Differential staining of western blots of avian egg white glycoproteins using diverse lectins

Avian egg white glycoproteins which differ in structure and carbohydrate composition, vary in their interactions with diverse lectins. Generally, wheat germ agglutinin (WGA) and concanavalin A (Con A) are used for the identification and separation of those of the chicken. In the present study, interactions of a battery of lectins, including: the above two, several galactophilic lectins (from Aplysia gonad (AGL), Erythrina corallodendron (ECorL), peanut (PNA) and Pseudomonas aeruginosa (PA-IL)), and fucose-binding lectins (from Ulex europaeus (UEA-I), Ulva lactuca (ULL) and P. aeruginosa (PA-IlL), which also binds mannose) with chicken, quail and pigeon egg white glycoproteins, were examined using both hemagglutination inhibition and Western blot analyses. The chicken egg white glycoproteins interacted most strongly with WGA, followed by Con A > AGL = PA-IlL. The quail glycoprotein order of affinities was: Con A > WGA = AGL = PA-IlL, while that of the pigeon was: AGL > PA-IL > WGA > Con A = PA-IlL. The blocking of the other lectins by the egg whites were insignificant. The results demonstrated the selectivity and efficiency of the five most reactive lectins for differential tagging of avian egg white glycoproteins and unveiled the profound heterogeneity of the latter, as well as the possible potential lectin usage for improving purification and quality control of the desired glycoproteins.     

Chemiluminescent Detection of Carbohydrates in the Tumoral Breast Diseases

Nowadays, there is an increase of investigations into the fibroadenoma, mainly because some studies have shown that the occurrence of fibroadenoma is linked to an increased risk of developing breast carcinoma. Currently, the chemiluminescence biomarkers are applied for validation methods and screening. Here, a lectin chemiluminescence is proposed as new histochemistry method to identify carbohydrates in mammary tumoral tissues. The lectins concanavalin A (Con A) and peanut agglutinin (PNA) conjugated to acridinium ester were used to characterize the glycocode of breast tissues: normal, fibroadenoma, and invasive duct carcinoma (IDC). The lectin chemiluminescence expressed in relative light units (RLU) was higher in fibroadenoma and IDC than in normal tissue for both lectins tested. The relationship RLU emission versus tissue area described a linear and hyperbolic curve for IDC and fibroadenoma, respectively, using Con A whereas hyperbolic curves for both transformed tissues using PNA. RLU was abolished by inhibiting the interaction between tissues and lectins using their specific carbohydrates: methyl-α-d-mannoside (Con A) and galactose (PNA). The intrinsic fluorescence emission did not change with combination of the lectins (Con A/PNA) to the acridinium ester for hydrophobic residues. These results represent the lectin chemiluminescence as an alternative of histochemistry method for tumoral diagnosis in the breast.     

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.     

Fluorescence Turn‐On Sensing of Lectins with Mannose‐Substituted Tetraphenylethenes Based on Aggregation‐Induced Emission

The synthesis of mannose‐substituted tetraphenylethenes (TPEs) and their aggregation‐induced emission (AIE) behavior, induced by interactions with concanavalin A (Con A), are reported. A mixture of the mannose‐TPE conjugates and Con A in a buffer solution displays an intense blue emission on agglutination within a few seconds, which serves as a “turn‐on” fluorescent sensor for lectins. The sensing is also selective: the conjugates act as a sensor for Con A, but do not sense a galactose‐binding lectin, PNA. Con A‐recognition is not affected even in the presence of other proteins in a mixture. The conjugates also exhibit high sensitivity to detect Con A. An increased sensitivity of the conjugates results if mannopyranoside substituents are linked to the TPE‐core unit with a flexible chain and/or when the number of mannose residues increases.     

