Purification and characterization of a lectin,BPL-3, from the marine green alga <Emphasis Type="Italic">Bryopsis plumosa</Emphasis>

A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails.     

Alternative ultrastructural localization of Dolichos biflorus lectin binding sites in proton secreting parietal cells of mice

Summary High amount of N-acetyl-d-galactosamine specific lectin binding sites were detected on the canalicular membranes of human parietal cells. Our present model investigations on mice showed that the intracellular distribution of the terminal N-acetyl-d-galactosamine containing glycoprotein highly depends on the actual functional state of the parietal cells. In the normal gastric mucosa 40%–60% of parictal cells react positively after staining with horseradish peroxidase or biotin labelled Dolichos biflorus lectin. Ultrastructurally lectin binding sites occur mainly on the basolateral membrane infoldings in fed animals, while they are present exclusively on the canalicular membranes of fasting mice, suggesting that the alternative appearance of lectin binding sites on the opposite membrane areas of parietal cells is tightly coupled to their main function, to H⁺ secretion.     

Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies

Summary Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, -methyl-d-mannosyl andN-acetyl-d-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars -methyl-d-mannoside andN-acetyl-d-glucosamine, respectively.     

Determination of the structure of sulfated tetra- and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. A fast atom bombardment-mass spectrometric and1H-NMR spectroscopic study

Alkaline borohydride reductive cleavage of hen ovomucin resulted in the release of a series of neutral and acidic oligosaccharide-alditols.¹H-NMR spectroscopy in combination with fast ion bombardment-mass spectrometry in negative ion mode were used for investigation of the structures of three oligosaccharide-alditols. The following structures were established: Abbreviations NeuAc N-acetyl-d-neuraminic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Gal-NAc-ol N-acetyl-d-galactosaminitol - NMR nuclear magnetic resonance - FAB-MS fast atom bombardmentmass spectrometry     

One-pot enzymatic production of 2-acetamido-2-deoxy-d-galactose (GalNAc) from 2-acetamido-2-deoxy-d-glucose (GlcNAc)

2-Acetamido-2-deoxy-d-galactose (GalNAc) is a common monosaccharide found in biologically functional sugar chains, but its availability is often limited due to the lack of abundant natural sources. In order to produce GalNAc from abundantly available sugars, 2-acetamido-2-deoxy-d-glucose (GlcNAc) was converted to GalNAc by a one-pot reaction using three enzymes involved in the galacto-N-biose/lacto-N-biose I pathway of bifidobacteria. Starting the reaction with 600 mM GlcNAc, 170 mM GalNAc was produced at equilibrium in the presence of catalytic amounts of ATP and UDP-Glc under optimized conditions. GalNAc was separated from GlcNAc using water-eluting cation-exchange chromatography with a commonly available cation-exchange resin.     

Differential distribution of lectin-binding glycoconjugates in the secretory granules of hamster oviductal ampulla during the estrous cycle: a quantitative cytochemical analysis

 High resolution lectin-gold cytochemistry was used to quantitatively analyze the distribution of glycoconjugates in the hamster oviductal ampulla during the five stages of the estrous cycle. Lectins binding to N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates in the secretory granules of ampullary epithelial secretory cells showed staining of equal intensity throughout the five different stages of the estrous cycle. In contrast, the labeling intensity of glycoconjugates which contain N-acetylglucosamine as terminal sugar residues reached its maximum around the time of ovulation, i.e., at proestrus. Glycoconjugates which carry fucose and mannose as terminal sugar residues appeared to be totally absent from the secretory granules of the oviductal ampulla during the estrous cycle. Together, electron microscopic observations combined with quantitative results indicate that N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates may be secreted into the ampullary lumen irrespective of the stage of the estrous cycle, whereas the secretion of certain N-acetylglucosamine-associated glycoconjugates is stage specific and reaches its peak at the time of ovulation. These findings suggest that, at the time of ovulation, the ampullary epithelium changes its secretory activity and contributes its secretory products to the zona pellucida of oocytes freshly released from the ovary. Accepted: 10 August 1998     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

Lectinhistochemical demonstration of galactose- and N-acetyl-d-galactosamine residues in cells of the rat anterior pituitary

