Heterogeneity in copper and glycan content of ceruloplasmin in human serum differs in health and disease

Crossed immunoelectrophoresis of human serum revealed two heterogeneity types of ceruloplasmin with different electrophoretic migration. The two types both consisted of peptides with Mr 150,000, 100,000 and 45,000, which were interpreted as native ceruloplasmin and two hydrolytic fragments. The two types were different in copper content, and one type could reversibly be changed into the other. The glycan microheterogeneity of ceruloplasmin was analyzed by crossed affinoimmunoelectrophoresis with free Lens culinaris agglutinin (LCA) and wheat germ agglutinin (WGA). A third of the ceruloplasmin molecules, both high and low copper type, bound to LCA and two thirds to WGA. The heterogeneity and the microheterogeneity of ceruloplasmin in two groups of patient sera were compared to sera from healthy individuals. The ceruloplasmin type with respect to copper content was a much better factor than either glycan microheterogeneity or total serum concentration in discriminating between the three groups.     

A simplified method for the preparation of WGA-Sepharose 4B and its use in the purification of MN blood group antigens

A simplified method for the preparation of wheat germ agglutinin(WGA)-Sepharose 4B by coupling highly purified WGA, prepared by improved affinity chromatography, with BrCN activated Sepharose 4B in a solution of high carbonate buffer is described. The amount of WGA linked to Sepharose 4B was 82.40% (3.07 mg WGA per ml Sepharose 4B). MN blood group antigens of human erythrocyte membranes purified with WGA-Sepharose 4B affinity chromatography showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The yield of the antigens from 400 mL fresh blood was 32-40 mg. The WGA-Sepharose 4B column could be used several times without loss of activity.     

Macroporous chitin affinity membranes for wheat germ agglutinin purification from wheat germ

Macroporous chitin membranes of controlled porosity and pore sizes have been prepared. They have good mechanical properties and allow high flow rates of protein solutions at low pressure drops. Because of the numerous N-acetyl-D-glucosamine (GlcNAc) moieties they contain, the chitin membranes can be used for the separation of some valuable proteins both as affinity ligands and support matrix, without further modification. Due to their high porosity and high adsorption surface area, the chitin membranes provide a larger number of accessible binding sites for the wheat germ agglutinin than the chitin beads do. The adsorption capacity for wheat germ agglutinin (180 mg/g chitin membrane) is about 20 times larger than that of chitin beads. Because of the numerous binding sites, multiple-point bindings are involved in the protein adsorption. For this reason, a strong eluant, namely a 1 M acetic acid aqueous solution, had to be used to efficiently recover the wheat germ agglutinin from the membrane. The wheat germ agglutinin was extracted from wheat germ with 0.05 M HCl, precipitated with ammonium sulfate, dialyzed against 0.01 M Tris–HCl buffer (pH 8.5), and purified on the chitin membrane. A high purity (>99%) wheat germ agglutinin with high yield (∼50 mg/100 g wheat germ) was obtained.     

Synthesis and lectin binding ability of glycosamino acid-calixarenes exposing GlcNAc clusters

Novel calix[4 or 8]arene-based glycoconjugates exposing terminal N-acetyl-D-glucosamine clusters have been synthesized using amino acid-calixarenes as building blocks. The obtained glycosamino acid-calixarenes 9b-14b have lectin-binding ability and amplified inhibitory effects on erythrocyte agglutination induced by wheat germ (Triticum vulgaris) agglutinin (WGA). The inhibitory ability is dependent on the presence of the spacer and on the shape and rigidity of the calixarene skeleton.     

Efficient wheat germ agglutinin purification with a chitosan‐based affinity chromatographic matrix

An efficient affinity chromatographic matrix based on chitosan for wheat germ agglutinin (WGA) purification was developed. The matrices assayed consisted of chitosan mini‐spheres cross‐linked with epichlorhydrin 45, 250 or 500 mM. The maximum adsorption capacity of pure WGA – calculated from the corresponding isotherms – was between 43.2 and 48.9 mg/g at pH 5.0 and between 16.6 and 27.6 mg/g at pH 8.5. However, the adsorption of agglutinin from wheat germ extract was higher at pH 8.5. In addition, 0.5 g of mini‐spheres cross‐linked with epichlorhydrin 250 mM adsorbed 94.5% of the WGA present in 5 mL of the concentrated extract. Acetic acid was able to elute 100% of the adsorbed WGA. The purity of the WGA obtained was greater than 95% and the purification factor was 56.8. The matrix was able to maintain an efficient performance of the purification process for three consecutive cycles. A new method to monitor the purification process by RP‐HPLC was developed.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells.

