Affinity For Glycoprotein Research Articles

A study on carbohydrate moieties of gills and air-breathing organs of the walking catfish Clarias batrachus (Linn.) following exposure to arsenic

The chronic sublethal (1 ppm) effects of an arsenic (As) salt, disodium arsenate heptahydrate (DSA) was examined on the carbohydrate moieties of the gills and air-breathing organs (ABO) of the walking catfish Clarias batrachus Linn. The glycogen content increased quantitatively in the gills during initial stages followed by a decrease after 7 days and throughout treatment. The fall was significant after 30–45 days exposure. This was also accompanied by an increase followed by a decrease in mucogenic activity of the gills. Glycoproteins (GPs) along with the mucous cells stained strongly for carboxylic groups and O-sulfated esters. Unlike the gills, the air-breathing organs showed an increase in glycogen content that surpassed the level of untreated control fish after prolonged exposure. The data indicate that As disturbed aerial respiration after chronic exposure. Mucogenic activity in the air-breathing organs fell markedly and always remained below the level of untreated control fish. Similar to the gills, the air-breathing organs also showed a strong affinity for GPs containing carboxylic groups and O-sulfated esters.

DSA affinity glycoproteome of human liver tissue

Due to the critical roles of glycoproteins in the activities of cells to tissues, mapping of liver glycoproteome may provide valuable basic information for finding disease marker proteins. In this study, Datura Stramonium Agglutinin (DSA) was chosen to enrich N-linked glycoproteins for its broader specificity with tri- or tetra-antennary complex type. DSA affinity glycoproteins’ profiles of human liver tissue were generated by two-dimensional electrophoresis (2-DE) followed by glycoprotein staining based on multiplexed proteomics (MP) technology. 64 ± 3 ( n = 3) protein spots were detected and 41 of glycoproteins were identified via peptide mass fingerprinting (PMF) using MALDI-TOF-MS/MS and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The detailed carbohydrate moiety of some glycoproteins might be concluded according to the literatures. The construction of DSA affinity glycoprotein database would contribute to the subsequent research.

Identification and analysis of α1,6‐fucosylated proteins in human normal liver tissues by a target glycoproteomic approach

alpha1,6-Fucose residues within the N-glycan core structures were commonly observed in many glycoproteins. Our previous studies showed that aberrantly alpha1,6-fucosylated glycoproteins might be associated with metastasis of hepatocellular carcinoma (HCC). Little is known about human normal liver tissues (HNLTs) in the literatures. In this study, a target glycoproteomic approach which consists of lectin-affinity chromatography, 2-DE, protein immunoprecipitation and lectin blot, and MALDI-MS/MS, was utilized to screen physiologically alpha1,6-fucosylated glycoproteins. Lens culinaris agglutinin (LCA)-affinity glycoprotein profiles of HNLT were established and analyzed, which allowed identification of 53 proteins by MS analysis, including haptoglobin precursor, alpha-enolase, etc. Gene ontology (GO) annotation proved that these proteins distribute predominately in organelle and play crucial roles in binding and catalytic reactions. The present methodology enabled the identification of all the specific subsets of glycoprotein, and the corresponding data could contribute to the finding of more aberrantly alpha1,6-fucosylated glycoproteins related to liver diseases.

Con A affinity glycoproteomics of normal human liver tissue

In order to establish the novel high throughput, high efficiency and low cost technological platform for the research of N-glycoproteomics, to resolve the significance of characteristic expression profile of glycoprotein and to find the proteins with biological functional importance, the glycoproteins with high-mannose core and the two antennary types were purified and enriched by the Con A affinity chromatography. Con A affinity protein expression profiles of normal human liver tissue were generated by using SDS-PAGE, two-dimensional electrophoresis (2-DE) followed by fast fluorescence staining based on multiplexed proteomics (MP) technology. 301 visible protein spots on the gel were detected and 85 of glycoproteins were further successfully identified via peptide mass fingerprinting (PMF) by a matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) and annotated to IPI databases. Identified glycoproteins definitely take part in the regulation of cell cycle and metabolic processes. The glycosylation sites were predicted with NetNGlyc 1.0 and NetOGlyc 3.1 software, meanwhile they were classified according to the geneontology methods. The construction of Con A affinity glycoprotein database of normal human liver tissue would contribute to the subsequent research.

