2-Aminopyridine--a label for bridging of oligosaccharides HPLC profiling and glycoarray printing.

2-Aminopyridine derivatives of oligosaccharides (OS-AP) were printed onto microchips by two different ways. The first method is based on direct covalent insertion of OS-AP in polyacrylamide gel 3D chip. The second method is based on conversion of OS-AP into more reactive OS-aminoalditol followed by covalent printing onto NHS-activated glass slides. This approach extends the range of saccharides suitable for covalent printing due to availability of commercial OS-AP and easy high-performance liquid chromatography separation of glycoprotein N-chains in form of AP derivatives.

Design of carbohydrate multiarrays.

Recently, microarray technology has increasingly been widely applied in glycobiology. This technology has rather evident potential advantages: unlimited number of carbohydrate ligands coated onto one small sized chip, enormously low consumption of both carbohydrate ligands and carbohydrate-binding proteins to be tested, etc. Literature data demonstrate that three approaches are used for glycoarray design. The first one is based on the physical adsorption of glycomolecules on a surface (as in a common ELISA), the second one-on covalent immobilization, and the third one-on a streptavidin-biotin system. In all of the described methods, carbohydrate ligands were placed on chips as a 2D monolayer and high sensitivity was achieved due to fluorescent detection. Notably, a tendency of stepping from model chips toward real multiarrays, where the number of carbohydrate ligands can be up to two hundred, has been observed the last 2 years, this already producing a number of interesting findings when studying carbohydrate-binding proteins. In 2005 new construction, 3D glycochip was described, where 150 mum diameter polyacrylamide gel elements serve as microreactors instead of 2D dots. As a result of the 3D placement of a ligand, two orders of magnitude increase of its density is possible, this providing principal signal improvement during fluorescent detection and increasing method sensitivity. At the same time, carbohydrate consumption is low, i.e., approximately 1 pmol per gel element. Copolymerization chemistry enables the immobilization of several glycomolecule classes to the gel, in particular, aminospacered oligosaccharides, polyacrylamide conjugates, and even 2-aminopyridine derivatives of oligosaccharides, which are widely used in the structural analysis of glycoprotein N-chains.

Hydrogel glycan microarrays.

The technology of hydrogel microchips manufacturing, which was developed previously for covalent immobilization of DNA and proteins, was applied for the preparation of glycochips and combined glyco/protein chips. Microchips consist of hydrogel drops separated with hydrophobic surface. Spacered amino-saccharides and polyacrylamide glycoconjugates were used for immobilization. Gel elements were approximately 1 nl in volume (150 microm in diameter and 25 microm in height), and the amount of covalently immobilized saccharide in the glycoarray was 0.4-1.7 pmol per gel element. Hydrogel glycan microchips were used for quantitative assay of antibodies against blood group antigens and assay of lectins with fluorescent detection. In all cases, only specific interaction with chip-immobilized saccharides was observed, whereas the background signal was very low. The detection limit of on-chip assays was comparable to that of the standard 96-well plate assays. Mixing of reaction solution allowed us to decrease the duration of the assays significantly: 2-3 h for incubation and development steps and 10 min for washing. A method for determination of association constants for binding of compounds with chip-immobilized ligands from the kinetics of their binding is proposed. Combined microchips containing different types of biomolecules can be designed and used for simultaneous detection of different compounds.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Uncharged P-selectin blockers.

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x). Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH2 at C-6 contacted the CH2 groups of the same Lys residue. In vivo, betaMan-SC6H4NO2- p blocked experimental inflammation better than SiaLe(x), but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe(x)-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect.

GlycoChip: multiarray for the study of carbohydrate-binding proteins.

Biotinylated glycoconjugates which were designed as oligosaccharides attached to 30 kDa polyacrylamide were coated on a microarray platform XNAonGOLD, which was developed earlier for an oligonucleotide assay. The specificity of antibodies to carbohydrate antigens was analyzed using the glyco-microarray. Comparison of the obtained results with those of common 96-well plate ELISA completely coincided with the found antibody specificities. However, parameters such as the analytical sensitivity of the method and the amount of biotinylated material coated on the microarray platform proved to be worse than expected.

Fluorescent carbohydrate probes for cell lectins.

