localization of three l -rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout ( Oncorhynchus mykiss ) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity.

BRA-2, a major C-type lectin in the hemolymph of the acorn barnacle Megabalanusrosa was detected in shell-associated proteins. Immobilized BRA-2 promoted CaCO3 crystallization as determined by an in vitro pH-drift mineralization assay. In nature, calcification occurs as wound repair in M. rosa after breakage of the shell plate. To observe this phenomenon, we inserted a rubber cube inside the mantle cavity of M. rosa through the basal plate, and kept the organisms in a tank for several weeks. The rubber cube was covered first with brownish-colored material, probably a melanin, and then with CaCO3 5 weeks after the insertion. In the CaCO3-associated proteins, BRA-3, a minor component of M. rosa hemolymph lectins, was present in addition to BRA-2. The presence of both the lectins and their ligands on the hemocyte was also observed. These results suggest that the C-type lectins of the M. rosa hemolymph participate in mineralization as well as defense in these organisms.

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the 16 sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species.

The interaction between bacteria and three L-rham-nose-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.

Tissue-specific expression of three L-rhamnose-binding lectins, named STL1, STL2, and STL3, in the steelhead trout (Oncorhynchus mykiss) was investigated. STL2 and STL3 mRNAs were restricted in the oocytes. In contrast, STL1 mRNA was detected only in the liver. The transcription of STL2 and STL3 started in previtellogenic oocytes. These results showed distinct expression profiles of rhamnose-binding lectins in the fish.

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli. Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val and Leu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity.

The amino acid sequence of a D-galactose-binding lectin isolated from the hemolymph of the acorn barnacle Balanus rostratus was determined. The lectin (BRL) (Mr 120K) is a multimeric protein whose subunit consists of 182 amino acids. The amino acid sequence was compared with those of multiple lectins (BRA-2, BRA-3) from Megabalanus rosa to explore the relationship between the structures and the inhibitory activity toward the crystal growth of calcium carbonate. Although BRL was 46% identical to BRA-2 and 15% identical to BRA-3, the lectin had no inhibitory activity, unlike BRA-2 and BRA-3. Both the number and the localization of acidic amino acid residues and their amide forms were different among them. Observations by scanning electron microscopy revealed modifications in the size and morphology of the calcium carbonate crystals grown in the presence of the lectins.

This study reports the isolation and partial characterisation of the ostrich serpin, alpha(2)AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich alpha(2)AP was purified using L-lysine-Sepharose chromatography, ammonium sulfate fractionation, and Super Q-650S and ostrich LBSI-Sepharose chromatographies. It revealed a M(r) of 84 K (thousand) and had one and two N-terminal amino acids in common with 11 of those of human and bovine alpha(2)AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human alpha(2)AP, DFP and EACA. Ostrich plasminogen was highly purified after L-lysine-Sepharose chromatography and showed a M(r) of 92 K, a total of 775 amino acids and its N-terminal sequence showed approximately 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a M(r) of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40 degrees C, respectively.

Congerin is a β-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.

Red soft coral (RSC; Dendronephthya nipponica, a marine coelenterate) causes spiny lobster fishermen living along the Pacific coast of Miyazaki Prefecture in Japan to develop occupational allergies, such as conjunctivitis, rhinitis, dermatitis and bronchial asthma. The aim of this study was to purify and to characterize RSC allergen, which causes occupational asthma in spiny lobster fishermen. The allergic responsiveness of spiny lobster fishermen to RSC was examined. The examinations included specific IgE production, skin test responses, lymphocyte stimulation tests and specific IgG production. We found that RSC has a strong sensitizing activity in humans at a molecular weight of 10 kD or more, while it has no IgE-producing activity at a molecular weight of less than 10 kD. Neither the nonatopic controls nor the atopic non-coral-allergic controls exhibited any RAST-binding activity to any fraction. For the purification and the identification of this new allergen component, repeated gel filtration of the RSC extract was performed on a Sephacryl S-200 column, followed by gel filtration on a Superose-6 column. The purified major allergen component Den n 1, which is separated on a Mono-Q column, showed intradermal responses, lymphocyte stimulating activity and specific IgG-producing activity in RSC-induced bronchial asthma patients. The 53-kD component was electroblotted on a polyvinylidene difluoride membrane. The N-terminal amino acid sequence of this new allergen component (Den n 1) was determined as Asp-Asp-Ile-Asn-Arg-Tyr-Ala-Phe-Asp-Asn-Lys-Ile-Asn- Asp-Lys-Leu-Phe-Asp-His-Trp-Gln-Ser.