Summary Lectins are a useful tool for identification of differently glycosylated hypophyseal hormones, prohormones and glycoconjugates without hormone function. -d-galactose and -N-acetyl-d-galactosamine (GalNAc) containing glycoconjugates were identified by light microscopy with biotinylated lectins in immunocytochemically localized cells of the anterior pituitary of the rat. Galactose, histochemically detectable by the peanut lectin (PNA), was found at penultimate position of the carbohydrate chain after removal of sialic acid. Galactose containing cells correspond to gonadotrophs and thyrotrophs located mainly in medioanterior regions of the pituitary. The lectins from the soybean (SBA) and horse gram (DBA) both specific for GalNAc residues, are bound to round and also polygonal cells corresponding again to gonadotrophs and thyrotrophs.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Detection and mapping of endogenous receptors for carrier-immobilized constituents of glycoconjugates (lectins) by labelled (neo)glycoproteins and by affinity chromatography in human adult mesencephalon,pons, medulla oblongata and cerebellum

Summary Different carrier-immobilized carbohydrate moieties were employed as tools to detect respective binding sites glycohistochemically and glycobiochemically. Besides ascertaining their presence the pattern of endogenous sugar receptors (lectins) in different regions of the human central nervous system was mapped to reveal any non-uniform expression. A strong and specific staining with biotinylated neoglycoproteins, exposing different sugar moieties as ligands, indicated the presence of sugar receptors in the nuclei, neuronal pathways and accessory structures such as ependyma cells, plexus chorioideus, intra- and extracerebral vessels and leptomeninges localized in the mesencephalon, in the pons, in the medulla oblongata and in the cerebellum. Significant differences were seen for various neuroanatomical regions like nerve cells in the basal and central regions of the nuclei pontis in the glycohistochemically detected level of expression of endogenous sugar receptors (lectins). The used approach with carbohydrate constituents of cellular glycoconjugates as ligands in search of specific receptors complemented studies on the localization of glycoconjugates with sugar-specific tools like plant lectins. Exemplary glycobiochemical investigations on the medulla oblongata and cerebellum were performed to investigate the molecular nature of sugar receptors detected glycohistochemically. Despite notable overall similarities, carbohydrate-binding proteins of differing molecular weight can be isolated from these two regions of the central nervous system, namely in the case of receptors with specificity to -galactoside termini, to N-acetyl-d-galactosamine and N-acetyl-d-glucosamine and to d-xylose. These combined glycohistochemical and glycobiochemical results serve as a guideline for exploring the physiological relevance of the detected regional differences.     

Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy--d-glucopyranoside and 2-acetamido-2-deoxy--d-xylopyranoside, two allyl 2-acetamido-2-deoxy--d-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy--d-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific forN-acetylglucosamine. We found that: 1) The inhibitory potency ofN-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3-or 4-OH group ofN-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.Abbreviations XyLNAc N-acetyl-d-xylosamine - BSA bovine serum albumin - NeuAc N-acetylneuraminic acid - GlcNAc34-BSA amidino-type neoglycoprotein [6] containing on the average 34N-acetylglucosaminyl residues per BSA molecule     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

An efficient method for N-acetyl-d-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system

N-Acetyl-d-neuraminic acid (Neu5Ac) is a precursor for producing many pharmaceutical drugs such as zanamivir which have been used in clinical trials to treat and prevent the infection with influenza virus, such as the avian influenza virus H5N1 and the current 2009 H1N1. Two recombinant Escherichia coli strains capable of expressing N-acetyl-d-glucosamine 2-epimerase and N-acetyl-d-neuraminic acid aldolase were constructed based on a highly efficient temperature-responsive expression system which is safe compared to chemical-induced systems and coupled in Neu5Ac production. Carbon sources were optimized for Neu5Ac production, and the concentration effects of carbon sources on the production were investigated. With 2,200 mM pyruvate as carbon source and substrate, 61.9 mM (19.1 g l⁻¹) Neu5Ac was produced from 200 mM N-acetyl-d-glucosamine (GlcNAc) in 36 h by the coupled cells. Our Neu5Ac biosynthetic process is favorable compared with natural product extraction, chemical synthesis, or even many other biocatalysis processes.     