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10(7) binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37 degrees C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

Recognition between a divalent sialyl molecule and wheat germ agglutinin

Structural divalency between a designed N-acetyl-neuraminic acid (NeuAc)-containing molecule and lectin wheat germ agglutinin (WGA) is investigated. The sialyl molecule was designed based on the NeuAc-WGA complex in the Protein Data Bank and featured polyethylene glycol linkers connecting to an aromatic scaffold. Our results elucidate the divalent recognition association constant between WGA and the multivalent-NeuAc molecules to be 10⁷ by surface plasmon resonance.     

Self-assembly of amphiphilic glycoconjugates into lectin-adhesive nanoparticles

This work describes the synthesis and self-assembly of carbohydrate-clicked rod-coil amphiphilic systems. Copper-catalyzed Huisgen cycloaddition was efficiently employed to functionalize the hydrophilic extremity of PEG-b-tetra(p-phenylene) conjugates by lactose and N-acetyl-glucosamine ligands. The resulting amphiphilic systems spontaneously self-assembled into nanoparticles when dissolved in aqueous media, as evidenced by dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The formation of highly monodisperse micelles having a mean diameter of 10 nm was observed for systems containing a PEG 900 core, and a decrease in the hydrophilic moiety (PEG 600) led to the formation of vesicles with a broader size distribution. The presence of carbohydrate residues on the surfaces of the micelles and their ability to establish specific interactions with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were further highlighted by light-scattering measurements, thus confirming the attractive applications of such sugar micelles in biosensor devices.     

Purification and characterization of a major glycoprotein in rat liver lysosomal membrane

A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.     

Interactions of enzymes and a lectin with a chitin-based graft copolymer having polysarcosine side chains

The molecular-recognition abilities of a water-soluble chitin derivative, chitin-graft-polysarcosine (2) were investigated using chitinase, lysozyme, and wheat germ agglutinin (WGA). The enzymatic degradabilities of 2 were evaluated using chitinase and lysozyme. The molecular weight of those compounds of 2 with a higher affinity toward water decreased rapidly, as compared with partially deacetylated chitin (1). The 1H NMR spectrum of the low-molecular-weight fraction, yielded after lysozymic hydrolysis, indicated that saccharide residues in the chitinous backbone were specifically recognized by the lysozyme, then beta-glycosidic linkages in the backbone were selectively hydrolyzed. Furthermore, the molecular-recognition ability of the chitinous backbone of graft copolymer 2 toward the lectin WGA was elucidated by the enzyme-linked lectin-binding assay (ELLA). It was revealed that the graft copolymer with a lower degree of substitution (DS) value efficiently interacted with WGA. Interestingly, a graft copolymer having longer polysarcosine side chains showed higher recognition ability toward WGA than that having short side chains.     

Voltammetric evaluation of the binding between wheat germ agglutinin and thionine/glucose-modified magnetic microbeads.

The binding between glucose residues and wheat germ agglutinin (WGA) on thionine/glucose-modified magnetic microbeads was evaluated using voltammetry. Thionine is an electroactive compound and has two amino groups. Thionine was immobilized to magnetic beads via cross-linking of the amino groups on the beads with an amino group on thionine. Glucose was bound to the other amino group of thionine via the formation of a Schiff base. The beads were only several micrometers in size the same size, as cells. WGA-binding to glucose on the bead surface blankets the thionine moiety. Thus, WGA-binding could be detected as a decrease in peak current of the thionine moiety.     

Voltammetric sensing of sugar by an electrode covered with wheat germ agglutinin/chitin film

The binding between wheat germ agglutinin (WGA) and N-acetylglucosamine at the electrode covered with chitin film was investigated with voltammetry. Chitin, β-1,4-poly-N-acetylglucosamine, is one of the biolpolymers which have a high biocompatibility. WGA is immobilized to the surface of chitin film by the affinity of WGA to N-acetylglucosamine residue of chitin. To investigate the binding event of WGA on the chitin modified electrode, N-acetylglucosamine labeled with an electroactive compound was prepared. The binding causes the changes in the electrode response of labeled sugar. The peak current of labeled sugar decreased due to the specific binding with WGA on the chitin film modified at the electrode. N-Acetylglucosamine was successfully determined by using the competitive reaction with labeled sugar to WGA on the chitin film electrode.     