Single step synthesis of carbohydrate monolithic capillary columns for affinity chromatography of lectins

Carbohydrate monolithic beds were synthesized in a single step in capillary columns to study affinity chromatography of lectins. In this method, carbohydrates (beta-galactose, beta-glucose, and alpha-mannose) with an easy to synthesize alkene terminated tetraethylene glycol spacer were used as functional monomers along the monomer 2-hydroxyethyl methacrylate (HEMA). As crosslinkers (+)-N,N'-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) were used. SEM showed the successful formation of monolithic beds in the capillary columns. The permeability of the columns was high. The specific interaction of the lectins Con A, Lens culinaris (LCA) and Arachis hypogaea (PNA) with the carbohydrate stationary phase was studied by frontal affinity chromatography (FAC). Con A and LCA were successfully eluted from the column using 0.1 M methyl-alpha-mannopyranoside and PNA with 0.1 M beta-galactose. Dissociation constants (Kd) for carbohydrate-lectin interactions were determined and compared with literature.     

Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera

In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.     

Distribution and translocation of photoreceptor Gbetagamma-phosducin system in medaka retina

Unlike other vertebrates, teleosts have rod- and cone-specific phosducins (PD-R and PD-C) in the retina. To evaluate the teleost Gbetagamma-PD systems, we isolated cDNAs encoding medaka Gbeta1 and GbetaC, which selectively expressed rods and cones. Immunohistochemical studies showed that the strong reactivity of GbetaC but not PD-C was detected in cone outer segments. In rod outer segments (ROS), PD-R reactivity was stronger in light-adapted retina than in dark-adapted retina. Western blot analyses of fractions torn from the cryosections showed that the PD-R concentration was low in dark-adapted ROS. It is suggested that PD-R is translocated to ROS and effectively downregulates the phototransduction cascade in light-adapted rods.     

Interaction of toxic lectin ricin with epithelial cells of rat small intestine in vitro.

To clarify the mechanism of oral toxicity of ricin, the interaction of ricin with the epithelial cells isolated from rat small intestine was compared in vitro with those of other plant lectins by two different determinations, i.e., viability and cytotoxicity. After incubation of the cells for 1 h at 37 degrees C with ricin, B-chain, castor bean hemagglutinin (CBH), soybean agglutinin (SBA), wheat germ agglutinin (WGA), concanavalin A (Con A), and peanut agglutinin (PNA), respectively, followed by staining with trypan blue, ricin and ricin B-chain as well as CBH and SBA were found to have effectively reduced the number of viable cells. On the contrary, only ricin inhibited protein synthesis in the cells and the effect was blocked by D-galactose. Additional experiments employing [125I]-labeled ricin strongly suggested that ricin was first bound via its B-chain to the galactosyl residues on the cell surface followed by internalization into cells as the whole 62 kDa molecule. These results infer first that ricin, as well as other lectins mentioned above, was able to reduce viability of the epithelial cells of rat small intestine by direct binding to the cell surface. The second effect, specific to ricin, was the inhibition of cellular protein synthesis.     

Tokay gecko photoreceptors achieve rod-like physiology with cone-like proteins

The retinal photoreceptors of the nocturnal Tokay gecko (Gekko gekko) consist exclusively of rods by the criteria of morphology and key features of their light responses. Unlike cones, they display robust photoresponses and have relatively slow recovery times. Nonetheless, the major and minor visual pigments identified in gecko rods are of the cone type by sequence and spectroscopic behavior. In the ongoing search for the molecular bases for the physiological differences between cones and rods, we have characterized the molecular biology and biochemistry of the gecko rod phototransduction cascade. We have cloned cDNAs encoding all or part of major protein components of the phototransduction cascade by RT-PCR with degenerate oligonucleotides designed to amplify cone- or rod-like sequences. For all proteins examined we obtained only cone-like and never rod-like sequences. The proteins identified include transducin alpha (Galphat), phosphodiesterase (PDE6) catalytic and inhibitory subunits, cyclic nucleotide-gated channel (CNGalpha) and arrestin. We also cloned cDNA encoding gecko RGS9-1 (Regulator of G Protein Signaling 9, splice variant 1), which is expressed in both rods and cones of all species studied but is typically found at 10-fold higher concentrations in cones, and found that gecko rods contain slightly lower RGS9-1 levels than mammalian rods. Furthermore, we found that the levels of GTPase accelerating protein (GAP) activity and cyclic GMP (cGMP) phosphodiesterase activity were similar in gecko and mammalian rods. These results place substantial constraints on the critical changes needed to convert a cone into a rod in the course of evolution: The many features of phototransduction molecules conserved between those expressed in gecko rods and those expressed in cones cannot explain the physiological differences, whereas the higher levels of RGS9-1 and GAP activity in cones are likely among the essential requirements for the rapid photoresponses of cones.     

