The elimination ofO-linked glycans from glycoproteins under non-reducing conditions

A simple procedure is described for the elimination ofO-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive -elimination ofO-glycans.     

Transport and posttranslational processing of the vacuolar enzyme α-mannosidase in jack-bean cotyledons

-Mannosidase (EC 3.2.1.24) is a vacuolar enzyme which occurs abundantly in the cotyledons of the jack-bean (Canavalia ensiformis (L.) DC). The mature enzyme is a tetramer with two polypeptides each of relative molecular mass (M_r) 66000 and M_r 44000. The enzyme has an interesting molecular structure because in its native form, it does not bind to concanavalin A (ConA) in spite of the presence of a high-mannose glycan. -Mannosidase is synthesized in the developing cotyledons of jack-beans at the same time as the abundant proteins canavalin and ConA. The enzyme is synthesized as a precursor which has an M_r of 110000 and is associated with the endoplasmic reticulum (ER). Antibodies against the deglycosylated subunits cross-react with the M_r-110000 precursor. Processing of the precursor to the constituent polypeptides occurs posttranslationally, probably in the protein bodies. Immunocytochemical evidence shows that -mannosidase is present in the ER and the Golgi complex of developing cells, and accumulates in the protein bodies.Labeling with [³H]glucosamine shows that after processing only the M_r-66000 polypeptide has glucosamine-containing glycans. The synthesis of these glycans is inhibited by tunicamycin, indicating that they are asparagine-linked oligosaccharides. Analysis of the glycans shows that there is a large glycan that is retained by ConA and a small glycan that is not retained by ConA. The large glycan is only partially sensitive to -mannosidase because of the presence of a terminal glucose residue. Cross-reaction of the large subunit with an antiserum directed against small, complex glycans of plant glycoproteins indicates that this polypeptide probably has a xylose-containing glycan. Pulse-chase experiments carried out in the presence of tunicamycin show that the presence of glycans is not required for transport of -mannosidase out of the ER-Golgi system.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - H L heavy, light subunit - IgG Immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

Detection of human asialo-α1-acid glycoprotein using a heterosandwich immunoassay in conjunction with the light addressable potentiometric sensor

Highly specific detection of human ₁-acid glycoprotein (AGP) and asialo-₁-acid glycoprotein (asialo-AGP) was made possible by use of a sandwich immunoassay. The glycoproteins were sandwiched between biotinylated and fluoresceinated polyclonal rabbit anti-human AGP antibodies. Additionally, asialo-AGP could be distinctly detected, apart from AGP, via the formation of a heterosandwich immunoassay using biotinylated polyclonal rabbit antihuman AGP and the lectin, fluoresceinated ricin toxin. Streptavidin was added to the formed immunocomplexes and the immunocomplexes captured on a biotinylated nitrocellulose membrane. The signal generator, urease conjugate of an anti-fluorescein antibody, was then bound to the complex on membrane. The rate of pH change under microvolume conditions (0.6 µl) was monitored using a silicon chip-based, light addressable potentiometer sensor. Results indicated that AGP and asialo-AGP can be detected to the 2 pg level when two antibodies are used to form the immunocomplex. Asialo-AGP can be detected down to 250 pg when the heterosandwich immunoassay is used; this assay exhibited no response up to 10 ng for native AGP or asialofetuin. Both immunoassays can be used to quantify the level of AGP and asialo-AGP in solution. Although the assay presented is very specific for AGP, asialo-AGP and terminal galactose, it is readily adaptable for the detection of any glycoprotein and terminal carbohydrate (or branched structure) by use of a protein-specific antibody and various lectins.     

The use of avidin-biotinylglycan as the model for in vitro glycoprotein processing

