Search

The aromatic compound 3-amino-4-hydroxybenzoic acid (3,4-AHBA) can be employed as a raw material for high-performance industrial plastics. The aim of this study is to produce 3,4-AHBA via a recombinant Streptomyces lividans strain containing griI and griH genes derived from Streptomyces griseus using culture medium with glucose and/or xylose, which are the main components in lignocellulosic biomass. Production of 3,4-AHBA by the recombinant S. lividans strain was successful, and the productivity was affected by the kind of sugar used as an additional carbon source. Metabolic profiles revealed that L aspartate-4-semialdehyde (ASA), a precursor of 3,4-AHBA, and coenzyme NADPH were supplied in greater amounts in xylose medium than in glucose medium. Moreover, cultivation in TSB medium with a mixed sugar (glucose/xylose) was found to be effective for 3,4-AHBA production, and optimal conditions for efficient production were designed by changing the ratio of glucose to xylose. The best productivity of 2.70 g/L was achieved using a sugar mixture of 25 g/L glucose and 25 g/L xylose, which was 1.5 times higher than the result using 50 g/L glucose alone. These results suggest that Streptomyces is a suitable candidate platform for 3,4-AHBA production from lignocellulosic biomass-derived sugars under appropriate culture conditions.

3-Phenyllactic acid (PhLA) is useful as a start-up material in the pharmaceutical and biorefinery industries. To enhance the production of PhLA from glucose using recombinant Escherichia coli, the effects of glucose concentration and oxygen limitation on PhLA production are assessed in a fed-batch system using dissolved oxygen (DO)-stat method. The highest titer of PhLA (7.3 g L -1 ) is observed with a high concentration of glucose and under oxygen-limited conditions (DO = 0 ppm). Under oxygen limitation, cell growth and the formation of acetate and l-phenylalanine (Phe) by-products after 72 h of cultivation are reduced by 30%, 70%, and 81%, respectively, as compared to that under high DO conditions (DO = 5 ppm). Gene expression levels are compared between low and high DO conditions by quantitative polymerase chain reaction (qPCR) analysis. Several genes in the glycolysis (gapA and pykA), pentose phosphate (tktA), and early shikimate pathways for PhLA biosynthesis (aroF, aroG, and aroH) are upregulated under oxygen limitation. The results suggest that oxygen limitation affects metabolism in the shikimate pathway at both metabolic and transcriptional levels and that controlling the DO level is critical for enhanced production of a variety of aromatic compounds through the shikimate pathway., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N -glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N -glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N -glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations., Competing Interests: The authors declare no conflict of interest.

The production of the bioplastic precursor 3-amino-4-hydroxybenzoic acid (3,4-AHBA) from sweet sorghum juice, which contains amino acids and the fermentable sugars sucrose, glucose and fructose, was assessed to address the limitations of producing bio-based chemicals from renewable feedstocks. Recombinant Corynebacterium glutamicum strain KT01 expressing griH and griI derived from Streptomyces griseus produced 3,4-AHBA from the sweet sorghum juice of cultivar SIL-05 at a final concentration (1.0 g l(-1)) that was 5-fold higher than that from pure sucrose. Fractionation of sweet sorghum juice by nanofiltration (NF) membrane separation (molecular weight cut-off 150) revealed that the NF-concentrated fraction, which contained the highest concentrations of amino acids, increased 3,4-AHBA production, whereas the NF-filtrated fraction inhibited 3,4-AHBA biosynthesis. Amino acid supplementation experiments revealed that leucine specifically enhanced 3,4-AHBA production by strain KT01. Taken together, these results suggest that sweet sorghum juice is a potentially suitable feedstock for 3,4-AHBA production by recombinant C. glutamicum., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The hepatitis B virus core (HBc) particle is known as a promising new carrier for the delivery of drugs and nucleic acids. However, since the arginine-rich domain that is located in the C-terminal region of the HBc monomer binds to the heparan sulphate proteoglycan on the cell surface due to its positive charge, HBc particles are introduced non-specifically into a wide range of cells. To avoid non-specific cellular uptake with the intent to control the ability of cell targeting, we individually replaced the respective arginine (R) residues of the arginine-rich domain located in amino acid positions 150-159 in glycine (G) residues., Results: The mutated HBc particles in which R154 was replaced with glycine (G) residue (R154G) showed a drastic decrease in the ability to bind to the heparan sulphate proteoglycan and to avoid non-specific cellular uptake by several types of cancer cells., Conclusions: Because this mutant particle retains most of its C-terminal arginine-rich residues, it would be useful in the targeting of specificity-altered HBc particles in the delivery of nucleic acids.

