Identification and characteristics of concanavalin A-binding neurospecific glycoproteins in human brain and brain tumors

Soluble and membrane-bound neurospecific Con A-binding glycoproteins from human brain and tumours were identified and characterized, using a procedure which included stepwise extraction with low and high ionic strength buffers, buffered. Triton X-100 and sodium deoxycholate followed by ConA-Sepharose column chromatography, SDS-PAAG electrophoresis and immunoblotting. Adsorbed antisera against different types of neurospecific glycoproteins were used. The bulk of neurospecific glycoproteins (11 and 13) were revealed in protein fractions extracted with low ionic strength buffers and Triton X-100. In astrocytomas and glyoblastomas, some neurospecific glycoproteins were absent. Some glycoproteins were found in tumours, but were absent in brain tissue. Soluble, 77 kD glycoprotein, 11 and 16 kD glycoproteins solubilized with high ionic strength buffers and intrinsic membrane-bound 51, 57, 61, 74 and 77 kD glycoproteins can be viewed as stable neurospecific markers in malignant brain tumours.     

Isolation of sulfated glycoproteins from human gastric juice with lysine-Sepharose

A procedure for the isolation of sulfated glycoproteins from human gastric juice is described. Sulfated glycoproteins are adsorbed on lysine-Sepharose at pH 2.0, where only the sulfate group of glycoproteins carries a negative charge. The method is simple and rapid, and moreover, the recovery of sulfate is excellent. The sulfated glycoproteins were readily separated from the glycoproteins and other components in gastric juice. The sulfate content of the isolated sulfated glycoproteins ranged from 4 to 15% and increased with increasing molarity of eluting salt.     

Mass spectrometric profiling of O-linked glycans released directly from glycoproteins in gels using in-gel reductive beta-elimination

Glycosylation is a widespread PTM of proteins; the carbohydrate moieties provide various functional, immunological and structural aspects of both eukaryotic and prokaryotic glycoproteins. Traditional strategies used to analyse glycoprotein O-glycans involve glycoprotein isolation, followed by glycan release using solution-phase base-catalysed beta-elimination. However, in a proteomics context, mixtures of proteins and glycoproteins are routinely separated using SDS-PAGE. We have therefore developed a method to enable the profiling of O-linked glycans directly from glycoproteins on gels. This is achieved using in-gel reductive beta-elimination followed by mass spectrometric analysis of the released glycans. Here we describe our demonstration of the feasibility of this approach, our development and optimisation of the procedure using bovine submaxillary gland glycoproteins as a standard, and then show its usefulness by applying the developed procedure to the analysis of the O-glycans from a glycoprotein band from a Coomassie-stained SDS-PAGE separation of a mixture of Mycobacterium avium capsular proteins and glycoproteins. The procedure has been shown to be applicable to both CBB- and silver-stained gels. The method offers a quick and easy way to identify the O-glycans from gel-separated glycoproteins within gel-based proteomics workflows.     

Fractionation and characterization of acidic oligosaccharides and glycopeptides from normal and pathological urines

A general procedure is described for the isolation of urinary acidic oligosaccharides and glycopeptides resulting from catabolism of glycoproteins. This procedure has been applied to normal urine and to urine from patients with diseases of the metabolism, including mucolipidosis and fucosidosis.     

Analysis of serine/threonine-linked oligosaccharides derived by alkaline-borohydride treatment of mucin glycoproteins electroblotted onto membranes: comparison of the saccharide profiles of the 390 kDa and 350 kDa forms of epitectin

Alkaline borohydride treatment is widely used for the release of carbohydrate moieties from O-glycosylated glycoproteins and mucins. We have adapted this procedure to micro quantities of glycoproteins blotted on membranes. After electrophoresis and transfer to nitrocellulose, nylon or polyvinylidene difluoride membrane, alkaline borohydride treatment was done directly on glycoprotein containing areas of membrane which were cut out with the aid of guide strips stained with Coomassie Blue or lectin-digoxigenin. In combination with standard saccharide fractionation techniques, this procedure can be used to characterize the oligosaccharides of mucins or mucin-type glycoproteins that are separated by gel electrophoresis from crude sources. Using this approach we have characterized the saccharides derived from the two species of epitectin, a malignancy-associated mucin type glycoprotein, isolated from metabolically labelled H.Ep2 cells.     