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.

The amino-acid sequence of α-amylase isolated from the pancreas of the ostrich, Struthio camelus was determined. The α-amylase (OPA) consisted of 497 amino acid residues with pyroglutamic acid at the N-terminus and no oligosaccharide. Amino acid identity between OPA and chicken, porcine and human pancreatic α-amylases individually, was found to be 88, 82 and 86%, respectively.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu2+-chelate Sepharose chromatography. Ostrich prothrombin exhibited a Mr of 72 800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin β-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

SUMMARY: Effects of culture conditions on the lectin level by a unicellular laboratory culture of Microcystis aeruginosa M228 (freshwater cyanobacterium) were investigated. The lectin level per cell at stationary phase increased two- or fourfold by lowering the light intensity from 45 or 90 μE/m2 per s to 12 μE/m2 per s, respectively, at 25°C as measured by the enzyme-linked immunosorbent assay method. The hemagglutinating activity against rabbit erythrocytes increased in response to the lectin level. The lectin level was threefold greater at 15°C than at 25°C, and nearly ninefold greater than at 30°C. Thus, although the specific growth rate of the cyanobacterium decreased with lowering light intensity and temperature, the lectin level per cell increased significantly. When the cyanobacterium was cultured at 15°C with a light intensity of 12 μE/m2 per s, the lectin level reached the maximum near the end of exponential phase, and then decreased toward stationary phase. The level near the end of exponential phase was nearly threefold greater than that of the culture incubated at 25°C with a light intensity of 45 μE/m2 per s. These findings show that the lectin production in M. aeruginosa M228 was induced by unfavorable conditions for the cell growth.

We have cloned and characterized the gene encoding Microcystis aeruginosa (strain M228) lectin (MAL). The gene contains 1551 nucleotides and an open reading frame for a protein of 517 amino acids with a predicted molecular weight of 55,159 Da. The carboxy-terminal region of MAL has three tandemly repeated homologous domains composed of 61 amino acids. These regions show similarity to the corresponding regions of the alpha-amylase of Clostridium beijerinckii (23% identity). The mal gene lies adjacent to an ORF that display homology to cytochrome P-450 and polyketide synthase. Southern hybridization showed that the genomic DNA of the strain M228 contained, in addition to MAL gene (mal), at least two other mal like gene.

Microcystis viridis NIES-102 strain, a unicellular freshwater bloom-forming cyanobacterium, showed transient hemagglutinating activity in laboratory culture during stationary phase under nonaeration conditions. However, the hemagglutinating activity which was inhibited with yeast mannan could not be observed during culture with aeration. A mannan-binding lectin named MVL was isolated with the assay of the hemagglutinating activity against rabbit erythrocytes from the cyanobacterium by successive hydrophobic and gel filtration chromatography. MVL was composed of a single polypeptide of 13 kDa. The gene (mvl) for MVL was cloned from a genomic DNA of NIES-102 strain as a template, and its sequence was determined. The deduced amino acid sequence showed that MVL consisted of 113 amino acid residues and was composed of two tandemly repeated homologous domains of 54 amino acid residues. MVL showed no sequence homology to any other lectins or proteins.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) was extracted from ostrich kidney cortex, intact kidney, ileum, pancreas, and liver. DPP IV was purified from the kidney cortex 373-fold by using homogenization, autolysis, diethylaminoethyl-Toyopearl 650 M, Cu 2+ -chelate affinity, and Mono Q chromatography. Autolysis conditions were investigated and optimized with regards to pH, incubation time, and Triton X-100 concentration. Nondenaturing gradient polyacrylamide gel electrophoresis (PAG) and sodium dodecyl sulfate-PAGE revealed molecular mass values of 270 K and 133 K for the native enzyme and its subunit, respectively. PAG-isoelectrofocusing indicated a pI of 4.7. The pH and temperature optimum were 8.0 and 45°C, respectively. The enthalpy of activation was computed as 20.91 kJ/mol. k cat and k cat / K m values were determined for various chromogenic substrates having the sequence of X-Pro-pNA. The effect of several metal ions on DPP IV activity was investigated. The N-terminus was blocked with an agent of unknown identity. The above data as well as the amino acid composition of ostrich DPP IV were compared with those of other mammalian and bacterial species. It appears that, in terms of physical characteristics, ostrich DPP IV correlated with those of other species, whereas chemically and kinetically it exhibited distinct characteristics.