Osmerus (Spirinchus) lanceolatus egg lectin (OLL) is a member of the rhamnose-binding lectin (RBL) family which is mainly found in aqueous beings. cDNA of OLL was cloned, and its genomic architecture was revealed. The deduced amino acid (aa) sequence indicated that OLL was composed of 213 aa including 95 aa of domain N and 97 aa of domain C. N and C showed 73 % sequence identity and contained both -ANYGR- and -DPC-KYL-peptide motifs which are conserved in most of the RBL carbohydrate recognition domains. The calculated molecular mass of mature OLL was 20,852, consistent with the result, and 20,677.716, from mass spectrometry. OLL was encoded by eight exons: exons 1 and 2 for a signal peptide; exons 3-5 and 6-8 for N- and C-domains, respectively. Surface plasmon resonance spectrometric analyses revealed that OLL showed comparable affinity for Galα- and β-linkages, whereas Silurus asotus lectin (SAL), a catfish RBL, bound preferentially to α-linkages of neoglycoproteins. The Kd values of OLL and SAL against globotriaosylceramide (Gb3) were 1.69 × 10⁻⁵ M for and 2.81 × 10⁻⁶ M, respectively. Thus, the carbohydrate recognition property of OLL is slightly different from that of SAL. On the other hand, frontal affinity chromatography revealed that both OLL and SAL interacted with only glycolipid-type oligosaccharides such as Gb3 trisaccharides, not with N-linked oligosaccharides. The domain composition of these RBLs and an analytical environment such as the "cluster effect" of a ligand might influence the binding between RBL and sugar chains.

For immunological experiments on glycogens, anti-glycogen antibodies are indispensable to capture, detect, and visualize sugar molecules. An anti-glycogen monoclonal antibody (IV58B6) and newly constructed antibody (ESG1A9mAb) have a common immunoglobulin type (IgM) and binding ability to glycogens, but overall possess different binding features. Therefore, they may prove useful for the construction of an advanced system of quantitative ELISA based on their molecular structures. For this purpose, detailed information on the carbohydrate-specificities of ESG1A9mAb and IV58B6 is first required, but their fine specificities for various types of glycogens have not been elucidated. To overcome this problem, we performed interaction analysis by ELISA of ESG1A9mAb and IV58B6 toward 15 glucose polymers, that is, 5 enzymatically-synthesized glycogens (ESGs), 6 natural source glycogens (NSGs), 3 enzymatically digested glycogens (EDGs), and soluble starch. To provide a more detailed analysis, we determined the association constants (K(a)) of the two antibodies toward these glycogens by surface plasmon resonance. The results indicated that the carbohydrate-binding properties toward NSGs of ESG1A9mAb and IV58B6 were similar, but markedly differed for ESGs and EDGs. ESG1A9mAb showed significant affinity for all the ESGs and NSGs tested, whereas IV58B6 had only slight affinity for ESGs, although the affinities were increased when the ESGs were enzymatically digested. This information should be helpful for the design of both in vitro and in vivo immunological assays., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lentinan (LNT), a β-glucan from the fruiting bodies of Lentinus edodes, is well known to have immunomodulatory activity. NO and TNF-α are associated with many inflammatory diseases. In this study, we investigated the effects of LNT extracted by sonication (LNT-S) on the NO and TNF-α production in LPS-stimulated murine RAW 264.7 macrophages. The results suggested that treatment with LNT-S not only resulted in the striking inhibition of TNF-α and NO production in LPS-activated macrophage RAW 264.7 cells, but also the protein expression of inducible NOS (iNOS) and the gene expression of iNOS mRNA and TNF-α mRNA. It is surprising that LNT-S enhanced LPS-induced NF-κB p65 nuclear translocation and NF-κB luciferase activity, but severely inhibited the phosphorylation of JNK1/2 and ERK1/2. The neutralizing antibodies of anti-Dectin-1 and anti-TLR2 hardly affected the inhibition of NO production. All of these results suggested that the suppression of LPS-induced NO and TNF-α production was at least partially attributable to the inhibition of JNK1/2 and ERK1/2 activation. This work discovered a promising molecule to control the diseases associated with overproduction of NO and TNF-α.