Synaptic inputs from rods and cones to horizontal cells in the tiger salamander retina

Synaptic inputs from rods and cones to horizontal cells (HCs) were studied in the flatmounted isolated retinas of the tiger salamander. Voltage-intensity relations, spectral sensitivities and response waveforms of the rod, the cone, and the HC under dark- and light-adapted conditions were examined. HCs receive mixed inputs from rods and cones in both dark- and light-adapted retinas. The relative rod/cone input in HCs depends on the intensity of stimulus and background illumination, and it varies from HC to HC.     

SYNAPTIC INPUTS FROM RODS AND CONES TO HORIZONTAL CELLS IN THE TIGER SALAMANDER RETINA

Synaptic inputs from rods and cones to horizontal cells (HCs) were studied in the flatmounted isolated retinas of the tiger salamander. Voltage-intensity relations, spectral sensitivities and response waveforms of the rod, the cone, and the HC under dark- and light-adapted conditions were examined. HCs receive mixed inputs from rods and cones in both dark- and light-adapted retinas. The relative rod/cone input in HC_s depends on the intensity of stimulus and background illumination, and it varies from HC to HC.     

Detection of isoforms of recombinant human erythropoietin by various plant lectins after isoelectric focusing

A screening method to determine the binding behavior of lectins toward recombinant human erythropoietin (rHuEPO) was developed. Twenty-three different lectins were tested for this purpose. rHuEPO isoforms were separated by isoelectric focusing using the International Olympic Committee (IOC) and World Anti-Doping Agency (WADA) accredited method for the direct detection of the prohibited doping substance erythropoietin (EPO). For the visualization of the rHuEPO isoforms lectins were used instead of antibodies. Optimization of the screening protocol enabled the detection of a maximum number of rHuEPO isoforms. By means of this protocol information about the binding properties of a lectin toward each individual rHuEPO isoform was accessible. All evaluated lectins showed significant differences in their binding behavior. The most intense response was obtained with WGA, DSL, PHA-E, LEL, PSA, and LCA. While WGA, DSL, PHA-E, and LEL were able to bind all isoforms detected by the standard antibody, LCA and PSA demonstrated a clear preference for rHuEPO isoforms located in the more basic region of the electropherogram. Further lectins tested were ConA, succWGA, PHA-L, RCA, SNA, MAA, STL, ECL, GSL-II, SJA, SBA, UEA-I, Jacalin, PNA, DBA, GSL-I, and VVA. Compared to the lectins mentioned above, they showed reduced sensitivity. Endogenous and recombinant EPO only differ in the composition of their N- and O-glycan moieties. As lectins possess the unique ability to recognize subtle differences in glycan substructures, they represent an interesting approach for their structural characterization. Furthermore, they might be useful for affinity enrichment/purification of rHuEPO in doping control.     

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.     

Molecular mechanisms characterizing cone photoresponses

In the vertebrate retina, rods mediate twilight vision and cones mediate daylight vision. Their photoresponse characteristics are different. The light-sensitivity of a cone is 10(2)-10(3) times lower than that of a rod. In addition, the photoresponse time course is much faster in cones. The mechanism characterizing cone photoresponses has not been known mainly because of the difficulty in isolating cones in large quantities to perform biochemistry. Recently, we developed a method to purify cones from carp retina using a density gradient, which made it possible to analyze the differences in the molecular mechanism of phototransduction between rods and cones. The results showed that signal amplification in cones is less effective, which explains the lower light-sensitivity of cones. The results also showed that visual pigment phosphorylation, a quenching mechanism of light-activated visual pigment, is much more rapid in cones than in rods. The rapid phosphorylation in cones is attributed to a very high total kinase activity in cones. Because of this high activity, cone pigment is readily phosphorylated even at very high bleaching levels, which probably explains why cone photoresponses recover quickly. Based on these findings, the molecular mechanisms of the differences in the photoresponse characteristics between rods and cones are outlined.     