Ostrich carboxypeptidases A and B were recently purified and characterized. The aim of this study was to isolate and purify, and partially characterize in terms of molecular weight, pI, amino acid composition and N-terminal sequencing, the precursor forms of carboxypeptidases from the ostrich pancreas. Inhibition studies with soybean trypsin inhibitor and activation studies with three proteases (bovine trypsin, bovine chymotrypsin and porcine elastase) were performed on crude ostrich acetone powder and the carboxypeptidase A and B activities were determined. SDS-PAGE was carried out after every incubation to monitor the rate and degree of conversion of a M(r) 66K component to procarboxypeptidase and carboxypeptidase A and B. The precursor forms were purified by Toyopearl Super Q and Pharmacia Mono Q chromatography. All three proteases converted the M(r) 66K component to procarboxypeptidases and carboxypeptidases over a set time interval, with carboxypeptidase A and B activities being detected in the acetone powder. Chymotrypsin was the preferred protease since it exhibited a more controlled activation of the procarboxypeptidases. The amino acid composition of procarboxypeptidase A revealed 525 residues. The N-terminal sequence of procarboxypeptidase A showed considerable homology when compared with several other mammalian sequences. M(r) and pI values of 52K and 5.23 were obtained for procarboxypeptidase A, respectively. This study indicated that ostrich procarboxypeptidase A is closely related to other mammalian procarboxypeptidase A molecules in terms of physicochemical properties.

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.

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 microg/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 as L-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 or Staphylococcus 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) of D-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 beta'-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (Ka) of STL2 to lipovitellin and beta' component were 3.44 x 10(6) and 4.99 x 10(6), 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.

Intestinal glycohydrolases are enzymes involved in assimilating carbohydrate for nutrition. The avian forms of these enzymes, in particular the maltase–glucoamylase complex (MG), are not well characterised. This study encompassed characterisation of these enzymes from ostrich intestines, and the first kinetic analysis of an avian MG. Proteolytically solubilised MG from ileal brush border membrane vesicles was purified by Sephadex G-200 gel filtration and Tris-affinity chromatography, while jejunal sucrase–isomaltase (SI) and MG were purified by Toyopearl-Q650 and phenyl–Sepharose chromatography. Amino acid sequences and compositions of enzyme subunits, resulting from SDS-PAGE, were determined. Kinetics of hydrolysis of linear oligosaccharides was studied. Ostrich MG and SI showed the highest activity in the jejunum, followed by the ileum and duodenum. No lactase or trehalase activity could be detected. The jejunal MG and SI, resulting from brush-border membrane vesicles, could not be separated during purification. However, a minor form of ileal MG was purified using Sephadex G-200 chromatography. Ileal MG contained three subunits of M r 145 000, 125 000 and 115 000. Although the N-terminal amino acid sequences bear no homology to SI, the M r 115 000 subunit shows homology to porcine MG in both sequence and amino acid composition. The pH optimum of maltose-, starch- and isomaltose-hydrolysing activity was 6.5 and that of sucrose-hydrolysing activity 5.5. The glycohydrolases were most active at 58°C, but were quickly denatured above 60°C. Sucrose- and starch-hydrolysing activities were more thermostable than maltose- and isomaltose-hydrolysing activities. Kinetic parameters ( K m , k cat and k cat / K m ) for the hydrolysis of maltooligosaccharides, starch and glycogen are reported for ileal MG. Maltotriose and maltotetraose displayed partial inhibition of ileal MG. The study revealed large similarities between ostrich SI and MG in charge, size, shape and hydrophobicity, based on their inseparability by several methods. Measurement of the specificity constants for maltooligosaccharide hydrolysis by ileal MG revealed less efficient hydrolysis of longer substrates as compared to maltose and maltotriose.

Microcystis aeruginosa, strain M228, a laboratory culture of freshwater cyanobacterium, showed hemagglutinating activity against rabbit, horse and human ABO erthrocytes. Crossed absorption tests revealed the presence of a single type of lectin in the extract of M228 strain cells. The lectin, termed MAL, was purified in combination with the affinity chromatography on acid-treated agarose gel and the gel permeation chromatography in an electrophoretically pure form. MAL was a glycoprotein containing 7.8% neutral sugars and was composed of a single polypeptide having a molecular weight of 57 kDa. Isoelectric point was estimated to be pH 6.4. Hemagglutinating activity of the lectin was inhibited effectively by N-acetyl- d -galactosamine and by glycoproteins. d -galactose and lactose also showed moderate inhibitory activity. The destruction of the hemagglutinating activity by a 2-mercaptoethanol treatment suggests the presence of intra-chain disulfide bond(s) essential for the activity in the molecule. The sequence of the amino-terminal region of MAL was determined as Val-Lys-Ala-Ser-Lys-Val-Ser-Thr-Ser-Gln-Ala-Gly-Ser-Lys-Glu-Lys-Lys-Ala.