Black soybean seed coat extract (BE) is a polyphenol-rich food material consisting of 9.2% cyanidin 3-glucoside, 6.2% catechins, 39.8% procyanidins, and others. This study demonstrated that BE ameliorated obesity and glucose intolerance by up-regulating uncoupling proteins (UCPs) and down-regulating inflammatory cytokines in C57BL/6 mice fed a control or high-fat diet containing BE for 14 weeks. BE suppressed fat accumulation in mesenteric adipose tissue, reduced the plasma glucose level, and enhanced insulin sensitivity in the high-fat diet-fed mice. The gene and protein expression levels of UCP-1 in brown adipose tissue and UCP-2 in white adipose tissue were up-regulated by BE. Moreover, the gene expression levels of major inflammatory cytokines, tumor necrosis factor-α and monocyte chemoattractant protein-1 were remarkably decreased by BE in white adipose tissue. BE is a beneficial food material for the prevention of obesity and diabetes by enhancing energy expenditure and suppressing inflammation.

Background: Galectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant (K(d))., Methods: In the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1-9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families., Results: An empirical [Galβ-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, "Galβ1-4-L-Fuc" [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to 'axial' 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration "Galβ-(syn)-gauche". The rule also worked perfectly for differentiation of galectins from other types of lectins., General Significance: The present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands., (2011 Elsevier B.V. All rights reserved.)

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galβ1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.

Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS-PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galbeta1-->4GlcNAcbeta1-->4Manbeta1-->4GlcNAcbeta1-->4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Previously, we developed an alpha2-6-sialic acid (Sia)-specific lectin (SRC) starting from an R-type galactose-specific lectin C-terminal domain. However, it showed relatively low affinity because of its monovalency. Here, we engineered a tandem repeat construct (SRC2) showing substantial affinity for alpha2,6-sialylated N-glycans (in the order of 10(-6)M in K(d)), almost comparable to a natural alpha2-6Sia-specific lectin from Sambucus sieboldiana (SSA). Notably, its binding to branched N-glycans was found to be more selective than SSA. Nevertheless, SRC2 showed no apparent hemagglutinating activity, while it exerted strong erythrocyte-binding activity. This unique feature will help flow cytometry analysis, where usual lectins including SSA agglutinate cells. Some other biochemical properties investigated for SRC2, e.g., high productivity in bacteria and easy release of captured glycoproteins with lactose have demonstrated versatility of this mutant protein as a powerful tool for sialoglycomics.

Lens culinaris lectin (LCA) is a useful probe for the detection in serum of a core-fucosylated alpha-fetoprotein, called AFP-L3 fraction, which is a well-known marker for the diagnosis and prognosis of hepatocellular carcinoma. Here we performed a systematic quantitative interaction analysis of LCA and its close homolog, Pisum sativum lectin (PSA), by frontal affinity chromatography with 143 pyridylaminated (PA) glycans including a series of core-fucosylated glycans. Both lectins showed binding affinity to core-fucosylated, mono- and bi-antennary N-glycans, but not to their tri- and tetra-antennary forms, indicating that the addition of the GlcNAc residue at the N-acetylglucosaminyltransferase IV position abrogates the binding affinity. However, their specificities are distinguishable: while LCA showed the highest affinity to the core-fucosylated, agalactosylated, bi-antennary N-glycan (K(a)=1.1 x 10(5) M(-1)), PSA showed the highest affinity to the core-fucosylated, trimannosyl structure (K(a)=1.2 x 10(5) M(-1)). Glycan-binding specificities of LCA and PSA were also analyzed by glycoconjugate microarray compared to other core-fucose-binding lectins from Aspergillus oryzae (AOL) and Aleuria auratia (AAL). LCA and PSA bound specifically to core fucose, whereas AOL and AAL exhibited broad specificity to fucosylated glycans. These results explain why LCA is appropriate as a specific probe for AFP-L3, which mainly contains a core-fucosylated, biantennary N-glycan, but not its highly branched forms.

