Blot analyses of glycoconjugates: enzyme-hydrazide--a novel reagent for the detection of aldehydes

A procedure for the general staining of glycoproteins and other glycoconjugates on protein blots has been developed. Aldehydes are formed on the sugars of glycoconjugates by periodate oxidation which then react with hydrazide groups of enzyme-hydrazides, a novel reagent designed for aldehyde detection. The bound enzyme-hydrazide is demonstrated histochemically. The new assay is advantageous over periodic-acid Schiff staining of gels as its reagents and signals are stable and the process is simple and expedient, and provides greater sensitivity.     

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots

The fluorescent hydrazide, Pro-Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff's (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2-4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro-Q Emerald 300 dye-labeled gels and blots may be poststained with SYPRO Ruby dye, allowing sequential two-color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid-range UV illumination. Pro-Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of alpha 1-acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro-Q Emerald 300 dye. Besides glycoproteins, as little as 2-4 ng of lipopolysaccharide is detectable in gels using Pro-Q Emerald 300 dye while 250-1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate-polyacrylamide gels, two-dimensional gels and on polyvinylidene difluoride membranes.     

Quantitation of glycoproteins on electroblots using the biotin-streptavidin complex

A method for the detection and quantitation of glycoproteins on nitrocellulose electroblots is described. Protein mixtures may be solubilized in sodium dodecyl sulfate prior to labeling, which is especially useful when dealing with membrane proteins. Mild periodate oxidation produces aldehydes on the oligosaccharide moieties which are then specifically condensed with biotin aminocaproyl hydrazide. After polyacrylamide gel electrophoresis and transfer of proteins onto nitrocellulose membranes, biotinylated glycoproteins are detected with enzyme-linked streptavidin and quantitated by densitometric scanning. As little as 1 ng of alpha 1-acid glycoprotein can be detected by this method. The use of mild oxidation conditions renders the method highly selective for the detection of sialic acid-containing glycoproteins.     

One-step purification of murine IgM and human alpha 2-macroglobulin by affinity chromatography on immobilized snowdrop bulb lectin

A new mannose-specific plant lectin (GNA) isolated from the snowdrop bulb was immobilized on Sepharose 4B and employed for the purification of certain glycoproteins with high-mannose type glycan chains. Murine IgM bound tightly to this column and was eluted with 0.1 M methyl alpha-D-mannoside whereas bovine and murine IgG were not bound. When a murine hybridoma serum containing IgM monoclonal antibody was applied to this column, highly purified IgM antibody was obtained after elution with methyl alpha-D-mannoside. On the contrary, human IgM was not bound by this column despite reports that it contains high-mannose type glycan chains. alpha 2-Macroglobulin was the sole glycoprotein present in human serum which was bound by the immobilized snowdrop lectin column. It appears that only glycoproteins containing multiple Man(alpha 1,3)Man units are bound to the immobilized lectin.     

Structural characterization of glycoprotein carbohydrate chains by using diagoxigenin-labeled lectins on blots

The carbohydrate structures of blotted glycoproteins can be analyzed by probing them with lectins. Here we describe a method where lectins conjugated with digoxigenin are used in combination with an anti-digoxigenin antibody AP conjugate as a very sensitive detection system for this type of analysis. The specificity of the lectins used, and the sensitivity of the detection system, provide valuable conclusions on the glycan structures. Only small amounts of glycoproteins are required for the analysis. The binding specificity of a set of lectins is demonstrated with various glycoproteins of defined carbohydrate structure. The application of these labeled lectins in combination with specific glycosidases for the characterization of the carbohydrate chains of recombinant tissue plasminogen activator and erythropoietin is presented.     