Lectin histochemistry on mucous substances of the taste buds and adjacent epithelia of different vertebrates

Summary In the present study carbohydrate residues in taste buds (TBs) and adjacent epithelial formations of a teleostean fish, a frog and the rabbit were detected by means of lectin histochemistry. Biotinylated lectins fromPisum sativum (PSA),Arachis hypogaea (PNA),Dolichos biflorus (DBA),Triticum vulgaris (WGA and succinylated WGA),Glycine max (SBA) andUlex europaeus (UEA I) have been applied. The lectins were bound to an avidin-biotin-peroxidase-complex (ABC) and visualized by diaminobenzidine/ H₂O₂. Most intensive reactivity was observed at the taste dise cells of the frog with DBA, S-WGA and SBA. PNA did not bind to the TBs of any of the animals tested. As shown in SBA preparations, sialic acid is present in a nonacylated and an acylated form in the mucosa of the frog's tongue. The TBs of the fish possess all the sugars we looked for except for the disaccharided-galactose-(1–3)--d-N-acetyl-galactosamine (Gal/GalNAc) and sialic acid. The TBs of the rabbit contain GalNAc, as detected with DBA, but not with SBA; and fucose (Fuc), mannose (Man) andN-acetyl-glucosamine (GlcNAc). As revealed by preincubation of the tissue sections with neuraminidase in TB cells of the rabbit, sialic acid masks Gal/GalNAc and GalNAc. These lectin-binding characteristics show that in the TBs of some selected representatives which belong to different vertebrate classes exist different mucous substances. These substances possess different binding characteristics to specific sugars, and this is possibly of particular interest to chemoreception phenomena.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.Parts of this investigation were presented at the 81. Versammlung der Anatomischen Gesellschaft, March 9–14, 1986 at Lübeck (Witt and Reutter 1986), and at the VII th Congress of the Europaen Chemoreception Research Organization, September 22–26, 1986, at Davos (Reutter and Witt 1987).Supported by the Deutsche Forschungsgemeinschaft (Re 225/9-1)     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

Novel Ca2+‐independent carbohydrate recognition of the C‐type lectins,SPL‐1 and SPL‐2, from the bivalve Saxidomus purpuratus

Novel Ca²⁺‐independent C‐type lectins, SPL‐1 and SPL‐2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL‐2 composed of two B‐chains) or distinct (SPL‐1 composed of A‐ and B‐chains) polypeptide chains, and show affinity for N‐acetylglucosamine (GlcNAc)‐ and N‐acetylgalactosamine (GalNAc)‐containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C‐type lectin family. X‐ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate‐recognition domain (CRD) of the C‐type lectin family. Nevertheless, these lectins (especially SPL‐2) showed Ca²⁺‐independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL‐2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate‐recognition motifs among the C‐type CRD (the QPD [Gln‐Pro‐Asp] and EPN [Glu‐Pro‐Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate‐binding specificities of individual A‐ and B‐chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α‐glycosidic linkages with slightly different specificities.     

The interaction between wheat germ agglutinin and membrane incorporated glycophorin A. An optical binding study

A novel integrated optical technique is used to monitor the kinetics of incorporation of glycophorin A (GPA) from solution into a planar dimyristoylphosphatidylcholine-cholesterol bilayer membrane, and the subsequent binding of wheat germ agglutinin (WGA) to the membrane-incorporated GPA. The technique significantly improves the attainable accuracy of kinetic measurements. The number of bound molecules can be determined to a precision of ca ± 80 mol µm^–2. Our results show that GPA incorporates spontaneously into the bilayer. Binding of WGA to GPA is optimal in the presence of human serum albumin, and can be reversed byN-acetyl-d-glucosamine. The kinetics of the binding are consistent with the presence of two classes of kinetically distinguishable binding sites with association rates of 2.0×10⁴ and 9.6×10² M^–1 s^–1, and dissociation rates of 2.7×10^–3 s^–1 and <10^–5 s^–1, respectively. A stoichiometry of 4 WGA monomers per GPA monomer was determined as characteristic of the overall binding interaction.Abbreviations DMPC dimyristoylphosphatidylcholine - GlcNAc N-acetyl-d-glucosamine - GPA glycophorin A - HSA human serum albumin - NeuNAc N-acetyl-d-neuraminic acid - TE transverse electric - TM transverse magnetic - WGA wheat germ agglutinin