Small-sized isolectins (9 KDa) from Hypnea japonica belong to a new lectin family. Here, we describe the carbohydrate-binding properties of the three isolectins (hypninA1, A2, and A3) and the amino acid sequence of hypninA3 (P85888). In frontal affinity chromatography with about 100 pyridylaminated oligosaccharides, the isolectins, which had no affinity for monosaccharides, commonly bound only core (alpha1-6) fucosylated N-glycans, and did not the other oligosaccharides examined, including (alpha1-2), (alpha1-3), and (alpha1-4) fucosylated glycans. The specific binding of hypninA3 with the fucosylated N-glycans (Ka; 0.52-7.58 x 10(6) M(-1)) was confirmed by surface plasmon resonance analyses on an immobilized glycoprotein with and without core (alpha1-6) fucose. Such specificity of hypninA is clearly distinct from those of other known fucose-binding lectins, making it a valuable tool for cancer diagnosis and quality control of medicinal antibodies. HypninA3 is a polypeptide composed of 90 amino acids containing four half-cystines.

The amino acid sequence of mannose-binding lectin, named DB1, from the yam (Dioscorea batatas, synonym Dioscorea polystachya) tubers was determined. The lectin was composed of two isoforms DB1(Cys86) and DB1(Leu86) consisting of 108 amino acid residues with 90% sequence homology between them. DB1 showed a high sequence similarity to snowdrop (Galanthus nivalis) bulb lectin, GNA; especially, the carbohydrate-binding sites of GNA were highly conserved in DB1. DB1 interacted with D-mannose residues of oligosaccharides, and the oligosaccharides carrying two mannose-alpha-1,3-D-mannose units showed high binding affinity. DB1 was examined for insecticidal activity against Helicoverpa armigera (Lepidoptera: Noctuidae) larvae at different stages of development. The rate of adults successfully emerging from pupae fed on DB1 was 33%, when incorporated into an artificial diet at a level of 0.01% (w/w). Although DB1 had no or marginal inhibitory effects on gut proteolytic and glycolic enzymes, the lectin strongly bound to larval brush border and peritrophic membrane detected by immunostaining. The results show that DB1 may fulfill a defense role against insect pests.

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of alpha1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of alpha1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K(d)) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for alpha1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K(a)) for alpha1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were alpha1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of alpha1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

To obtain lectins without tedious purification steps, we developed a convenient method for a one-step purification of lectins using sugar-immobilized gold nano-particles (SGNPs). Proteins in crude extracts from plant materials were precipitated with 60% ammonium sulphate, and the precipitate was re-dissolved in a small volume of phosphate buffer. The resultant solution was then mixed with appropriate SGNPs under an optimized condition. After incubating overnight at 4 degrees C, lectins in the mixture formed aggregate with SGNPs, which was visually detected and easily sedimented by centrifugation. The aggregate was dissolved by adding inhibitory sugars, which were identical to the non-reducing sugar moieties on the SGNPs. According to SDS-PAGE and MS of thus obtained proteins, it was found that SGNPs isolated lectins with a high purity. For example, a protein isolated from banana using Glcalpha-GNP (alpha-glucose-immobilized gold nano-particle) was identified as banana lectin by trypsin-digested peptide-MS finger printing method.