In an attempt to evaluate the effects of the protein matrix on the specificity of glycoprotein processing in Golgi membranes, we have developed a model neoglycoprotein consisting of biotinylated glycans bound noncovalently to avidin (Chen, V. J., and Wold, F. (1986) Biochemistry 25, 939-444) with which the protein effect on processing can be evaluated as the difference in substrate efficiency between a free biotinylated glycan and the same biotinylated glycan bound to avidin. The avidin (streptavidin)-glycan complex stability was found to be proper for the experimental design; the complex remains intact for extended periods of incubation at the concentrations used, but the glycan can be completely liberated and recovered by heating the complex at 95 degrees C for 10 min in the presence of a 10-fold molar excess of biotin. By measuring the relative rates of [14C]sugar incorporation into the free and bound substrates it was demonstrated that the protein indeed influences the processing reactions; under conditions where free glycans such as biotinyl-Asn-Glc-NAc2-Man5 and 6-(biotinamido)hexanoyl-Asn-Glc-NAc2-Man5 could be converted to the biantennary products R-Asn-GlcNAc2-Man3-GlcNAc2-Gal2-sialyl2 in the presence of UDP-GlcNAc, UDP-Gal and CMP-sialic acid and Golgi enzymes, the avidin-bound derivative without the extension arm gave only low levels of product and the streptavidin-bound one remained unaltered. The presence of the extension arm in the substrates significantly improved the yield of some products in the complex, apparently by reducing or eliminating the avidin inhibition of the early steps, but not of the late ones. There are consequently two types of effect of the protein matrix on processing efficiency. One is expressed only when the glycan is close to the protein surface and affecting primarily early steps (mannosidases and GlcNAc transferases). The other is apparently independent of the proximity of the glycan core and the protein, and affects primarily late steps, in particular the incorporation of the second sialic acid residue into a biantennary complex glycan.     

Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry

Characterization of protein-carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH-glycan fractions are added, and bound BACH-glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH-glycan ligands on streptavidin-coated surfaces provide more information on glycan-CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH-glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins.     

Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P₂, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5-[-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P₂. Activation of the GTPase activity of dynamin II by pure POPC was then shown.     

Constitutive overexpression of a ripening-related pepper endo-1,4-β-glucanase in transgenic tomato fruit does not increase xyloglucan depolymerization or fruit softening

The ripening-related pepper endo-1,4--D-glucanase (EGase) CaCel1 was over-expressed in transgenic tomato plants under the control of the constitutive 35S promoter to investigate the effects on plant growth and fruit softening of high levels of a potential cell wall-degrading activity. In transgenic fruit, recombinant CaCel1 protein was associated with a high-salt putative cell wall fraction, and extractable CMCase activity was increased by up to 20-fold relative to controls. However, the effects of high levels of EGase activity on fruit cell wall metabolism were relatively small. The largest consequence observed was a decrease of up to 20% in the amount of matrix glycans in a 24% KOH-soluble fraction consisting of polysaccharides tightly bound to cellulose. This decrease was confined to polysaccharides other than xyloglucan, did not affect the size distribution of remaining molecules, and was not correlated with a corresponding increase in glycans in a 4% KOH-soluble fraction loosely bound to cellulose, suggesting that the missing polymers had been degraded to fragments small enough to be lost from the extracts. The amount of matrix glycans in the 4% KOH-soluble fraction was not substantially changed, but the size distribution showed a small relative increase in the amount of polymers in a peak eluting close to a linear dextran marker of 71 kDa. This could be due either to an increase in the amount of polymers of this size, or to a loss from the extract of other polymers present in peaks of higher molecular weight. Transgenic fruit were not softer than controls but appeared the same or slightly firmer at both green and red developmental stages, and no differences in plant vegetative growth were observed. CaCel1 did not cause depolymerization of tomato fruit xyloglucan in vivo, but differences in the amount or molecular weight profile of other matrix glycans were observed. The data suggest that degradation of a proportion of matrix glycans other than xyloglucan does not result in fruit softening, and that fruit softening is not limited by the amount of EGase activity present during ripening.     

Sensitive Nonradioactive Method for Screening Compounds Interacting with Neuronal α7 Cholinoreceptor

A sensitive nonradioactive method for detection of substances interacting with the neuronal 7-type nicotinic acetylcholine receptor (AChR) was proposed. The method uses biotinylated -cobratoxin (Bt-CTX) and is based on the ability of the N-terminal ligand-binding extracellular domain (LBED) of AChR to interact with -cobratoxin (CTX) as does the whole receptor. LBED produced by heterologous expression of a gene fragment of the 7 subunit of AChR from the rat brain in Escherichia coli cells was sorbed in wells of a 96-well plate and incubated with Bt-CTX. The specifically bound Bt-CTX was determined by staining with streptavidin–peroxidase complex. The ability of other compounds to interact with 7-AChR was checked according to the degree with which they inhibit the Bt-CTX binding to LBED. Nicotine, carbamylcholine, d-tubocurarin, anabaseine, conotoxin ImI, and neurotoxin II were used as model compounds. The sensitivity of this method was comparable with that of the radioligand method (up to 10 pmol).     

