Changes of alpha1-acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively

The relationship between variations of alpha1-acid glycoprotein (orosomucoid, AGP) microheterogeneity detected from isoelectric focusing (IEF) patterns and clinical stage of acute inflammation based on serum C-reactive protein (CRP) levels and interleukin-6 (IL-6) levels was investigated. Serum samples were obtained from healthy subjects, and from patients with esophageal or stomach carcinoma before and after operation. Samples without neuraminidase treatment were used for AGP microheterogeneity analysis, and samples with neuraminidase treatment for AGP heterogeneity analysis. In AGP microheterogeneity, nine bands were detected in the range of pI 3.18-3.57 in sera obtained from healthy subjects. In patients, AGP microheterogeneity changed the first day after operation; the percentage of bands surrounding pI 3.5 increased, and the highest value appeared in sera taken the first or second day after operation and then decreased quickly. These bands showed reactivity for concanavalin A (Con A). The increase in Con A-reactive AGP occurred later than the increase in IL-6, and occurred earlier than the increase in CRP. On the seventh day after operation, the percentage of bands around pI 3.2 increased. These bands showed the reactivity for Datura stramonium agglutinin. On the other hand, in samples with neuraminidase treatment, little change of AGP heterogeneity was observed in most samples, which did not reflect the stage of inflammation. These findings suggested that AGP microheterogeneity detection was a useful marker for the clinical stage of inflammation.     

Combined use of lectin affinity chromatography and endo-beta-galactosidase to study polylactosamine sequences isolated from haemopoietic cell surfaces

The trypsin-sensitive glycopeptides from cell surfaces of a multipotential murine haemopoietic cell line (DE) have been studied using serial lectin affinity chromatography on columns of immobilized lentil lectin (LCA), concanavalin A (Con A), and wheat-germ agglutinin (WGA). WGA-binding material consisted of glycopeptides that failed to bind to LCA and Con A. Step elution from the WGA-column with 0.01-, 0.1-, 0.5- and 1.0 M N-acetyl-D-glucosamine yielded four affinity classes of glycopeptide (WGA-W, WGA-I, WGA-S and WGA-SS respectively). WGA-W, WGA-I and WGA-S contained both alkali-stable (N-linked) and alkali-labile (O-linked) carbohydrate on high molecular weight glycopeptides. The WGA-SS fraction contained only N-linked carbohydrate. N-linked glycopeptides isolated from each WGA-binding class differed in molecular size, relative N-acetylneuraminic acid content and affinity for Ricinus communis 120 agglutinin. endo-beta-Galactosidase digestion showed that these glycopeptides contained polylactosamine-type glycans. Gel filtration profiles of the enzyme treated materials were different for each WGA-binding population suggesting variation in branching patterns and/or substitution with fucose residues. Affinity chromatography has shown that the WGA binding molecules are the major glycopeptide group at DE cell surfaces.     

Comprehensive native glycan profiling with isomer separation and quantitation for the discovery of cancer biomarkers

Glycosylation is highly sensitive to the biochemical environment and has been implicated in many diseases including cancer. Glycan compositional profiling of human serum with mass spectrometry has already identified potential biomarkers for several types of cancer and diseases; however, composition alone does not fully describe glycan stereo- and regioisomeric diversity. The vast structural heterogeneity of glycans presents a formidable analytical challenge. We have developed a method to identify and quantify isomeric native glycans using nanoflow liquid chromatography (nano-LC)/mass spectrometry. A microfluidic chip packed with graphitized carbon was used to chromatographically separate the glycans. To determine the utility of this method for structure-specific biomarker discovery, we analyzed serum samples from two groups of prostate cancer patients with different prognoses. More than 300 N-glycan species (including isomeric structures) were identified, corresponding to over 100 N-glycan compositions. Statistical tests established significant differences in glycan abundances between patient groups. This method provides comprehensive, selective, and quantitative glycan profiling.     

Microheterogeneity of orosomucoid in pathological conditions

Studies of orosomucoid (alpha 1-acid glycoprotein) in human serum have revealed that orosomucoid is a mixture of molecules with differences in the glycan chains. This microheterogeneity has been studied using crossed affinoimmuno-electrophoresis with the lectin concanavalin A which binds to biantennary glycans. The relative proportions of the three orosomucoid subtypes are altered in various pathological conditions independently of the total serum orosomucoid concentration. There are reproducible differences in microheterogeneity patterns between some pathological conditions: Acute tissue injury or inflammation results in a high proportion of orosomucoid with biantennary glycans. Conditions with increased estrogen levels are associated with a high proportion of orosomucoid with tri- or tetraantennary glycans and a low total serum orosomucoid concentration. Chronic inflammation also seems to be associated with a high proportion of orosomucoid with tri- or tetraantennary glycans but with a high total serum concentration of orosomucoid. Other diseases, such as cancer, can not be associated with any specific microheterogeneity pattern. The microheterogeneity pattern in these conditions seems to be determined by disease activity and unspecific inflammation in surrounding tissues.     