The Jacalin-related lectin (JRL) family comprises galactose-binding-type (gJRLs) and mannose-binding-type (mJRLs) lectins. Although the documented occurrence of gJRLs is confined to the family Moraceae, mJRLs are widespread in the plant kingdom. A detailed comparison of sugar-binding specificity was made by frontal affinity chromatography to corroborate the structure-function relationships of the extended mJRL subfamily. Eight mJRLs covering a broad taxonomic range were used: Artocarpin from Artocarpus integrifolia (jackfruit, Moraceae), BanLec from Musa acuminata (banana, Musaceae), Calsepa from Calystegia sepium (hedge bindweed, Convolvulaceae), CCA from Castanea crenata (Japanese chestnut, Fagaceae), Conarva from Convolvulus arvensis (bindweed, Convolvulaceae), CRLL from Cycas revoluta (King Sago palm tree, Cycadaceae), Heltuba from Helianthus tuberosus (Jerusalem artichoke, Asteraceae) and MornigaM from Morus nigra (black mulberry, Moraceae). The result using 103 pyridylaminated glycans clearly divided the mJRLs into two major groups, each of which was further divided into two subgroups based on the preference for high-mannose-type N-glycans. This criterion also applied to the binding preference for complex-type N-glycans. Notably, the result of cluster analysis of the amino acid sequences clearly corresponded to the above specificity classification. Thus, marked correlation between the sugar-binding specificity of mJRLs and their phylogeny should shed light on the functional significance of JRLs.

Binding specificities of mouse macrophage galactose-type C-type lectin 1 (MGL1/CD301a) and 2 (MGL2/CD301b) toward various oligosaccharides were compared by frontal affinity chromatography. MGL1 preferentially bound oligosaccharides containing Lewis(X) (Le(X)) trisaccharides among 111 oligosaccharides tested, whereas MGL2 preferentially bound globoside Gb4. The important amino acids for the preferential bindings were investigated by pair-wise site-directed mutagenesis at positions 61, 89, 97, 100, 110-113, 115, 124, and 125 in the soluble recombinant carbohydrate recognition domains (CRD) prepared in Escherichia coli and purified with galactose-Sepharose. Mutations of Val, Ala, Thr, and Phe at positions 61, 89, 111 and 125 on MGL1 CRD caused reductions in Le(X) binding. Mutations of MGL2 CRD at Leu, Arg, Arg, and Tyr at positions 61, 89, 115 and 125 were implicated in the preference for beta-GalNAc. Le(X) binding was observed with MGL2 mutants of Arg89Ala and Arg89Ala/Ser111Thr. MGL1 mutants of Ala89Arg and Ala89Arg/Pro115Arg showed beta-GalNAc bindings. Molecular modeling illustrated potential direct molecular interactions of Leu61, Arg89, and His109 in MGL2 CRD with GalNAc.

Galectins are a family of beta-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small beta-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.

A rhamnose-binding lectin, named SFL, was isolated from the eggs of ayu (sweet fish, Plecoglossus altivelis) by affinity and ion-exchange chromatographies. SFL revealed 287 amino acid residues with 3 tandemly repeated domains, and contained 8 half-Cys residues in each domain. The lectin was shown to have a highly specific binding affinity to globotriaosylceramide (Gb3) by frontal affinity chromatography using 100 oligosaccharides. SFL was localized in several tissues and serum of both male and female ayu, such as gill, liver, ovary, testis, intestine, stomach, brain, kidney and serum. The lectin agglutinated the spores of the microsporidian Glugea plecoglossi, which is a pathogen of ayu. Although SFL bound to glycoproteins and glycolipids of G. plecoglossi spores, Gb3 could not be detected in either of them. The results suggest that SFL could interact with various glycoconjugates of pathogens to play a role in the adhesion of microorganisms invading in the body.