Purification of clenbuterol-like β 2-agonist drugs of new generation from bovine urine and hair by α 1-acid glycoprotein affinity chromatography and determination by gas chromatography–mass spectrometry

The control of illegal use of clenbuterol and other β 2-agonist drugs as growth promoters in the European Union countries has led to outlaw practices for synthesizing new concept molecules, showing similar biological activity but not detectable by test methods usually employed to perform the official monitoring programmes. The synthesis schemes of some β 2-agonist compounds, formally derived from clenbuterol, were found out by Italian detective authorities. These compounds were synthesised ex novo in our laboratories: then, both their molecular structures and biological activities were characterised. In this paper, we describe different strategies for purifying some β 2-agonist drugs of new concept, more hydrophobic than clenbuterol. A two-step clean up procedure, prior to gas chromatography–mass spectrometry analysis, was developed for the multi-residue determination of these β 2-agonists from bovine hair and urine. The purification strategy we chose was based on adsorption solid phase extraction and, subsequently, on specific molecular recognition by affinity chromatography. The affinity columns were homemade by coupling bovine α 1-acid glycoprotein, a plasmatic acceptor for basic drugs, to a chromatographic support; their effectiveness for purifying new β 2-agonists was discussed. The data about method recoveries and repeatability were also reported.

Complex carbohydrate specificity of lectin from fruiting body of Ganoderma lucidum. A surface plasmon resonance study

The thermodynamics and kinetics of binding of glycans and glycoproteins to Ganoderma lucidum lectin was studied using surface plasmon resonance. The lectin showed highest affinity for asialo triantennary N glycan (Ka = 3.52 x 10(5)) among the glycans tested. There was a several fold increase in affinity for glycoproteins compared to their corresponding glycans and coincident increase in contribution from enthalpy (DeltaH), suggesting the involvement of hydrogen bonding in the interaction as well as involvement of protein-protein interactions. Increased affinity also showed increase in unfavorable negative binding entropy (DeltaS) which was compensated with higher enthalpy. The glycoproteins showed faster association rates (k(1)) and the activation energy (E(1)) in the association process was much lower for the glycoproteins than glycans, resulting in their faster associations. These observations elaborate the role of protein matrix in lectin-glycoconjugate interaction.

Identification and analysis of altered α1,6-fucosylated glycoproteins associated with hepatocellular carcinoma metastasis

Tumor metastasis might be associated with the expression levels of cellular glycoproteins and the alteration of their glycan parts. In order to screen the aberrantly alpha1,6-fucosylated glycoproteins related to hepatocellular carcinoma (HCC) metastasis, a high-throughput glycomic approach which consisted of 2-DE, electronic transfer of proteins, lectin affinity blot and precipitation, and MALDI-TOF-MS/MS, was established. Lens culinaris agglutinin (LCA) affinity glycoprotein profiles of higher and lower metastatic HCC cell lines were compared and analyzed. Seven out of 34 identified glycoproteins were differentially displayed; they were cytokeratin 8 (CK8), annexin I, annexin II, heterogeneous nuclear ribonucleoprotein A/B, PDZ and LIM domain 1, RNA-binding motif protein 4, and poly(rC)-binding protein 1. On comparison with Hep3B, CK8 showed a higher affinity to Ricinus communis agglutinin 1 (RCA-I) and LCA, and annexin I presented a higher affinity to LCA and Con A by the lectin-binding assay. Furthermore, the up-regulation of CK8, annexin I, and annexin II were found by Western blot and immunofluorescence analysis in higher metastatic HCC cell lines. This implied that the alteration of CK8, annexin I, and annexin II both in their expression levels and their glycan parts might be related to metastatic ability, and play a critical role in the process of HCC metastasis.