Affinity electrophoresis for diagnosis of cancer and inflammatory conditions

Affinity electrophoresis, with concanavalin A and Lens culinaris agglutinin as ligands, was applied to study the microheterogeneity of serum proteins, with special emphasis on alpha 1-fetoprotein and alpha 1-acid glycoprotein, two proteins of potential clinical value. A total of 602 samples of serum from patients with various neoplastic and inflammatory conditions were evaluated. Affinity electrophoresis provided useful information for differential diagnosis of cancers of various origins including hepatomas, metastatic liver cancers and yolk sac tumors. The method also proved indispensable for the detection of intercurrent infection in patients with rheumatoid arthritis, systemic lupus erythematosus and scaled burns.     

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co- and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined.     

Heterogeneous nature of human complement factor B: an electrophoretic approach for the analysis of its oligosaccharide chain structure and its physiological breakdown products

Factor B is a glycoprotein which plays an essential role in the alternative pathway of complement activation. It carries the proteolytic activity of the convertases, and its physiological breakdown products Ba and Bb have some effects on the cells of the immune system. Human factor B exhibits a microheterogeneity and five isoforms are present in serum. The nature and origin of the microheterogeneity was investigated by using electrophoretic techniques. Treatments of factor B with neuraminidase and glycopeptidase F show that this microheterogeneity is mainly due to differences in its sialic acid content, varying from seven to eleven residues per molecule, and resulting in different oligosaccharide structures. However, deglycosylated factor B reveals a residual, nonallotypic variation in the Bb region of the polypeptide backbone. We confirm the presence of four asparagine-linked oligosaccharide chains of the complex type in native factor B, two of which are located in the Ba fragment, and the two others in the Bb fragment. The prevalent isoform of the native protein carries two sialic acid residues per oligosaccharide chain. Biosynthesis experiments show that the microheterogeneity of secreted factor B from HepG2 cells is acquired during the processing of its glycans. However, in vitro-secreted factor B is more heterogeneous than the serum protein. We propose a structural model for the microheterogeneity of the native protein and its physiological fragments. We discuss as well the feasibility of electrophoretic techniques to deal with microheterogeneity analysis.     

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.     

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.     

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.     

Speciation of protein-binding zinc and copper in human blood serum by chelating resin pre-treatment and inductively coupled plasma mass spectrometry

A method for the speciation of zinc and copper binding with proteins in human serum was explored by chelating resin (Chelex-100) pre-treatment and inductively coupled plasma mass spectrometry (ICP-MS). It was shown by a SEC (size-exclusion chromatography)-ICP-MS system that albumin-zinc and albumin-copper (loosely-bound species) could be selectively removed from serum by adsorption on the Chelex-100 resin after the chelating resin pre-treatment, while alpha 2-macroglobulin-zinc and ceruloplasmin-copper (firmly-bound species) remained in the serum. The zinc and copper bound with alpha 2-macroglobulin and ceruloplasmin, respectively, were then determined by ICP-MS after batch treatment of the serum samples with the Chelex-100 resin. In addition, the total concentrations of zinc and copper were also determined by ICP-MS after a 20-fold dilution with 0.1 M HNO3. The albumin-zinc and -copper were estimated as the differences between the concentrations of total and firmly-bound species. The present batch pre-treatment method was applied to the speciation analysis of zinc and copper binding with proteins in sera donated from 25 healthy volunteers as well as from a pregnant woman and a myelodysplastic syndrome patient. The observed concentrations of alpha 2-macroglobulin-zinc and ceruloplasmin-copper were in the ranges 109-202 ng ml-1 (12.4-31.3% of total zinc) and 513-880 ng ml-1 (90.6-99.7% of total copper), respectively. The present method is simple (only addition of the chelating resin and centrifugation is required) and reproducible (average RSD = 2% for alpha 2-macroglobulin-zinc and 1% for ceruloplasmin-copper in intra-assay measurements, and 5% for alpha 2-macroglobulin-zinc and 4% for ceruloplasmin-copper in inter-assay measurements), and there is less risk of contamination during separation.     

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.     

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes

Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.     