Fluorescein labeled carbohydrate (Glyc) probes were synthesized as analytical tools for the study of cellular lectins, i.e. SiaLe(x)-PAA-flu, Sia2-PAA-flu, GlcNAc2-PAA-flu, LacNAc-PAA-flu and a number of similar ones, with PAA a soluble polyacrylamide carrier. The binding of SiaLe(x)-PAA-flu was assessed using CHO cells transfected with E-selectin, and the binding of Sia2-PAA-flu was assessed by COS cells transfected with siglec-9. In flow cytometry assays, the fluorescein probes demonstrated a specific binding to the lectin-transfected cells that was inhibited by unlabeled carbohydrate ligands. The intense binding of SiaLe(x)-PAA-3H to the E-selectin transfected cells and the lack of binding to both native and permeabilized control cells lead to the conclusion that the polyacrylamide carrier itself and the spacer arm connecting the carbohydrate moiety with PAA did not contribute anymore to the binding. Tumors were obtained from nude mice by injection of CHO E-selectin or mock transfected cells. The fluorescent SiaLe(x)-PAA-flu probe could bind to the tumor sections from E-selectin positive CHO cells, but not from the control ones. Thus, these probes can be used to reveal specifically the carbohydrate binding sites on cells in culture as well as cells in tissue sections. The use of the confocal spectral imaging technique with Glyc-PAA-flu probes offered the unique possibility to detect lectins in different cells, even when the level of lectin expression was rather low. The confocal mode of spectrum recording provided an analysis of the probe localization with 3D submicron resolution. The spectral analysis (as a constituent part of the confocal spectral imaging technique) enabled interfering signals of the probe and intrinsic cellular fluorescence to be accurately separated, the distribution of the probe to be revealed and its local concentration to be measured.

Tk, a new colon tumor-associated antigen resulting from altered O-glycosylation.

Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.

[Inhibitory activity of monomeric and polymeric selectin ligands]. / Ingibitornaia aktivnost' monomernykh i polimernykh ligandov selektinov.

The ability of tetrasaccharides (SiaLex, SiaLea, HSO3Lex), their conjugates with polyacrylamide (40 kDa), and several other monomeric and polymeric substances to block selectins has been compared with that of polysaccaride fucoidan. Two assay systems were used: one was constructed on the base of recombinant E-, P-, and L-selectins; the other was a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 microM with the recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-o-sulfate in addition to one of the above mentioned oligosaccharides, were the most potent synthetic blockers. Compared with the most potent of the known inhibitors, fucoidan, bi-ligand glycoconjugate HSO3Lea-PAA-sTyr, displayed in vitro similar activity in blocking L-selectin, while its activity towards P-selectin was ten times lower. All the synthetic polymers tested were able to inhibit neutrophil extravasation to inflammation site, acting in concentration about 10 mg/kg. Thus, the effect of SiaLex is considerably more effective in vivo than in vitro, whereas heavily charged fucoidan and bi-ligand neoglycoconjugate acted in converse manner.

Detection of a potential receptor for the H-blood-group antigen on rat colon-carcinoma cells and normal tissues.

Up-regulation of the synthesis of carbohydrate tumor-associated antigens terminated by the disaccharide Fucalpha1-2Gal is frequent in colon carcinoma and associated with poor prognosis. There is evidence that Fucalpha1-2Gal (H-disaccharide) structures increase cancer-cell motility and tumorigenicity by as-yet unknown mechanisms. Using polyacrylamide-based neoglycoconjugates, we looked for a potential receptor for this disaccharide, and observed that a neoglycoconjugate probe containing the H-disaccharide could bind rat colon-carcinoma cells in a dose-dependent manner, whereas very little binding was evidenced when a probe containing glucose was used. Binding of the H-disaccharide probe could be inhibited by the free H-disaccharide as well as by unlabeled neoglycoconjugates containing a terminal H-disaccharide. The best inhibitor was the H-type-1 trisaccharide neoglycoconjugate. Histochemical detection of the potential H-receptor was performed on rat normal tissues and in situ 1,2-dimethylhydrazine-induced colon carcinomas. A strong binding of the H-disaccharide probe was evidenced on most tumors that could be partly inhibited by the trisaccharide Fucalpha1-2Galbeta1-4Glc and by the unlabeled H-disaccharide neoglycoconjugate, indicating carbohydrate specificity of the binding. Staining of normal colonic mucosa was much weaker. Strong staining was also observed on some normal tissues, such as the spleen or lymph nodes, while others, such as lungs or liver, were negative. Probes containing glucose or the Lewis-a trisaccharide did not stain tumors or normal tissues. These results provide preliminary evidence for the existence of H-specific binding sites, the number of which increases in colon carcinoma.