Identification of a probable new adrenergic agonist by nuclear magnetic resonance and mass spectrometry

In animal production, it is consolidated the synthesis and the illegal use of growth promoters of new generation, able to skip routine screening and confirmatory analysis. In this work it is reported the nuclear magnetic resonance (NMR) and the mass spectrometry identification of a probable new adrenergic drug found in a feed premix. The substance was selectively purified on alpha 1 acid glycoprotein affinity columns; then its structure was first achieved by recording the 13C NMR spectrum that gave the total number of carbons of the molecule, successively sorted by DEPT experiments into quaternary, CH, CH2, and CH3 groups. However, the complete assignments of all resonances were derived from the bi-dimensional analysis and the crucial indications from the 1H–13C reverse experiments. Further characterisation was performed by atmospheric pressure chemical ionisation both in positive and negative ion mode, matching the molecular ion and the fragmentation pattern with those of most recently described new adrenergic agonists. After the loss of a ter-butylic group, the structure shows an internal symmetry along with the presence of Chlorine clusters. The proposed formula of the compound, the 8,8′-diamino-9,9′-dichloro-1-terbutyl-1,1′,4,4-tetrahydro-5H,5′H-2,2′-bi-1-benzazepine-5,5′-dione, partially resembles that of Zilpaterol for the presence of a heterocyclic ring; Further work is in progress to characterise the structure–activity relationship.

Development of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in milk after alfa‐1‐acid glycoprotein affinity chromatography

In this work we present a method for confirmatory analysis of chloramphenicol (CAP) in bovine and buffalo raw milk. CAP is extracted in acetonitrile and purified by affinity chromatography on an alpha-1-acid glycoprotein (AAG) column, then is identified and determined by ion-trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified at the minimum required performance limit (MRPL) of 0.30 ppb, by monitoring the [M-H]- ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in Decision 2002/657/EC for confirmation of prohibited veterinary drugs. The trueness and within-day and between-day repeatability data are also reported. Moreover, the loading capacity of affinity columns towards CAP was tested. This method, based on the molecular recognition between drug and AAG during the purification step to improve sample cleanup, represents a quantitative and repeatable procedure for confirmatory analysis, and fits the requirements for the routine official control of CAP residues in raw milk.

Fucose Depletion from Human IgG1 Oligosaccharide Enhances Binding Enthalpy and Association Rate Between IgG1 and FcγRIIIa

Depletion of fucose from human IgG1 oligosaccharide improves its affinity for Fcγ receptor IIIa (FcγRIIIa). This is the first case where a glycoform modification is shown to improve glycoprotein affinity for the receptors without carbohydrate-binding capacity, suggesting a novel glyco-engineering strategy to improve ligand–receptor binding. To address the mechanisms of affinity improvement by the fucose depletion, we used isothermal titration calorimetry (ITC) and biosensor analysis with surface plasmon resonance. ITC demonstrated that IgG1–FcγRIIIa binding was driven by favorable binding enthalpy (Δ H) but opposed by unfavorable binding entropy change (Δ S). Fucose depletion from IgG1 enhanced the favorable Δ H, leading to the increase in the binding constant of IgG1 for the receptor by a factor of 20–30. The increase in the affinity was mainly attributed to an enhanced association rate. A triple amino acid substitution in IgG1, S298A/E333A/K334A, is also known to improve IgG1 affinity for FcγRIIIa. ITC demonstrated that the amino acid substitution attenuated the unfavorable Δ S, resulting in a three- to fourfold increase in the binding constant. The affinity enhancement by the amino acid substitution was due to a reduced dissociation rate. These results indicate that the mechanism of affinity improvement by the fucose depletion is quite distinct from that by the amino acid substitution. Defucosylated IgG1 exhibited higher antibody-dependent cellular cytotoxicity (ADCC) than S298A/E333A/K334A-IgG1, showing a correlation between IgG1 affinity for FcγRIIIa and ADCC. We also examined the effect of FcγRIIIa polymorphism (Val158/Phe158) on IgG1–FcγRIIIa binding. The Phe to Val substitution increased FcγRIIIa affinity for IgG1 in an enthalpy-driven manner with the reduced dissociation rate. These results together highlight the distinctive functional improvement of affinity by IgG1 defucosylation and suggest that engineering of non-interfacial monosaccharides can improve glycoprotein affinity for receptors via an enthalpy-driven and association rate-assisted mechanism.