India ink staining of proteins on nylon and hydrophobic membranes

India ink was found to be an acceptable stain for proteins blotted or dotted onto positively charged nylon or hydrophobic membranes. The hydrophobic membrane, Immobilon, was an outstanding matrix for binding proteins and displayed low levels of background staining. The least amount of protein detected by india ink staining was between 1.0 and 10 ng. India ink staining of proteins on nylon membranes is an easy, inexpensive, and quick method for the unequivocal detection of both standards and unknowns in the same blot. However, inks, ink concentrations, fixing conditions, staining times, pH, washing conditions, and membrane lots all need to be controlled to achieve maximum sensitivity for protein detection following india ink staining.     

A high resolution PAS stain for polyacrylamide gel electrophoresis

An improved PAS method for the detection of glycoproteins after electrophoresis on acrylamide or urea-acrylamide gels is described. Stronger oxidizing conditions and a more controlled washing for the removal of periodic acid resulted in both increased staining intensity and band resolution. The method will not stain protein and it appears that a 2–3 μg sample of bound or precipitable carbohydrate would easily be detected. Mucopolysaccharides were not detectable by this method.     

Biocytin hydrazide--a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology

Biocytin hydrazide (BCHZ), a new, water-soluble, long-chained, biotin-containing hydrazide, was synthesized and used for the selective nonradioactive detection of glycoconjugates. Procedures were developed for labeling glycoconjugates on blots. The method involves either chemical (periodate-induced) or enzymatic (via galactose oxidase) oxidation of glycoconjugates, the resultant aldehyde groups are then labeled with biocytin hydrazide, followed by interaction with an avidin-based enzyme probe. Since the biotin-containing reagent is a relatively small, charged molecule, the primary labeling step may be carried out on intact cells and on membrane preparations as well as on blotted samples. On blots, the labeling pattern was similar for both periodate- and galactose oxidase-induced biotinylation procedures. In contrast, periodate-induced labeling of either erythrocyte membranes or cells (prior to blotting) produced an altered labeling pattern. Combined enzyme-induced biotinylation of membranes or cells resulted in a pattern similar to that observed for the direct staining of blots. Using galactose oxidase on human erythrocyte membranes, the procedure was sensitive enough to selectively label the Band 3 lactosaminoglycoprotein.     

Enzyme-based detection of glycoproteins on blot transfers using avidin-biotin technology

Avidin-biotin technology has been employed for the improved nonradioactive detection of glycoproteins on blots. Periodate oxidation of samples on blots converts the glycoprotein-based carbohydrate residues to the corresponding aldehydes. The latter undergo interaction with preformed complexes consisting of either avidin hydrazide or streptavidin hydrazide combined with biotinylated alkaline phosphatase. The sensitivity of the new assay exceeds the previously described enzyme hydrazide method by a factor of at least 10. The approach can be rendered selective for sialoglycoproteins, and approximately 12 sugar-containing bands could be observed in erythrocyte membrane preparations. Problems of nonspecific binding and high levels of background label were alleviated using a nonglycosylated basic protein (lysozyme) for quenching.     

Immunochemical detection of glycosphingolipids on thin-layer chromatograms.

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM1, GD1A, and GT1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations.     

N-Linked Glycan Chains on S-Allele-Associated Glycoproteins from Nicotiana alata

The products of the self-incompatibility locus of flowering plants are glycoproteins. The specificity of different alleles at this locus might be expressed through differences in either amino acid sequences or by the glycan substituents. We have investigated the numbers of N-linked glycan chains on the S-glycoproteins and obtained information on their structure by enzymic cleavage with N-glycanase and endo-[beta]-N-acetylglucosaminidase H. In addition to there being variation in the numbers of chains on the S-glycoproteins, each glycoprotein appears to consist of a spectrum of "glycoforms" bearing chains of differing type and fine structure. This microheterogeneity in N-linked glycan chains may be functionally significant.     

Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivalis) bulb

Carbohydrate binding properties of a new plant lectin (GNA) isolated from snowdrop bulbs were studied using the technique of quantitative precipitation, hapten inhibition, and affinity chromatography on immobilized lectin. Purified GNA precipitated highly branched yeast mannans but did not react with most glucans. Hapten inhibition experiments showed that D-mannose is an inhibitor of GNA-mannan interaction but neither N-acetyl-D-mannosamine nor D-glucose is an inhibitor. Hapten inhibition with various sugars showed that GNA requires the presence of equatorial hydroxyl groups at the C-3 and C-4 positions and an axial group at the C-2 position of the D-pyranose ring. A nonreducing terminal D-mannose residue is necessary for the interaction of oligosaccharides, and oligosaccharides with terminal Man(alpha-1-3)Man units showed the highest inhibitory potency (10-30 times greater than D-mannose) among the manno-oligosaccharides tested. The presence of the hydrophobic p-nitrophenyl aglycone increased the affinity of D-mannose only slightly. Immobilized GNA bound yeast mannan but did not bind glycogen. The behavior of glycoproteins with high mannose type glycan chains depended on the density and the structure of their glycan chains. Glycopeptides which carry Man(alpha 1-3)Man units were retarded on the immobilized GNA column whereas those lacking this unit or with hybrid type glycan chains were not retarded on the column.     

Glycomic profiling of developmental changes in bovine testis by lectin histochemistry and further analysis of the most prominent alteration on the level of the glycoproteome by lectin blotting and lectin affinity chromatography

The emerging concept of the sugar code attributes functional significance to oligosaccharides of cellular glycoconjugates by protein (lectin)-carbohydrate interactions. Hence it follows that monitoring of glycan expression (glycomic profiling) is not only valuable to delineate characteristic (phenomenological) changes in the cell's glycosylation but will also come up with the localization of epitopes with potential in biorecognition. It is for this purpose that we have set up a panel of 16 markers (plant lectins and a carbohydrate-specific antibody). The selection met two criteria: a) to be able to detect the common constituents of natural glycans; and b) to place emphasis on detection of neutral carbohydrate units at the spatially accessible branch ends of glycan chains, which are known to be active as ligands for endogenous lectins in situ. Next, we incorporated recent insights into the importance of epitope clustering to turn less abundant oligosaccharides into potent ligands into our study design. To be able to focus on such high-affinity sites, we performed systematic titration studies aimed at defining the probe concentration at which carbohydrate-independent background staining is minimal while still yielding a clear signal. These requirements were met by marker concentrations of 1.25-2.5 microg/ml. Under these conditions, we defined cell-type- and differentiation-dependent changes in bovine testis. Sertoli cells lacked reactivity, whereas gonocytes were differentially reactive with the tested markers. The extent of staining intensity was subject to developmental changes, preferentially for Gal/GalNAc presentation and in this group most prominently with the galactoside-specific lectin from Viscum album L. (mistletoe). Of interest in this context, this lectin is known as a potent mitogen and signal inductor as well as haemagglutinin. The Gal/GalNAc-dependent signals decreased markedly in the course of development and staining was completely lost in the case of mistletoe lectin 12 weeks after gestation. Spermatids of adult testis presented respective glycan epitopes. In contrast to this developmental course of staining, endothelial cells either maintained a constant signal intensity or revealed a signal increase during development for Gal/GalNAc-specific lectins. Their binding of concanavalin A and the two phyto-haemagglutinins (PHA-E/L), which were not or only weakly reactive for gonocytes, served as inherent activity control. Based on lectin blot analysis with the mistletoe lectin as the marker which detected the most prominent change, the glycoprotein patterns from fetal and adult tissue specimens were qualitatively different, rendering changes in expression of the protein part of glycoproteins more likely than remodeling a glycoprotein's glycan chains. Methodologically, results of this procedure were compared to data obtained with lectin affinity chromatography and the combination of the two procedures. Differences in the profiles were discovered that can be assigned to the disparate ways to process the detergent extracts. When access to sample quantity is limited, as is possible in the case of fetal tissue, direct lectin blotting is recommended.     

Accessibility of the carbohydrate moiety of membrane-bound rhodopsin to enzymatic and chemical modification

Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.     

Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background. This can be achieved by the selective isolation of a subproteome, such as N-glycosylated proteins, from the original sample. The present protocol includes the development and optimization of SRM assays associated with each peptide of interest and the qualification of assays in the biological matrix to establish the limits of detection and quantification. The protocol also describes the enrichment of formerly N-glycosylated peptides relying on periodate oxidation of glycan moieties attached to the proteins, their immobilization on solid supports through hydrazide chemistry, proteolysis and enzymatic release of the formerly N-glycosylated peptides.     

Selective labeling of functional groups on membrane proteins or glycoproteins using reactive biotin derivatives and 125I-streptavidin

Amino groups, sulfhydryl groups or oxidation-induced aldehydes on erythrocyte membrane proteins and/or glycoproteins, were reacted with biotinyl N-hydroxysuccinimide ester (BNHS), 3-(N-maleimido-propionyl) biocytin (MPB) or biocytin hydrazide (BCHZ), respectively. The detergent-lysed biotinylated samples were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were transferred onto nitrocellulose membranes. The blot was then incubated with a solution containing 125I-streptavidin, and processed for autoradiography. The advantages of this approach over previously reported procedures for labeling the three functional groups include the following: extremely high sensitivity; short exposure times of autoradiograms and relatively low levels of radioactivity; single-step radiolabeling procedures subsequent to processing and handling of gels and no background labeling in control samples.     

Restricted N-glycan Conformational Space in the PDB and Its Implication in Glycan Structure Modeling

Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan''s crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.     

Optimization of evanescent-field fluorescence-assisted lectin microarray for high-sensitivity detection of monovalent oligosaccharides and glycoproteins

Lectin microarray is an emerging technique, which will accelerate glycan profiling and discovery of glycan-related biomarkers. One of the most important stages in realizing the potential of the technique is to achieve sufficiently high sensitivity to detect even the low concentrations of some target glycoproteins which occur in sera or tissues. Previously, we developed a lectin microarray based on an evanescent-field fluorescence-assisted detection principle that allows rapid profiling of glycoproteins. Here, we report optimization of procedures for lectin spotting and immobilization to improve the sensitivity and reproducibility of the lectin microarray. The improved microarray allows high-sensitivity detection of even monovalent oligosaccharides that generally have a low affinity with lectins (K(d)>10(-6) M). The LOD observed for RCA120, a representative plant lectin, with asialofetuin, and an asialo-biantennary N-glycan probe were determined to be 100 pg/mL and 100 pM, respectively. With the improved lectin microarray system, closely related structural isomers, i.e., Le(a) and Le(x), were clearly differentiated by the difference in signal patterns on relevant multiple lectins, even though specific lectins to detect these glycan structures were not available. The result proved a previously proposed concept of lectin-based glycan profiling.     

Analysis of the proteins,glycoproteins and glycosaminoglycans of fibroblast adhesions to substratum

The focal adhesion preparations which remain attached to a glass substratum when fibroblast bodies are removed by a gentle stream of buffer have been analysed by gel electrophoresis coupled with other selective methods of analysis. The results are consistent with the presence of three classes of macromolecular components. (i) Muscle and associated proteins amongst which actin was abundant with significant amounts of tropomyosin, some myosin and traces of α-actinin. Some vimentin was present but no vinculin. We detected a major new protein component, as yet unidentified, with a molecular weight in the region of 50 000–55 000 which is not desmin or tubulin and could have an important function at the focal adhesion. (ii) Glycoproteins which are a specialised subset of those in the whole plasma membrane and included a family which bind ricin and therefore contain β-galactose end groups, together with a series having carbohydrate chains which bound neither ricin nor concanavalin A. The relative proportion of ricin-binding glycoproteins compared to concanavalin A-binding glycoproteins was higher than in whole plasma membranes. (iii) Glycosaminoglycans, with hyaluronate identified as the major component by column chromatography and its susceptibility to Streptomyces hyaluronidase.     

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.