Thiol‐ene and thiol‐yne‐based synthesis of glycodendrimers as nanomolar inhibitors of wheat germ agglutinin

Alkene and alkyne functional polyester‐based dendrimers of generation 1 to 4 have been prepared and reacted under free‐radical conditions with 2‐acetamido‐2‐deoxy‐1‐thio‐β‐D ‐glucose (GlcNAc‐SH). As the alkene‐dendrimers underwent the addition of one thiyl radical per ene group whereas each yne group of alkyne‐dendrimers was saturated by two thiyl radicals, a collection of glycodendrimers with glycan density ranging from six to ninety‐six GlcNAc per dendrimer was obtained. The recognition properties of the prepared glycodendrimers toward the wheat germ agglutinin (WGA) were evaluated by enzyme‐linked lectin assay (ELLA). The eight glycodendrimers were excellent ligands showing IC₅₀ values in the nanomolar range and relative potencies per sugar unit up to 2.27 e⁶ when compared to monosaccharidic GlcNAc used as monovalent reference. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 2422–2433     

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.     

Mass spectrometric study of N‐glycans from serum of woodchucks with liver cancer

Woodchucks have been a preferred lab animal model of chronic hepatitis B viral infection. The model recapitulates the disease progression of HBV infection to hepatocellular carcinoma (HCC) and has documented similarities in protein glycosylation with human HCC. This study examined N‐glycans in serum of animals with(out) HCC. Oligosaccharides were released enzymatically using PNGaseF from total serum or from serum partially fractionated by extraction. Two different extraction procedures – reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and solid‐phase extraction (SPE) on a cation‐exchange/reversed‐phase STRATA‐XC cartridge – were used with the purpose of confirming glycosylation profiles. Oligosaccharides were analyzed by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) after derivatization with phenylhydrazine and/or permethylation. Characteristic fragment ions produced under MS/MS conditions allowed discrimination between isomeric structures of oligosaccharides, including those sialylated with two types of acidic residues. The complementary methods allowed structural characterization of oligosaccharides from various N‐glycan classes. Furthermore, to validate results, glycosylation profiles of woodchuck sera were compared to glycans obtained from mouse serum on the same conditions. In summary, we have identified 40 N‐glycan structures in the serum of woodchucks and some types of oligosaccharide structures appeared to increase in HCC samples following protease digest. The study provides improved tools for the characterization of N‐glycans from total serum in the progression of liver disease. Copyright © 2009 John Wiley & Sons, Ltd.     

Voltammetric behaviors of wheat-germ agglutinin on a chitin-modified carbon-paste electrode.

The voltammetric behavior of wheat-germ agglutinin (WGA) on a chitin-modified carbon-paste electrode (CPE) was investigated using glucose labeled with an electroactive compound. WGA usually consists of two subunits, each with two binding sites for sugars. WGA was immobilized on the electrode surface by selective binding to a N-acetylglucosamine residue of chitin. Because glucose also combines with WGA, the glucose was coupled with electroactive daunomycin to evaluate the binding. When a WGA-labeled glucose complex was formed, the electroactive moiety became electroinactive. The binding caused a decrease in the peak current of the labeled glucose. In a measurement of only daunomycin used as a label, the peak current in a solution with WGA was similar to that in a solution without WGA. Therefore, it is clear that the labeled glucose was held in the remaining binding site of WGA on the electrode surface. Thus, a CPE modified with chitin would be powerful as a reaction field between sugar and lectin.     

HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals

In contrast with conventional drugs, biopharmaceuticals are highly complex molecules with remarkable heterogeneity. Protein glycosylation is an inherent source of this heterogeneity and also affects the safety, efficacy, and serum half-life of therapeutic glycoproteins. Therefore analysis of the glycan pattern is an important issue for characterization and quality control in the biopharmaceutical industry. In this publication we describe a complete workflow for the analysis of protein N-glycans. The sample-preparation procedure, consisting of the release of the N-glycans by PNGase-F, followed by fluorescence labeling with 2-aminobenzamide and removal of excess label, was optimized to avoid alteration of the glycan sample. Subsequently, labeled glycans were analyzed by hydrophilic-interaction liquid chromatography (HILIC) with fluorescence detection. The developed method was validated for analysis of antibody N-glycans. To demonstrate the accuracy of the method an antibody sample was additionally analyzed by an orthogonal method. The antibody was digested with lysyl endopeptidase and the (glyco-)peptides were analyzed by RP-HPLC–MS. The consistency of the results between these two methods demonstrates the reliability of the glycan analysis method introduced herein.     

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.     

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.