Purification and partial characterization of an acid β-fructosidase from sweet-pepper (Capsicum annuum L.) fruit

The present study describes the biochemical characteristics of an acid -fructosidase (EC 3.2.1.26) purified from the fruit of sweet pepper (Capsicum annuum L.). The soluble form, which constitutes more than 95% of the total activity at pH 4.5, hydrolyzes sucrose, raffinose, and stachyose. Its pH and temperature optima are 4.5 and 55 °C, respectively. Metal cations such as Ag⁺ and Hg²⁺ strongly inhibit its activity, suggesting the presence of at least one sulfhydryl group at the catalytic site. After purification of the enzyme by means of ammonium sulfate fractionation, gel chromatography (diethyl-aminoethyl-Sephacel, hydroxylapatite, concanavalin A-Sepharose), and preparative gel electrophoresis, the purified enzyme was shown to be a 42 kDa glycoprotein interacting specifically with concanavalin A. After complete chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight of the constitutive polypeptide was estimated to be 39 kDa. The enzyme glycans were characterized using both affino- and immunodetection. The enzyme has at least two N-linked oligosaccharide sidechains, one of the high-mannose type, and the other of the complex type. The high-mannose glycan has a low molecular weight (1 kDa), and is responsible for the interaction between the enzyme and concanavalin A. The complex-type glycan has an estimated molecular weight of 2 kDa. It contains one 1 2-linked xylose residue, probably one fucose residue 1 3-linked to the chitobiose unit, and no terminal galactose residue. The two glycans, associated to the 39 kDa polypeptide, constitute the acid -fructosidase of the sweet-pepper fruit.Abbreviations F -fructosidase - ConA concanavalin A - DEAE diethylaminoethyl - DTNB dithionitrobenzoic acid - endo F endo--N-acetylglucosamidase F - endo H endo--N-acetylglucosamidase H - NEM N-ethylmaleimide - PCMB parachloromercurobenzoate - PNGase glycopeptide-N-glycosidase - TFMS trifluoromethane sulfonic acid This work was partly supported by a grant from the Commission Permanente de Coopération Franco-Québécoise to L. Faye, and S. Yelle. D. Michaud was a recipient of a graduate scholarship from the Natural Science and Engineering Research Council of Canada.     

Differentiation-dependent glycosylation of gp190, an oncofetal crypt cell antigen expressed by Caco-2 cells

gp190 is a glycoprotein expressed on the cell surface of several human colon carcinoma cells in culture, on epithelial cells of fetal colon, but not on the normal mucosa of adult colon; thus it is referred to as an oncofetal crypt cell antigen. We report the characterisation of O[emsp4 ]-linked glycans carried by gp190 synthesised by [³H]glucosamine-labelled Caco-2 cells at the confluence (undifferentiated cells) and at three weeks of postconfluence (differentiated cells). By using a specific monoclonal antibody, gp190 was isolated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mobility of gp190 from differentiated cells was found to be lower than that from undifferentiated cells, suggesting a more extensive glycosylation process in the former glycoprotein. The major results of the glycan characterisation have been as follows: (i) gp190 carries mainly, if not exclusively, O-linked glycans with the core-2 structure; (ii) the elongation with N-acetyllactosamine units of the Gal1,4GlcNAc1,6(Gal1,3)GalNAc tetrasaccharide predominates in gp190 synthesised by differentiated cells, whereas the direct 2,3sialylation of the tetrasaccharide is prevalent in gp190 synthesised by undifferentiated cells. The increment in the core-2 1,6GlcNAc-transferase activity under the Caco-2 differentiation process may be relevant in producing the larger occurrence of polylactosaminoglycans in gp190 from differentiated cells. Since no change in the activity of the 2,3sialyltransferases upon cell differentiation was observed, we suggest that the lower 2,3sialylation in gp190 synthesised by polarised cells might be due to a changed transit-rate through the distal Golgi apparatus.     

Bound conformations for ligands for G-protein coupled receptors

The conformation of the C-terminus of the -subunit of transducin, the G-protein of vision, has been determined by transfer NOE when bound to activated (MII) rhodopsin. One hundred three new NOE constraints are apparent when light is shown on a mixture of rhodopsin bilayers and the undecapeptide. Analogs of the -peptide with covalent constraints were designed restricting the bound conformation; they stabilize MII thus supporting the deduced structure. The NMR structure of a complex of the intracellular loops of rhodopsin facilitates docking of the -peptide and also shows proximity of residues known by mutational analysis to interact to generate the activated rhodopsin-transducin interface. This constrains the location of transmembrane helices in the structure of activated rhodopsin. Methods for the prediction of affinity have been used to estimate the relative binding constants of peptide analogs with the loop complex and show strong correlation with experimental data. Various models of the rhodopsin-transmembrane helical segments have been computationally fused with distance geometry to determine the overall model which best fits the experimental data on the rhodopsin-transducin interface.     