Lectin recognition of liposomes containing neoglycolipids. Influence of their lipidic anchor and spacer length

Eleven neoglycolipids were synthesized with a membrane anchor (cholesterol or Guerbet alcohols) attached, via a hydrophilic spacer, to a potential lectin-recognized sugar moiety (N-acetylglucosamine: GlcNAc). Neoglycolipids G24-0, G28-0 and G32-0, with a C₂₄, C₂₈ and C₃₂ Guerbet alcohol residue, had no spacer, while neoglycolipids G24-3, G28-3, G32-3 contained a triethoxy spacer. Cholesteryl neoglycolipids chol-1 to chol-4 and chol-7 contained one to four and seven ethoxy units. All the cholesteryl neoglycolipids were incorporated into liposomes, while, for the Guerbet derivatives, the presence (or not) of a spacer and the length of their alkyl chains played an important role for obtaining mixed liposomes. The abilities of liposomes to be recognized by wheat germ agglutinin (WGA) were measured as an increase of the absorbance at 450 nm. Significant WGA-induced aggregations were obtained with liposomes containing neoglycolipids chol-3, chol-4, chol-7, G28-3 and G32-3. As neoglycolipid G24-3 was not recognized, the accessibility of its GlcNAc moiety for WGA depended not only on the spacer length but on the nature of the anchor. The WGA-induced aggregations increased with increasing lectin concentrations until a maximum value, which depended on the nature of the neoglycolipid. The highest aggregation obtained with G28-3 and G32-3 occurred for a WGA–neoglycolipid mole ratio superior to the ratio observed with chol-3, chol-4 and chol-7. A preincubation of WGA with free GlcNAc did not inhibit the aggregation between WGA and mixed liposomes, demonstrating a greater affinity of WGA for the neoglycolipid GlcNAc than for free GlcNAc.     

Analytical high-performance affinity chromatography of ovalbumin-derived glycopeptides on columns of concanavalin A-and wheat germ agglutinin-immobilized gels

Concanavalin A (Con A) or wheat germ agglutinin (WGA) was immobilized on a silica-based support, and the chromatographic behaviours of a series of dansylated ovalbumin-derived glycopeptides on small columns of the resultant gels were compared. These columns had high contents of lectins, and allowed differentiation of these glycopeptides. This method was rapid and reproducible, and enabled sensitive detection of these fluorescent glycopeptides. The structural requirement of these glycopeptides to manifest affinity to the immobilized lectins is also discussed, based on binding constants obtained from their retention times.     

Preparation of a high-performance multi-lectin affinity chromatography (HP-M-LAC) adsorbent for the analysis of human plasma glycoproteins

We report on the preparation of an improved multi-lectin affinity support for HPLC separations. We combined the selectivity of three different lectins: concanavalin A (ConA), wheat germ agglutinin (WGA), and jacalin (JAC). Each lectin was first covalently immobilized onto a polymeric matrix and then the three lectin media were combined in equal ratios. The beads were packed into a column to produce a mixed-bed multi-lectin HPLC column (high-performance multi-lectin affinity chromatography (HP-M-LAC)) for fast chromatographic affinity separations. The support was characterized with respect to kinetics of immobilization, ligand density, and binding capacity for human plasma glycoproteins. A high lectin density (15 mg/mL of beads) was found to be optimal for the binding of glycoproteins from human plasma. A single clinical sample can be fractionated in less than 10 min per run, making this a useful sample preparation tool for proteomics/glycoproteomics studies associated with disease abnormalities.     

Voltammetric evaluation for the binding of wheat germ agglutinin to glucosamine-modified magnetic microbead

Binding of wheat germ agglutinin (WGA) on glucosamine-modified magnetic microbeads was investigated with voltammetry. A magnetic bead was considered as a cell, and the beads with amino groups were modified with the sugar by using a cross-linking reagent. To evaluate the binding, glucose labeled with an electroactive daunomycin was prepared as a probe. After WGA and the beads were mixed in 0.1 M phosphate buffer (pH 7.0), the labeled glucose was added to the solution. The binding was monitored from the changes in the electrode response of labeled glucose because the labeled glucose was held to the binding site of WGA for the sugar. In contrast, other lectin not having the binding site to glucosamine or glucose was incubated with the glucosamine-modified beads. As a result, the change of peak current was not observed. Therefore, it is clear that the binding of WGA to glucosamine moiety on the bead surface selectively takes place. This method would be powerful for evaluation of interaction between protein and sugar chain existing at cell surface.     