Determination of carbohydrate specificity of monoclonal antibodies against MUC1.

The carbohydrate specificity of 57 MAbs submitted to the ISOBM TD-4 Workshop on MUC1 were investigated by two versions of ELISA, direct binding and inhibition of binding. The following free saccharides and their polyacrylamide conjugates (Sug-PAA) were used: tetrasaccharides--SiaLe(x), Sia--Le(a); trisaccharides--Le(x), 3'HSO3Le(x), Le(a), 3'HSO3Le(a), 3'SiaLac, Atri, Btri; a number of disaccharides including TF, Hdi, SiaTn, LactNAc, and monosaccharides. It was shown that MAbs 143 and 167 interacted only with SiaLe(x), MAbs 127 and 128 only with Le(x). Antibodies 123 and 164 interacted preferably with Le(a) but also recognized Le(c). Antibody 151 recognized alpha GalNAc (Tn) and cross-reacted with beta GalNAc. Antibody 157 displayed high affinity to Atri and Atetr (type 1). Neither anti-TF nor anti-SiaTn antibodies were revealed.

Carbohydrate-based probes for detection of cellular lectins.

Carbohydrate (spacered saccharide residue, Glyc) probes with various tags were synthesized as analytical tools for study of cellular lectins, i.e., Glyc-polyacrylamide-3H, Glyc-PAA-biotin, Glyc-PAA-fluorescein (flu), and Glyc-PAA-digoxigenin, where PAA is a soluble polyacrylamide carrier of approximately 30 kDa. Binding of all types of probes, where Glyc is the sialyl Lewis X (SiaLeX) tetrasaccharide or a blank saccharide, was assessed using Chinese hamster ovary (CHO) cells either transfected with the E-selectin cDNA or mock-transfected. High binding of SiaLeX-PAA-3H to E-selectin-transfected cells and absence of binding to control cells (both native and permeabilized) allowed the conclusion that the polyacrylamide carrier and the spacer arm do not contribute significantly to the binding. The biotinylated probe showed a high level of nonspecific binding in cell enzyme-linked assays. A similarly built digoxigenin-labeled probe was significantly better. In flow cytometry assays, the fluorescein probe demonstrated a specific binding to E-selectin-transfected cells of a similar level to that given by an anti-E-selectin antibody. In addition, it could be inhibited by the anti-E-selectin antibody, further demonstrating specificity. Tumors were obtained from nude mice by injection of CHO E-selectin or mock-transfected cells. The fluorescent SiaLeX-PAA-flu probe could bind to tumor sections from E-selectin-positive CHO cells, but not from control CHO cells. These probes can thus be used to reveal specifically complex carbohydrate-binding sites on cells either in culture or on tissue sections.

Further refinement of the description of the ligand-binding characteristics for the galactoside-binding mistletoe lectin, a plant agglutinin with immunomodulatory potency.

The galactoside-binding lectin from mistletoe (Viscum album L.) is a biological response modifier, eliciting e.g. enhanced secretion of cytokines. This immunological activity warrants the further analysis of its ligand-binding properties with special attention paid to blood group epitopes. To avoid the microheterogeneity and complexity of naturally occurring glycoproteins, chemically strictly defined neoglycoconjugates and a panel of synthetic oligosaccharides were employed in solid-phase assays for direct binding and assessment of the relative inhibitory capacity. Since label incorporation into the lectin, although performed under protective conditions, or surface immobilization by adsorption to plastic may affect its affinity characteristics, the extent of neoglycoconjugate binding in the absence of any interfering substance and in the presence of oligosaccharides was determined comparatively with labeled and with immobilized lectin. In principle, these two factors could be excluded to markedly alter binding features. In addition to lactose, the blood group determinants H and B were strongly reactive. A fucose residue can thus especially be accommodated to the binding site when linked to the non-reducing unit. N-Acetyllactosamine was nearly as potent as an inhibitor as lactose. Lec and the A determinant were notably inferior to the other ABH blood group epitopes. Le(a) and Le(x) and their sialylated derivatives displayed only very weak binding capacity. Among the two natural isomers of sialyllactose, the alpha 2,6-form displayed a higher level of inhibitory capacity than the alpha 2,3-derivative. Isomeric variants of the Thomsen-Friedenreich antigen, too, reduced lectin binding to the lactose-carrying polymer. Their capacities were surpassed by those of the H and the B determinants and a related form of the latter, the P1 epitope. An overlap of specificity with the immunomodulatory human galectin-3 is thus measurable for H/B-like structures. The documented differential reactivity of the mistletoe lectin to blood group oligosaccharides may have a bearing on the responsiveness of blood group-positive cell populations.