Lectin Variation in Members of <i>Rhizoctonia</i> Species

Isolates from two multinucleate Rhizoctonia species, namely Rhizoctonia solani and Rhizoctonia circinata, and binucleate Rhizoctonia spp., were evaluated for the presence of lectins, using human erythrocytes. Isolates from R. solani had similar lectins across the anastomosis groups. Agglutination profiles, however, revealed individual preferences for human blood types but with a general preference for type A over B and O. R. solani lectins had a general affinity for N-acetyl-D-galactosamine and mucin. Also, some isolates from the binucleate Rhizoctonia AG-DII, had lectins that showed strong affinity for glycoproteins, fetuin and mucin. There was no lectin activity in members of R. circinata. The lectins in members of the genus Rhizoctonia varied in content and character across the species sampled but had similar affinity for mucin. Furthermore, 62% of the self-anastomosing and 35% of the non-self anastomosing isolates tested showed lectin activity. The presence of lectins in both self-anastomosing and non-self anastomosing isolates suggests that lectins may not be directly involved in the recognition process in hyphal anastomosis.

Imparting bone affinity to glycoproteins through the conjugation of bisphosphonates.

To develop a novel means of conjugating bisphosphonates onto the carbohydrate moieties of glycoproteins to enhance protein affinity to bone. 1-Amino-1,1-diphosphonate methane (aminoBP) was conjugated onto the carbohydrate moietites of oxidized fetuin by using 4-(maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (MMCCH). Bone affinity of the resulting conjugates was compared to proteins obtained from another means of conjugation, whereby aminoBP was conjugated onto fetuin's lysine moieties by using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). The use of the MMCCH resulted in the conjugation of up to seven aminoBPs per molecule of fetuin. These conjugates gave a 2.6-, 2.0-, 30.5-. and 1.84-fold increased affinity for untreated, ashed, demineralized bone and hydroxyapatite, respectively, as compared to conjugates from the SMCC reaction. Both conjugates exhibited a pH-independent, equally slow degradation in adult bovine serum-containing media. The use of the MMCCH chemistry to conjugate aminoBP onto fetuin was feasible. Furthermore, the described processes of conjugation resulted in amino-BP-dependent increase in the glycoprotein's affinity to various bone matrices in a manner that exceeds the affinity produced by the previously established method, which used SMCC.

Synthesis of RGD Analogs as Potential Vectors for Targeted Drug Delivery

RGD analogs bind to integrin receptors with high affinity and therefore have the potential to be used as vectors for the targeted delivery of pharmaceutical agents to designated sites. Critical to this application is the ability to synthesize RGD analogs with different side chain functional groups that allow for the ready tethering of pharmaceutical agents without sacrificing their affinity for the target receptor significantly. A series of RGD analogs intended to be used as delivery vectors of pharmaceutical agents were prepared and evaluated for their ability to inhibit platelet aggregation by binding to glycoprotein IIb/IIIa. Among them, compound 11 showed the lowest IC50 against platelets activated by ADP. It was found that such RGD analogs could tolerate side chain modification fairly well with various functional groups attached such as amide, amine, ester, protected amine and poly(ethylene glycol). The fact that the compound with a side chain modification of poly(ethylene glycol) retained high affinity for glycoprotein IIb/IIIa (IC50 150 nM) suggests the feasibility of tethering fairly large pharmaceutical agents to such RGD analogs without significant sacrifice of their affinity to the intended receptor.