Plug-and-Play Pairing via Defined Divalent Streptavidins

Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4 Å resolution and trans-divalent streptavidin to 1.6 Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.     

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

We have shown that recombinant forms of VP8* domains of the human rotavirus outer capsid spike protein VP4 from human neonatal strains (N155(G10P[11]) and RV3(G3P[6]) and a bovine strain (B223) recognize unique glycans within the repertoire of human milk glycans. The accompanying study by Yu et al.², describes a human milk glycan shotgun glycan microarray that led to the identification of 32 specific glycans in the human milk tagged glycan library that were recognized by these human rotaviruses. These microarray analyses also provided a variety of metadata about the recognized glycan structures compiled from anti-glycan antibody and lectin binding before and after specific glycosidase digestions, along with compositional information from mass analysis by matrix-assisted laser desorption ionization-mass spectrometry. To deduce glycan sequence and utilize information predicted by analyses of metadata from each glycan, 28 of the glycan targets were retrieved from the tagged glycan library for detailed sequencing using sequential disassembly of glycans by ion-trap mass spectrometry. Our aim is to obtain a deeper structural understanding of these key glycans using an orthogonal approach for structural confirmation in a single ion trap mass spectrometer. This sequential ion disassembly strategy details the complexities of linkage and branching in multiple compositions, several of which contained isomeric mixtures including several novel structures. The application of this approach exploits both library matching with standard materials and de novo approaches. This combination together with the metadata generated from lectin and antibody-binding data before and after glycosidase digestions provide a heretofore-unavailable level of analytical detail to glycan structure analysis. The results of these studies showed that, among the 28 glycan targets analyzed, 27 unique structures were identified, and 23 of the human milk glycans recognized by human rotaviruses represent novel structures not previously described as glycans in human milk. The functional glycomics analysis of human milk glycans provides significant insight into the repertoire of glycans comprising the human milk metaglycome.     

Separation and characterization of the complexes constituting the cellulolytic enzyme system of Clostridium thermocellum

Clostridium thermocellum, strain JW20 (ATCC 31449) when growing in cellulose produces a cellulolytic enzyme system, that at the early stage of the fermentation is largely bound to the substrate. As cellulose is consumed the bound enzyme is released as free enzyme to the culture fluid. The bound enzyme fraction extracted with distilled water from the cellulose contains two major components, a large complex (M_r100×10⁶) and a small complex M_r4.5×10⁶) which were separated by gel filtration and sucrose solved by affinity chromatography into a complex that binds to the column and into a non-bindable mixture of proteins. All four fractions have endo--glucanase activity but only the two bound complexes and the free bindable complex hydrolyze crystalline cellulose with cellobiose as the main product. These three complexes are qualitatively similar in that they each contain about 20 different polypeptides (M_r values from 45,000 to 200,000) of which about ten are major components. However, the relative amounts of some of the peptides in the complexes differ. At least four polypeptides of the complexes have endo--glucanase activity.Abbreviations CM cellulose, carboxymethyl cellulose - CMCase carboxymethyl cellulase cosidered endo--1,4-glucanase - SDS sodium dodecyl sulfate - YAS yellow affinity substance - YAS-cellulose yellow affinity substance-cellulose complex     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.     

Insight into the Regulation of Glycan Synthesis in Drosophila Chaoptin Based on Mass Spectrometry

Background

A variety of N-glycans attached to protein are known to involve in many important biological functions. Endoplasmic reticulum (ER) and Golgi localized enzymes are responsible to this template-independent glycan synthesis resulting glycoforms at each asparagine residues. The regulation mechanism such glycan synthesis remains largely unknown.

Methodology/Principal Findings

In order to investigate the relationship between glycan structure and protein conformation, we analyzed a glycoprotein of Drosophila melanogaster, chaoptin (Chp), which is localized in photoreceptor cells and is bound to the cell membrane via a glycosylphosphatidylinositol anchor. Detailed analysis based on mass spectrometry revealed the presence of 13 N-glycosylation sites and the composition of the glycoform at each site. The synthetic pathway of glycans was speculated from the observed glycan structures and the composition at each N-glycosylation site, where the presence of novel routes were suggested. The distribution of glycoforms on a Chp polypeptide suggested that various processing enzymes act on the exterior of Chp in the Golgi apparatus, although virtually no enzyme can gain access to the interior of the horseshoe-shaped scaffold, hence explaining the presence of longer glycans within the interior. Furthermore, analysis of Chp from a mutant (RNAi against dolichyl-phosphate α-d-mannosyltransferase), which affects N-glycan synthesis in the ER, revealed that truncated glycan structures were processed. As a result, the distribution of glycoforms was affected for the high-mannose-type glycans only, whereas other types of glycans remained similar to those observed in the control and wild-type.