One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis

A novel one-pot procedure for the nonreductive release of O-linked glycans from glycoproteins and the simultaneous derivatization of released glycans with 1-phenyl-3-methyl-5-pyrazolone (PMP) is described. Unlike the traditional reductive β-elimination, which produces alditols, this new method employs PMP/ammonia aqueous solution as the reaction medium. The O-glycans are released from glycoproteins and derivatized with PMP nonreductively, specifically, and quantitatively. Samples can be easily purified from ammonia, excess PMP, and peptide residues by evaporation, chloroform extraction, and solid-phase extraction (SPE) column fractionation for HPLC, CE, or MS analysis. The procedure has been elaborated with two purified glycoproteins, porcine stomach mucin and bovine fetuin, and successfully applied to O-glycan profiling of a challenging biological specimen, healthy human plasma. This new procedure has shown methodological significance in O-glycan analysis.     

Affinity of bronchial secretion glycoproteins and cells of human bronchial mucosa for Ricinus communis lectins.

The coupling of Ricinus communis lectins to Sephadex G 25 was used in order to study mucins and other glycoproteins from human bronchial secretion. The major part of human bronchial mucins and other glycoproteins such as immunoglobulins A, bronchotransferrin and alpha1-antichymotrypsin were isolated by this procedure. A parallel study of human bronchial mucosa was achieved with peroxidase labeled Ricinus communis lectins; this study characterized goblet cells and mucous cells which contain mucins, and serous cells which are involved in the synthesis or the secretion of the other glycoproteins.     

Comparison of glycoproteins by two-dimensional mapping of glycosylated peptides

We describe here a two-dimensional mapping procedure which is capable of resolving glycopeptides isolated by lectin affinity chromatography from radioiodinated tryptic digests of glycoproteins. Glycopeptide maps were successfully produced for the model proteins alpha 1-acid glycoprotein and fetuin, as well as for the two surface glycoproteins gp90 and gp45 from equine infectious anemia virus (EIAV). Differences were detected in the glycopeptide maps obtained for the gp90 and gp45 components from two antigenically distinct strains of EIAV, demonstrating the ability of this procedure to detect variations in glycosylation in closely related glycoproteins. Thus this glycopeptide mapping technique provides a simple, rapid method to study changes in glycopeptides requiring only micrograms of glycoprotein.     

Identification of glycoproteins on nitrocellulose membranes and gels

In this article we describe a procedure for the detection of glycoproteins on gels employing the periodic acid-Schiff’s reagent. In addition, a number of staining protocols and direct binding ELISA, employing antibodies and lectins, are described for the identification and quantitation of glycoproteins after their immobilization by dot, slot, or Western blotting onto nitrocellulose membranes. We document, in detail, the conditions (i.e., the effect of solvent and detergents) for the immobilization of one specific family of O-linked glycoproteins, namely mucins. However, taking into account our suggestions, these procedures should be applicable to other types of glycoprotein.     

Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin

An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.     

Identification and partial characterization of five major membrane glycoproteins of BHK fibroblasts

Summary Five major membrane glycoproteins of the BHK-B4 hamster fibroblast plasma membrane have been identified by binding specific rabbit antibodies to the cell surface and by recovering the detergent solubilized immunocomplexes with Protein A-Sepharose immunoadsorption. These glycoproteins, designated as gp45, gp65, gp95, gp130 and gp140, are exposed at the cell surface since: (i) they were accessible to antibodies in intact viable cells; (ii) they were radioiodinated by the lactoperoxidase-glucose oxidase procedure; and (iii) they were cleaved by proteolytic enzymes in conditions affecting only the cell surface. Among these glycoproteins the gp130 is the predominant component and its exposed portion is characterized by lack of sensitivity to trypsin cleavage. Glycoproteins of different molecular weight, but immunologically related to the major hamster membrane glycoproteins, have been detected at the surface of both rat and mouse fibroblasts.     