Ricinus communis agglutinin I (RCA120) is considered a versatile tool for the detection of galactose-containing oligosaccharides. However, possible contamination by the highly toxic isolectin 'ricin' has become a critical issue for RCA120's continued use. From a practical viewpoint, it is necessary to find an effective substitute for RCA120. For this purpose, we examined by means of frontal affinity chromatography over 100 lectins which have similar sugar-binding specificities to that of RCA120. It was found that Erythrina cristagalli lectin (ECL) showed the closest similarity to RCA120. Both lectins prefer Gal beta1-4GlcNAc (type II) to Gal beta1-3GlcNAc (type I) structures, with increased affinity for highly branched N-acetyllactosamine-containing N-glycans. Their binding strength significantly decreased following modification of the 3-OH, 4-OH and 6-OH of the galactose moiety of the disaccharide, as well as the 3-OH of its N-acetylglucosamine residue. Several differences were also observed in the affinity of the two lectins for various other ligands, as well as effects of bisecting GlcNAc and terminal sialylation. Although six other Erythrina-derived lectins have been reported with different amino acid sequences, all showed quite similar profiles to that of ECL, and thus, to RCA120. Erythrina lectins can therefore serve as effective substitutes for RCA120, taking the above differences into consideration.

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

Previous studies indicated that sclerotes of the phytopathogenic Ascomycete Sclerotinia sclerotiorum contain a lectin that based on its molecular structure, specificity and N-terminal amino acid sequence could not be classified yet into any lectin family. Using a combination of molecular cloning, frontal affinity chromatography and molecular modelling the identity of the S. sclerotiorum agglutinin (SSA) was analyzed. Molecular cloning demonstrated that SSA shares no sequence similarity with any known fungal lectin or protein. The lectin is synthesized as a 153 amino acid polypeptide without signal peptide and undergoes apart from the removal of the N-terminal methionine no further processing. Frontal affinity chromatography revealed that the binding site of SSA primarily accommodates a non-reducing terminal GalNAc with a preference for the alpha- over the beta-anomer. SSA also strongly interacts with both glycolipid type glycans with terminal non-reducing Gal or GalNAc and galactosylated N-glycans. SSA shares a residual sequence similarity with part of the non-toxin haemagglutinin HA33/A from Clostridium botulinum. Molecular modeling using the three-dimensional structure of HA33/A as a template indicated that SSA can fold into a similar beta-trefoil domain. Though these results should be interpreted with care it is tempting to speculate that the Sclerotiniaceae lectins thus appear to be structurally related to the ricin-B superfamily. All evidence suggests that SSA represents a novel family of fungal lectins with a unique sequence and sugar-binding properties. Taking into account that orthologues of SSA are fairly common within the family Sclerotiniaceae but could not be identified in any other fungal species one can reasonably conclude that SSA-type lectins are confined to a small taxonomic group of the Ascomycota.

The argasid tick Ornithodoros moubata is a vector of various viral and borrelian diseases in animals and humans. We report here molecular characterization and oligosaccharide-binding properties of a novel galectin (OmGalec) from this tick. OmGalec consisted of 333 amino acids with a predicted molecular weight of 37.4 kDa. Its amino acid sequence did not contain a signal peptide or transmembrane domain. It possessed tandem-repeated carbohydrate recognition domains, in which the typical motifs important for carbohydrate affinity were conserved. OmGalec was expressed both transcriptionally and translationally at all stages of the tick life cycle and in multiple organs and was abundant in hemocytes, midguts, and reproductive organs, which are of importance in immunity, interaction with pathogens, and development, respectively, suggesting that OmGalec is a multifunctional molecule. The oligosaccharide affinity profile analyzed by applying an automated frontal affinity chromatography system revealed that rOmGalec showed a general feature of the galectin family, i.e. significant affinity for lactosamine-type disaccharides, Galbeta1-3(4)Glc(NAc), via recognition of 4-OH and 6-OH of galactose and 3 (4)-OH of Glc(NAc). Its preference for type I saccharides and alpha1-3GalNAc-containing oligosaccharides might provide clues for identifying its ligands and its potential multiple functions. Our results may contribute to the elucidation of galectin functions in the development and immunity of arthropods and/or vector and pathogen interaction and provide valuable information for the development of novel tick control strategies.

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.