Noncovalent fluorous interactions for the synthesis of carbohydrate microarrays

A surface patterning method based on noncovalent immobilization of fluorous-tagged sugars on fluorous-derivatized glass slides allows the facile fabrication of carbohydrate microarrays. To expand the scope of these arrays, the first syntheses are reported of arabinose, rhamnose, lactose, maltose, and glucosamine tagged with a single C₈F₁₇-tail for ease of purification as well as array formation. Screening of these carbohydrate microarrays against lectins from Triticum vulgaris (WGA) and Arachis hypogaea (PNA) demonstrate that the noncovalent fluorous–fluorous interaction is sufficient to retain not only mono- but also disaccharides under the biological assay conditions.     

Salamander blue-sensitive cones lost during metamorphosis

The tiger salamander lives in shallow water with bright light in the aquatic phase, and in dim tunnels or caves in the terrestrial phase. In the aquatic phase, there are five types of photoreceptors--two types of rods and three types of cones. Our previous studies showed that the green rods and blue-sensitive cones contain the same visual pigment and have the same absorbance spectra; however, the green rods have a larger photon-catch area and thus have higher light sensitivity than the blue-sensitive cones. Here we show that after metamorphosis, the terrestrial salamander looses the blue-sensitive cones, while the density of the green rods increases. Moreover, the size of the green rod outer segments is increased in the terrestrial phase, compared to that in the aquatic phase. This switch from the blue-sensitive cones to the green rods may represent an adaptation to the dim light environment of the terrestrial phase.     

Carbohydrate decoration of microporous polypropylene membranes for lectin affinity adsorption: comparison of mono- and disaccharides

Carbohydrates (saccharides) are ubiquitous on the extracellular surface of living cells and mediate a myriad of biological recognition and signaling processes. Carbohydrate decoration of polymer surfaces with covalent attachment of saccharides offers a new realm of opportunities to mimic cellular events such as protein recognition and binding. We describe the carbohydrate decoration (surface glycosylation) of poly(2-hydroxyethyl methacrylate)-grafted microporous polypropylene membranes (poly(HEMA)-g-MPPMs) with mono- and disaccharides. Galactose, lactose, glucose, and maltose were covalently attached on the surfaces of poly(HEMA)-g-MPPMs and were compared in detail. The process was verified by solid state (13)C NMR spectra. Membranes with high binding degree (BD) of saccharide ligands on the surfaces were facilely prepared from poly(HEMA)-g-MPPMs with high grafting degree (GD) of poly(HEMA). For poly(HEMA)-g-MPPM with the same GD of poly(HEMA), the BD of disaccharides is lower than that of monosaccharides and the disaccharide-decorated MPPMs are more hydrophilic than the monosaccharide-decorated ones. The carbohydrate-decorated MPPMs prepared from galactose, lactose, glucose, and maltose (denoted as MPPM-Gal, MPPM-Lac, MPPM-Glc and MPPM-Mal, respectively) recognize and adsorb specifically one of the two lectins, concanavalin A (Con A) and peanut agglutinin (PNA). As the BD of saccharide increases, the "glycoside cluster effect" plays a primary role in lectin adsorption. MPPM-Lac has enhanced affinity to PNA as compared with MPPM-Gal having similar BD of saccharide., on the other hand, MPPM-Mal shows no enhanced affinity to Con A in comparison with MPPM-Glc as the BD of saccharide is above 0.9 μmol/cm(2), where the "glycoside cluster effect" occurs.     