Determination of new β2-Agonists drugs by α1 glycoprotein affinity chromatographyglycoprotein affinity chromatography

The illicit use of new β2-agonists drugs in meat production is prompting the European Union to improve analytical techniques and testing methods, to authoritatively demonstrate the abuse of such compounds. In this paper, we describe the preparation of α1-acid glycoprotein (AAG) columns for purification by affinity chromatography of clenbuterol and new β2-agonists from bovine urine. HPLC-UV/DAD analyses showed no significant matrix interference in binding, thus indicating the specificity of such a clean-up procedure. Such AAG columns were able to effectively bind a broader range of β2-agonists, compared to immuno-affinity columns. Our approach could improve the clean-up step within a multi-residue analytical strategy.

Fast detection of a (CA)18 microsatellite repeat in the IgE receptor gene by capillary electrophoresis with laser-induced fluorescence detection.

The optimum separation conditions of polymerase chain reaction (PCR) products have been found for high-speed capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. DNA fragments obtained after PCR amplification of the region covering the (CA)18 microsatellite repeat in nitron 5 of the gene for FcERIbeta, a high affinity glycoprotein receptor for IgE, located on chromosome 11 (11q13), were analyzed with the aim of investigating the repeat polymorphism. The results of polyacrylamide slab gel electrophoresis (PAGE), agarose gel electrophoresis, CE with absorbance detector and CE with LIF are compared. The CE with LIF proved to shorten analysis time by a factor of 100 when compared to slab gel electrophoresis. CE-LIF utilizes a short capillary with an effective length of 6.3 cm and electric field strength from 100 to 550 V/cm. The respective PCR products of sizes from 116 to 210 base pairs (bp) were analyzed in 3 min.

The E-selectin-ligand ESL-1 is located in the Golgi as well as on microvilli on the cell surface.

Neutrophils and subsets of lymphocytes bind to E-selectin, a cytokine inducible adhesion molecule on endothelial cells. The E-selectin-ligand-1 (ESL-1) is a high affinity glycoprotein ligand which participates in the binding of mouse myeloid cells to E-selectin. The sequence of mouse ESL-1 is highly homologous to the cysteine rich FGF receptor (CFR) in chicken and the rat Golgi protein MG160. We have analysed the subcellular distribution of ESL-1 by indirect immunofluorescence, flow cytometry, various biochemical techniques and by immunogold scanning electron microscopy. We could localize ESL-1 in the Golgi as well as on the cell surface of 32Dc13 cells and neutrophils. Cell surface staining was confirmed by cell surface biotinylation and by cell surface immunoprecipitations in which antibodies only had access to surface proteins on intact cells. In addition, ESL-1(high) and ESL-1(low) expressing cells, sorted by flow cytometry, gave rise to high and low immunoprecipitation signals for ESL-1, respectively. Based on immunogold labeling of intact cells, we localized ESL-1 on microvilli of 32Dc13 cells and of the lymphoma cell line K46. Quantitative evaluation determined 80% of the total labeling for ESL-1 on microvilli of K46 cells while 69% of the labeling for the control antigen B220 was found on the planar cell surface. These data indicate that ESL-1 occurs at sites on the leukocyte cell surface which are destined for the initiation of cell contacts to the endothelium.