Retention of the glycocalyx after cell detachment by EGTA.

Chinese hamster ovary cells were examined ultrastructurally following several detachment procedures. Alterations in the surface glycoproteins were observed by using ruthenium red in the fixation procedure. Trypsin removed a major portion of the cells glycocalyx and formed spherical cellular configurations. EGTA detached cells were also spherical, however, their glycocalyx appeared to remain although redistributed over the cell surface. Kinetic studies showed no alterations in subsequent population doubling times following either detachment procedure. EGTA may thus represent the current method of choice for cell detachment if preservation of the surface glycoproteins is of interest.     

Application of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral and amino sugars in glycoproteins

A method is described for simultaneous gas chromatographic analysis of neutral sugars and hexosamines in glycoproteins. Sugars are hydrolyzed with the aid of Dowex 50-X2 (H⁺) resin and the resin bound glucosamine and galactosamine are deaminated with NaNO₂ to the coresponding neutral 2,5-anhydrohexoses. Hexoses and 2,5-anhydrohexoses are then quantitated as the corresponding alditol acetates. Application of the procedure to several different glycoproteins is presented.     

Solubilization of glycoproteins from enveloped viruses by detergents

The effects of some known ionic and nonionic detergents as well as that of a novel nonionic detergent MESK on various enveloped viruses were investigated. It was found that nonionic detergens (MESK, Triton X-100, octyl-beta-D-glucopyranoside) selectively solubilize glycoproteins of enveloped viruses. The most mild selective action is exerted by the nonionic detergent MESK. Using this detergent, pure preparations of glycoproteins of influenza, parainfluenza, equine Venezuela encephalomyelitis, rabies, vesicular stomatitis and herpes viruses were obtained. The procedure of isolation of purified glycoproteins includes incubation of viral suspensions with MESK, removal of subviral structures by centrifugation and purification of glycoproteins from detergent admixtures by dialysis. Purified glycoproteins retain their native structure and a high biological activity and immunogenicity. MESK seems to be due a perspective tool in the production of subunit vaccines.     

Comparative glycoproteomics based on lectins affinity capture of N-linked glycoproteins from human Chang liver cells and MHCC97-H cells

We present here an effective technique for the large-scale separation and identification of N-linked glycoproteins from Chang liver cells, the human normal liver cells. To enrich N-linked glycoproteins from the whole cells, a procedure containing the lysis of human liver cells, the solubilization of total proteins, lectin affinity chromatography including Concanavalin A and wheat germ agglutinin was established. Furthermore, captured N-linked glycoproteins were separated by 2-DE, and identified by MS and database searching. Finally, we found 63 N-glycoproteins in Chang liver cells. In addition, using the above method, we identified 7 remarkably up-regulated glycoproteins from MHCC97-H cells, highly metastatic liver cancer cells, compared to Chang liver cells. These up-regulated glycoproteins were associated with liver cancer and might be used as biomarkers for tumor diagnosis. Results showed that we established a high-throughput proteomic analysis for separating N-linked glycoproteins from human liver cells. This strategy greatly improved the glycoprotein analysis method associated with proteome-wide glycosylation changes related to liver cancer. Our work was part of the HUPO Human Liver Proteome Project (HLPP) studies and was supported by CHINA HUPO.     