Structure and Dynamics of Molecular Rods in Membranes: Application of a Spin‐Labeled Rod

Molecular rods consisting of a hydrophobic backbone and terminally varying functional groups have been synthesized for applications for the functionalization of membranes. In the present study, we employ a spin‐labeled analogue of a recently described new class of molecular rods to characterize their dynamic interactions with membranes. By using the different approaches of ESR and NMR spectroscopy, we show that the spin moiety of the membrane‐embedded spin‐labeled rod is localized in the upper chain/glycerol region of membranes of different compositions. The rod is embedded within the membrane in a tilted orientation to adjust for the varying hydrophobic thicknesses of these bilayers. This orientation does not perturb the membrane structure. The water solubility of the rod is increased significantly in the presence of certain cyclodextrins. These cyclodextrins also allow the rods to be extracted from the membrane and incorporated into preformed membranes. The latter will improve the future applications of these rods in cellular systems as stable membrane‐associated anchors for the functionalization of membrane surfaces.     

Synthesis and Spectroscopic Characterization of Fluorophore‐Labeled Oligospiroketal Rods

Fluorescence probes consisting of well‐established fluorophores in combination with rigid molecular rods based on spirane‐type structures were investigated with respect to their fluorescence properties under different solvent conditions. The attachment of the dyes was accomplished by 1,3‐dipolar cycloaddition between alkynes and azides (‘click’ reaction) and is a prime example for a novel class of sensor constructs. Especially, the attachment of two (different) fluorophores on opposite sides of the molecular rods paves the way to new sensor systems with less bulky (compared to the conventional DNA‐ or protein‐based concepts), nevertheless rigid spacer constructs, e.g., for FRET‐based sensing applications. A detailed photophysical characterization was performed in MeOH (and in basic H₂O/MeOH mixtures) for i) rod constructs containing carboxyfluorescein, ii) rod constructs containing carboxyrhodamine, iii) rod constructs containing both carboxyfluorescein and carboxyrhodamine, and iv) rod constructs containing both pyrene and perylene parts. For each dye (pair), two rod lengths with different numbers of spirane units were synthesized and investigated. The rod constructs were characterized in ensemble as well as single‐molecule fluorescence experiments with respect to i) specific rod? dye and ii) dye? dye interactions. In addition to MeOH and MeOH/NaOH, the rod constructs were also investigated in micellar systems, which were chosen as a simplified model for membranes.     

Detection of carbohydrates using new labeling reagent 1-(2-naphthyl)-3-methyl-5-pyrazolone by capillary zone electrophoresis with absorbance (UV)

Surface plasmon resonance (SPR) was used to screen the interaction between a variety of affinity ligands and hemagglutinin (HA) from human influenza virus, with the aim of identifying low affinity ligands useful for the development of a rapid bioanalytical sensor. Three sialic acid-based structures and four lectins were evaluated as sensor ligands. The sialic acid-based ligands included a natural sialic acid-containing glycoprotein, human alpha1-acid glycoprotein (alpha1-AGP), and two synthetic 6'-sialyllactose-conjugates, with varying degree of substitution. The interaction of HA with the four lectin-based ligands, concanavalin A (Con A), wheat germ agglutinin (WGA), Maackia amurensis lectin (MAL), and Sambucus nigra agglutinin (SNA), showed a wide variation of affinity strengths. Affinity and kinetics data were estimated. Strong affinities were observed for Con A, WGA, alpha1-AGP, and a 6'-sialyllactose-conjugate with a high substitution degree, and low affinities were observed for MAL and a 6'-sialyllactose-conjugate with low substitution. The main objective, to identify a low affinity ligand which could be used for on-line monitoring and product quantification, was met by a 6'-sialyllactose-ovalbumin conjugate that had 0.6 mol ligand per mol carrier protein. The apparent affinity of this ligand was estimated to be 1.5+/-0.03 microM (K(D)) on the SPR surface. Vaccine process samples containing HA were analyzed in the range 10-100 microg HA mL(-1) and correlated with single-radial immunodiffusion. The coefficient of variation on the same chip was between 0.010 and 0.091.