Secretory component, the receptor for polymeric immunoglobulin, has nothing to do with β-galactosyltransferase in human milk

Secretory component (SC) in external secretions is a soluble form of the polymeric immunoglobulin-receptor that is expressed on the cell membrane of mucosal epithelial cells. β-(1–4)galactosyl transferase (β-GT) is an enzyme that transfers galactose to non-reducing N-acetylglucosamine residues on various glycoproteins and is present in a soluble form in secretions as well as in a membrane-bound form. β-GT is considered to have affinity for glycoproteins, including IgA in secretion. It has been claimed that these two proteins are related to or identical with each other. In the present study, we defined that the SC and the β-GT are each independent molecules by the following facts; (1) both molecules are separable either by antibody-affinity chromatography, conventional ion-exchange or molecular exclusion chromatography, (2) conventionally purified SC from human milk contained neither enzymatic activity or antigenic determinants of the β-GT, (3) recombinant β-GT does not show reactivity with antibodies to SC, and (4) the SC showed no reactivity with antibody to β-GT.

Post-receptor events associated with thrombin-induced platelet activation

Thrombin is by far the most potent platelet agonist. Potentially this reflects multiple intracellular processes involved in transmitting the activation signal from the initial contact with a receptor, or binding site, to the final platelet response. Platelet membranes have two putative receptors: the high affinity glycoprotein Ib, whose function remains to be clarified, and the moderate affinity autoproteolytic receptor. The autoproteolytic receptor is a member of a family of receptors, with seven transmembrane domains, which interact with GTP-binding proteins. Distal to the membrane, several forms of phospholipase C are activated and roles for both heterotrimeric and low molecular mass GTP-binding proteins have been presented. Phospholipase C acts on inositol phospholipids to generate inositol trisphosphate and diacylglycerol, both of which function as second messengers in thrombin-induced platelet activation. Inositol trisphosphate mobilizes internal calcium stores and this is accompanied, and enhanced, by an influx of calcium from the external milieu. Diacylglycerol and calcium both serve to regulate the activity of multiple protein kinases which, in turn, mediate the phosphorylated state of numerous proteins. Phosphorylation can occur on serine, threonine or tyrosine residues of target proteins and the phosphorylated state of these proteins determines the final activation of the platelet.

Post-receptor events associated with thrombin-induced platelet activation

Thrombin is by far the most potent platelet agonist. Potentially this reflects multiple intracellular processes involved in transmitting the activation signal from the initial contact with a receptor, or binding site, to the final platelet response. Platelet membranes have two putative receptors: the high affinity glycoprotein Ib, whose function remains to be clarified, and the moderate affinity autoproteolytic receptor. The autoproteolytic receptor is a member of a family of receptors, with seven transmembrane domains, which interact with GTP-binding proteins. Distal to the membrane, several forms of phospholipase C are activated and roles for both heterotrimeric and low molecular mass GTP-binding proteins have been presented. Phospholipase C acts on inositol phospholipids to generate inositol trisphosphate and diacylglycerol, both of which function as second messengers in thrombin-induced platelet activation. Inositol trisphosphate mobilizes internal calcium stores and this is accompanied, and enhanced, by an influx of calcium from the external milieu. Diacylglycerol and calcium both serve to regulate the activity of multiple protein kinases which, in turn, mediate the phosphorylated state of numerous proteins. Phosphorylation can occur on serine, threonine or tyrosine residues of target proteins and the phosphorylated state of these proteins determines the final activation of the platelet.

Biochemical characterization of binding sites for dihydropyridine and ω-conotoxin in brain of adult chicken

Binding studies using the calcium channel blockers ω-conotoxin and dihydropyridine revealed a rather equal amount of binding sites in brain from adult chicken. The ω-conotoxin binding sites could be solubilized using digitonin, without substantial loss, whereas a great decrease in dihydropyridine binding sites was observed, indicating that both types of binding sites have different sensitivity to solubilization. In contrast to ion exchange chromatography where both binding sites comigrated, glycoprotein affinity chromatography led to a different partition of the binding sites in the flow through and eluate fractions. Our results indicate that both types of calcium channel blockers bind to different targets in adult chicken.