Wheat germ agglutinin modified liposomes for the photodynamic inactivation of bacteria

Photodynamic inactivation (PDI) of bacteria is a promising approach for combating the increasing emergence of antibiotic resistance in pathogenic bacteria. To further improve the PDI efficiency on bacteria, a bacteria-targeting liposomal formulation was investigated. A generation II photosensitizer (temoporfin) was incorporated into liposomes, followed by conjugation with a specific lectin (wheat germ agglutinin, WGA) on the liposomal surface. WGA was successfully coupled to temoporfin-loaded liposomes using an activated phospholipid containing N-hydroxylsuccinimide residue. Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were selected to evaluate the WGA modified liposomes in terms of bacteria targeted delivery and in vitro PDI test. Fluorescence microscopy revealed that temoporfin was delivered to both kinds of bacteria, while flow cytometry demonstrated that WGA- modified liposomes delivered more temoporfin to bacteria compared to nonmodified liposomes. Consequently, the WGA- modified liposomes eradicated all MRSA and significantly enhanced the PDI of P. aeruginosa. In conclusion, the WGA- modified liposomes are a promising formulation for bacteria targeted delivery of temoporfin and for improving the PDI efficiency of temoporfin on both Gram-positive and Gram-negative bacterial cells.     

Quantification, 2DE analysis and identification of enriched glycosylated proteins from mouse muscles: Difficulties and alternatives

One of the problems with 2DE is that proteins present in low amounts in a sample are usually not detected, since their signals are masked by the predominant proteins. The elimination of these abundant proteins is not a guaranteed solution to achieve the desired results. The main objective of this study was the comparison of common and simple methodologies employed for 2DE analysis followed by MS identification, focusing on a pre‐purified sample using a wheat germ agglutinin (WGA) column. Adult male C57Black/Crj6 (C57BL/6) mice were chosen as the model animal in this study; the gastrocnemius muscles were collected and processed for the experiments. The initial fractionation with succinylated WGA was successful for the elimination of the most abundant proteins. Two quantification methods were employed for the purified samples, and bicinchoninic acid (BCA) was proven to be most reliable for the quantification of glycoproteins. The gel staining method, however, was found to be decisive for the detection of specific proteins, since their structures affect the interaction of the dye with the peptide backbone. The Coomassie Blue R‐250 dye very weakly stained the gel with the WGA purified sample. When the same gel was stained with silver nitrate, however, MS could positively assign 12 new spots. The structure of the referred proteins was not found to be prone to interaction with Coomassie blue.     

Interaction of wheat germ agglutinin with an N-acetylglucosamine-carrying telomer brush accumulated on a colloidal gold monolayer

A disulfide-carrying telomer with many pendent N-acetylglucosamine (GlcNAc) residues (Cys-PMHGlcNAc) was obtained by photo-polymerization of 1-(6'-methacryloylaminohexyl)-2-N-acetoamido-2-deoxy d-glucopyranoside) (MHGlcNAc) using a benzyl N,N-diethyldithiocarbamoyl (BDC) derivative that shows abilities of initiation, transfer, and termination (iniferter). The disulfide-carrying telomer was accumulated on a monolayer of colloidal Au on a glass substrate, and the interaction of wheat germ agglutinin (WGA) with GlcNAc residue at the polymer brush-solution interface was examined by using the localized surface plasmon resonance (LSPR) technique. For comparison, an amphiphile carrying many pendent GlcNAc residues was also prepared with MHGlcNAc and a lipophilic radical initiator and was incorporated in a phospholipid liposome to examine interaction of the GlcNAc residue with WGA on the liposome surface using turbidity measurements. Both the colloidal gold optical device and the liposome showed a concentration-dependent specific binding of WGA, and the GlcNAc-carrying liposome had a detection limit of 100 nM for WGA, whereas that of the colloidal gold device was 10nM. The sugar-carrying telomer-coated device examined here is not only useful as a simple biosensor chip but is also expected to expand our knowledge of bio-related phenomena at the liquid-telomer brush interfaces on a colloidal Au.     

Lectin affinity chromatography as a tool to differentiate endogenous and recombinant erythropoietins

This work exploits the combination of the lectin affinity chromatography (LAC) with an ultra-sensitive immunochromatographic assay to differentiate several types of erythropoietin (EPO). The chromatographic behaviours of different commercial types of recombinant human EPO (rhEPO), EPO analogues (Aranesp) and urine human EPO (uhEPO) from healthy individuals on eight lectin-Sepharose columns, have been worked out. Results show that when using wheat germ agglutinin (WGA)-Sepharose columns, a careful desorption regime starting with very low concentration (2mM) of the competitive sugar N-acetylglucosamine (GlcNAc) makes it possible to efficiently distinguish endogenous EPO from recombinant EPO and EPO analogues.