Conclusions/Significance

These results indicate that glycan processing depends largely on the backbone structure of the parent polypeptide. The information we obtained can be applied to other members of the LRR family of proteins.     

Preparative affinity electrophoresis of different glycoforms of serum glycoproteins: Application for the study of inflammation-induced expression of sialyl-Lewisx groups onα 1-acid glycoprotein (orosomucoid)

During acute inflammation, human ₁-acid glycoprotein (AGP) is subject to marked changes in branching of its glycans, its degree of fucosylation and the expression of sialyl-Lewis^x)(SLe^x) groups. To be able to study these changes in glycosylation in more detail, a procedure was developed to isolate the different glycoforms of AGP in milligram amounts by preparative affinity electrophoresis (AE) with a free lectin as fractionating agent. The method was applied to isolate differently fucosylated forms of AGP with the fucose-specific lectinAleuria aurantia (AAL). AGP was separated into one non-reactive (AO) and four reactive (A1-A4) fractions. It was found that, in particular, the highly fucosylated fractions A3 and A4 contained the inflammation-induced SLe^x groups of AGP. Analysis by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A (Con A) of these different glycoforms of AGP showed that the presence of tri-and/or tetraantennary glycans, instead of diantennary glycans, was associated with a higher degree of fucosylation. Identical results were obtained by subjecting Con A-fractionated forms of AGP to CAIE with AAL as the affinocomponent. It is expected that this method of preparative AE can generally be applied to other glycoproteins, which can be separated in different glycoforms by CAIE using lectins.Part of this work was published in abstract form,Glycoconjugate J 1993;10; 317.     

A monoclonal antibody for terminal β-galactose. Use in analysis of glycosphingolipids

A monoclonal antibody (mAb 8281) specific for the terminal -galactose (Gal) of glycosphingolipids (GSL) and glycoproteins was produced from mice immunized with lipid extract from fresh acute lymphocytic leukemia (ALL) cells. Immuno-thin layer chromatography (ITLC) and competition assays with purified neutral GSL standards, free sugars, and synthetic neoglycoproteins showed mAb 8281 to be strongly reactive with LacCer, GalCer and Gal--O-(CH₃)₂S(CH₃)₂-CONH-(Gal--O-CETE) linked to bovine serum albumin (BSA). The penultimate sugar also played a role in binding. The antibody was not reactive with carbohydrates with terminal Gal structures and unrelated terminal moieties. Indirect immunoperoxidase staining and flow cytometry with mAb 8281 demonstrated positive staining on numerous tissues, including smooth muscle, gastrointestinal mucosa, lymph node B cells and monocytes. ITLC analysis of the GSL composition of fresh B cell neoplasms using mAb 8281 confirmed the presence of lactosylceramide and galactosylceramide in neoplasms of varying stages of differentiation. Because of its specificity for terminal Gal carbohydrate residues, mAb 8281 may be useful in structural and functional analyses of GSL.     

Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1

Introduction: Protein glycosylation is recognized as an important post-translational modification, with specific substructures having significant effects on protein folding, conformation, distribution, stability and activity. However, due to the structural complexity of glycans, elucidating glycan structure-function relationships is demanding. The fine detail of glycan structures attached to proteins (including sequence, branching, linkage and anomericity) is still best analysed after the glycans are released from the purified or mixture of glycoproteins (glycomics). The technologies currently available for glycomics are becoming streamlined and standardized and many features of protein glycosylation can now be determined using instruments available in most protein analytical laboratories.

Areas covered: This review focuses on the current glycomics technologies being commonly used for the analysis of the microheterogeneity of monosaccharide composition, sequence, branching and linkage of released N- and O-linked glycans that enable the determination of precise glycan structural determinants presented on secreted proteins and on the surface of all cells.

Expert commentary: Several emerging advances in these technologies enabling glycomics analysis are discussed. The technological and bioinformatics requirements to be able to accurately assign these precise glycan features at biological levels in a disease context are assessed.