Frontal affinity chromatography using fluorescence detection (FAC-FD) is a versatile technique for the precise determination of dissociation constants (Kd) between glycan-binding proteins (lectins) and fluorescent-labeled glycans. A series of glycan-containing solutions is applied to a lectin-immobilized column, and the elution profile of each glycan (termed the 'elution front', V) is compared with that (V0) for an appropriate control. Here we describe our standard protocol using an automated FAC system (FAC-1), consisting of two isocratic pumps, an autosampler, a column oven and two miniature columns connected to a fluorescence detector. Analysis time for 100 sugar-protein interactions is approximately 10 h, using as little as 2.5 pmol of pyridylaminated (PA) oligosaccharide per analysis. Using FAC-FD, we have so far obtained quantitative interaction data of >100 lectins for >100 PA oligosaccharides.

Structural glycomics plays a fundamental role in glycoscience and glycotechnology. In this paper, a novel strategy for the structural characterization of glycans is described, in which MS2 analysis involving a LIFT-TOF/TOF procedure is combined with frontal affinity chromatography (FAC). As model compounds, 20 neutral pyridylaminated (PA) oligosaccharides were chosen, which included four groups of structural isomers differing in sequence, linkage, position, or branching features. By depicting significant diagnostic ions on MS2, most of the analyzed oligosaccharides were successfully differentiated, while two pairs of linkage isomers, i.e., LNT/LNnT, and LNH/LNnH were not. For subsequent analysis by FAC, 14 lectins showing significant affinity to either LNT (type 1) or LNnT (type 2) were screened, and a galectin from the marine sponge Geodia cydonium (GC1) and a plant seed lectin from Ricinus communis (RCA-I) were used for determination of type 1 and 2 chains, respectively. With these specific probes, both of the isomeric pairs were unambiguously differentiated. Furthermore, a pair of triantennary, asparagine-linked oligosaccharide isomers could also be successfully differentiated. Thus, the combination of MS2 and FAC is a practical alternative for the structural characterization of complex glycans.

Agaricus bisporus agglutinin (ABA) is known as a useful lectin to detect T-antigen (Core1) disaccharide (Galbeta1-3GalNAcalpha) and related O-linked glycans. However, a recent X-ray crystallographic study revealed the presence of another intrinsic sugar-binding site, i.e., for GlcNAc. To confirm this possibility, detailed analysis was performed using two advanced methods: lectin microarray and frontal affinity chromatography (FAC). In the lectin microarray, intense signals were observed on ABA spots for both N-glycanase-treated and O-glycanase/beta1-4galactosidase-treated Cy3-labeled asialofetuin. This indicates substantial affinity for both O-linked and agalactosylated (GlcNAc-exposed) N-linked glycans. A further approach by FAC using 20 pNP and 130 PA-oligosaccharides demonstrated that ABA bound to Core1 (K(d) = 3.4 x 10(-6) M) and Core2 (1.9 x 10(-5) M) but not to Core3 and Core6 O-linked glycans. It also showed substantial affinity to mono-, bi-, and tri-antennary agalactosylated complex-type N-linked glycans (K(d) > 1.8 x 10(-5) M). These results establish ABA as a lectin having dual sugar-binding sites with distinct specificity, i.e., for Gal-exposed O-linked glycans and GlcNAc-exposed N-linked glycans.

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.

Frontal affinity chromatography (FAC) is a quantitative method that enables sensitive and reproducible measurements of interactions between lectins and oligosaccharides. The method is suitable even for the measurement of low-affinity interactions and is based on a simple procedure and a clear principle. To achieve high-throughput and efficient analysis, an automated FAC system was developed. The system designated FAC-1 consists of two isocratic pumps, an autosampler, and a couple of miniature columns (bed volume, 31.4 microl) connected in parallel to either a fluorescence or an ultraviolet detector. By use of this parallel-column system, the time required for each analysis was reduced substantially. Under the established conditions, fewer than 10 hrs are required for 100 interaction analyses, consuming as little as 1 pmol pyridylaminated oligosaccharide for each analysis. This strategy for FAC should contribute to the construction of a lectin-oligosaccharide interaction database essential for future glycomics. Overall features and practical protocols for interaction analyses using FAC-1 are described.