Two-dimensional electrophoresis of surface glycoproteins of normal BHK cells and ricin resistant mutants

The surface glycoproteins of baby hamster kidney (BHK) cells were iodinated by lactoperoxidase and submitted to a two-dimensional electrophoresis procedure involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension. After autoradiography a complex but reproducible pattern was obtained. The technique was then applied to the study of three ricin-resistant mutant clones with reduced rates of cell-cell and/or cell-substratum adhesion. Abnormal patterns were observed in all three mutant clones indicating different mechanisms of ricin resistance and identifying glycoproteins which may be involved in cellular interactions.     

Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

Preparative method of isolating influenza virus glycoproteins

Two preparative methods for isolation of biologically active glycoproteins from influenza virus A - hemagglutinin and neuraminidase, were elaborated. A three-step procedure involves solubilization of glycoproteins with nonionic (Triton N-101, TN) or cationic (cetylpyridinium chloride, CPC) detergents, separation from degraded virions by centrifugation, and removal of detergents and lipids by precipitation of the glycoproteins with butanol (TN), or, alternatively, precipitation of CPC upon cooling. Using virion concentration of approximately 1 mg/ml and optimal weight ratio of detergent to virus (protein) of approximately 20:1 (for TN) and 1:1 (for CPC), the glycoproteins were obtained with the overall yield of 70-80%. The isolated glycoproteins exhibit the same immunological and enzymatic activities as intact virus A/Texas/77 and A/Leningrad/80.     

Heterogeneity of hydroxyproline-containing glycoproteins in protoplasts from a Vinca rosea suspension culture

Protoplasts prepared from a Vinca rosea suspension culture regeneratedcell walk in culture within 24 hr. Glycoproteins containinghydroxyproline were isolated from the protoplasts prior to regenerationof the cell walls. The glycoproteins were extracted, withouta degradation procedure, with 0.05 M sodium acetate buffer,pH 5.6, containing 0.3 M NaCl. Newly synthesized hydroxyprolinein protoplasts labelled with proline appeared almost exclusivelyin the glycoproteins. In this system, the hydroxylation of prolineand the incorporation of arabinose into glycoproteins were suppressedby protein synthesis inhibitors. Inhibition of hydroxylationby the removal of Fe²⁺ also caused suppression of arabinoseincorporation into glycoproteins. These observations suggestthat glycoproteins are precursors of extensin, a hydroxyproline-richglycoprotein contained in the cell wall of plants. The precursorswere heterogeneous in size and charge as determined by gel filtrationand ion exchange chromatography. (Received February 16, 1979; )     

Correlation Between Epstein-Barr Virus Membrane Antigen and Three Large Cell Surface Glycoproteins

A correlation between Epstein-Barr virus membrane antigen (MA) and three surface glycoproteins has been established on the basis of radio-immunoprecipitation and immunoabsorption experiments. For radio-immunoprecipitation, Epstein-Barr virus-infected cells were radiolabeled either with neuraminidase-galactose oxidase tritiated borohydride, a procedure highly specific for surface glycoproteins, or with a general tritiated amino acid mixture. Intact cells were incubated with MA(-) or MA(+) human sera, washed free of unbound immunoglobulins, and then lysed with Nonidet P-40. The antigen-antibody complexes were bound to protein A-Sepharose and after elution with sodium dodecyl sulfate were analyzed by acrylamide gel electrophoresis in sodium dodecyl sulfate. MA(+) sera specifically precipitated three glycoproteins with molecular weights of 236,000, 212,000, and 141,000 from B95-8 cells induced with 12-O-tetradecanoylphorbal-13-acetate (TPA) and from Raji cells superinfected with P3HR-1 virus. These glycoproteins were not detected on Epstein-Barr virus-negative Ramos cells treated with TPA or on B95-8 cells treated simultaneously with TPA and phosphonoacetic acid. Soybean lectin-Sepharose bound all three glycoproteins, and lectin-Sepharose-bound glycoproteins from TPA-induced P95-8 cells absorbed MA-specific antibody from MA(+) human sera. The data strongly suggest that either all three glycoproteins have MA determinants or they are part of a complex in which one or more of the components constitute the reactive antigen.