[Synthesis of thioacetamide analogs of blood group specific trisaccharide A and its fragments]. / Sintez tioatsetamidnykh analogov gruppospetsificheskogo trisakharida krovi A i ego fragmentov.

The determinant trisaccharide of the blood group A, GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta, was modified by treatment of the trisaccharide acetate with phosphorus pentasulphide to convert its acetamide fragment determining the serological difference between antigenic determinants A and B into the thioacetamide derivative. Thionated analogues of the A disaccharide and of GalNAc alpha-glycoside were obtained in a similar way. Interaction of three monoclonal anti-A antibodies with modified antigens was studied using ELISA, the O-->S replacement allowing to evaluate the contribution of the acetamido carbonyl group into the antigen-antibody interaction.

Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides.

Several types of polymeric glycoconjugates, N-substituted polyacrylamides, have been synthesized by the reaction of activated polymers with omega-aminoalkylglycosides: (i) (carbohydrate-spacer)n-polyacrylamide, 'pseudopolysaccharides'; (ii) (carbohydrate-spacer)n-phosphatidylethanolaminem-polyacrylamide, neoglycolipids, derivatives of phosphatidylethanolamine; (iii) (carbohydrate-spacer)n-biotin-polyacrylamide, biotinylated probes; (iv) (carbohydrate-spacer)n-polyacrylamide-(macroporous glass), affinity sorbents based on macroporous glass, covalently coated with polyacrylamide. An almost quantitative yield in the conjunction reaction makes it possible to insert in the conjugate a predetermined quantity of the ligand(s). Pseudopolysaccharides proved to be a suitable form of antigen for activation of polystyrene and poly(vinyl chloride) plates (ELISA) and nitrocellulose membranes (dot blot), being advantageous over traditional neoglycoproteins. Polyvalent glycolipids insert well in biological membranes: their physical properties, particularly solubility, can be changed in a desired direction. Biotinylated derivatives were used as probes for detection and analysis of lectins.

[Epitope specificity of hemagglutinating monoclonal anti-B antibodies]. / Epitopnaia spetsifichnost' gemaggliutiniruiushchikh monoklonal'nykh anti-B-antitel.

Fine epitope specificity of ten monoclonal antibodies (MA) agglutinating red blood cells B was studied. Three methods were used: 1) inhibition of MA binding to natural antigen by synthetic oligosaccharides (OS) and their polyacrylamide conjugates, 2) direct MA binding to a series of synthetic OS-polyacrylamide conjugates differing in carbohydrate epitope density, 3) direct MA binding to the affinity sorbents. It is shown that all antibodies studied prefer trisaccharide B determinant Gal alpha 1-3(Fuc alpha 1-2) Gal independently of their ability to discriminate serological subgroups of B erythrocytes (B, Bweak, B3). The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells B subgroups is discussed. Of an interest is that MAs which are able to agglutinate any B subgroups also bing the synthetic tetrasaccharide Gal alpha 1-3(Fuc alpha 1-2)Gal beta 1-3GalNAc, a B type 3 determinant.

[Epitope specificity of hemagglutinating monoclonal anti-A-antibodies]. / Epitopnaia spetsifichnost' gemaggliutiniruiushchikh monoklonal'nykh anti-A-antitel.

Fine epitope specificity of three anti-A monoclonal antibodies (MA) 1H410, 3F9, and 44F9 was studied by: 1) direct MA binding to synthetic oligosaccharides (OS) linked to polyacrylamide matrix, and 2) inhibition of MA binding to natural antigen by synthetic OS and their polyacrylamide conjugates. It has been established that the antigen binding site of MA 1H10 is specific for tetrasaccharide A (type 3), whereas MAs 3F9 and 44F9 recognize trisaccharide A, the contribution of alpha-L-fucosyl residue being insignificant in the case of 44F9 binding. The correlation of the MAs epitope specificity with their ability to agglutinate red blood cells of A1 and weak A subgroups is discussed.