The properties of 22 synthetic peptides containing histidine, which were designed on the basis of the antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, were examined with regard to their antioxidative activity against the peroxidation of linoleic acid and the scavenging effects on active oxygen and free radical species. The antioxidative activities of these peptides in an emulsion oxidation system using 2,2'-azobis(2-amidinopropane) dihydrochloride as a radical initiator correlated well within an aqueous system. Although the histidine-containing peptides had a quenching activity on singlet oxygen, they did not show antioxidative activity in an 2,2'-azobis(2,4-dimethylvaleronitrile)-induced oxidation system or scavenging effects on 1,1-diphenyl-2-picrylhydrazyl radical and superoxide. The metal-ion chelating activities and the hydrophobicities of these peptides showed no direct correlation with their antioxidative activities. Leu-Leu-Pro-His-His was modified with a hydroxyl radical in an aqueous ethanol system during the peroxidation of linoleic acid.

Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.

The proteasome (EC 3.4.99.46) is a high molecular mass (approximately 700 kDa) multisubunit enzyme complex which is the focus of worldwide research in order to identify the structure, mechanism of action and specificity of the complex. The purpose of the present study was to investigate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysing (PGPH) activities of ostrich liver proteasome. The proteasome was purified from ostrich liver by employing ammonium sulphate fractionation, followed by three sequential chromatographic steps on Toyopearl Super Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns. Temperature and pH optima were examined and the effect of inhibitors, detergents, fatty acids and cations on the peptidase activities was determined. Ostrich proteasome exhibited a relative M(r) of approximately 665,000 using non-denaturing gradient PAGE and dissociated into the characteristic "ladder" associated with the proteasome subunits during SDS-PAGE. The pH optima for the peptidase activities were found to be slightly alkaline (tryptic activity) and neutral (chymotryptic-like and PGPH activities). Ostrich liver proteasome was found to be activated in terms of the PGPH activity by fatty acids and SDS, whereas the chymotryptic and tryptic-like activities were differentially inhibited. Ostrich proteasome, in its inhibition by monovalent cations, was similar to the proteasomes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. The yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic properties which ostrich liver proteasome exhibited, it can be safely concluded that it corresponds well with the proteasomes isolated from many other sources.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20-40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.

Proteases consisting of various endo- and exo-proteases were prepared by homogenizing Aspergillus oryzae mycelia using a French-press. The hydrolysis of 1% soybean proteins was carried out at 35°C and pH 7.0. The back-pressure, flux and rotational speed of the reactor affected the hydrolysis rate and productivity. The reactor system could be operated in a continuous process by replenishing the substrate solution, with the concentration adjusted to maintain 0.43% substrate in the reaction vessel. Permeate flux gave a constant product as shown by molecular weight distribution and amino acid composition.

Antioxidative activities of 28 synthetic peptides, which were designed based on an antioxidative peptide (Leu-Leu-Pro-His-His) derived from proteolytic digests of a soybean protein, against the peroxidation of linoleic acid in an aqueous system were measured by the ferric thiocyanate method. The results for the hydroperoxide levels derived from linoleic acid agreed with those obtained by reversed-phase high-performance liquid chromatography. The deletion of the C-terminal His decreased the activity, whereas the deletion of the N-terminal Leu had no effect. In the peptide sequence, His and Pro played important roles in the antioxidative activity and, among the peptides tested, Pro-His-His was the most antioxidative. The activity decreased on substitution of the second His with d-His. Introduction of Tyr to the positions of Pro or His did not increase the activities of the corresponding peptides. Antioxidative peptides showed synergistic effects with nonpeptidic antioxidants as observed in soybean protein h...

During the past several decades, intensive investigations have been conducted to clarify the biochemical and physiological properties of lectins from marine organisms, including cyanobacteria, algae, and invertebrates and fish. These investigations have revealed that lectins are highly diversified in terms of not only structural aspects but also functional aspects, including unique carbohydrate-binding specificities. Lectins are still being intensively investigated to understand their biological roles in cell recognition and biodefense as well as to employ them as valuable tools for studying complex carbohydrates in solution and on cell surfaces. Here, we review the structures and activities of lectins from marine organisms and their applications as carbohydrate recognition molecules and medicinal agents with antitumor and antiviral activities.