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Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs., (© 2016 AlphaMed Press.)

Stem cells have a unique ability to self-renew and differentiate into diverse cell types. Currently, stem cells from various sources are being explored as a promising new treatment for a variety of human diseases. A diverse set of functional and phenotypical markers are used in the characterization of specific therapeutic stem cell populations. The glycans on the stem cell surface respond rapidly to alterations in cellular state and signaling and are therefore ideal for identifying even minor changes in cell populations. Many stem cell markers are based on cell surface glycan epitopes including the widely used markers SSEA-3, SSEA-4, Tra 1-60, and Tra 1-81. We have now discovered by mRNA analysis that a novel glycosyltranferase, epidermal growth factor (EGF) domain-specific O-linked GlcNAc transferase (EOGT), is highly expressed in stem cells. EOGT is responsible for adding O-linked N-acetylglucosamine (O-GlcNAc) to folded EGF domains on extracellular proteins, such as those on the Notch receptors. We were able to show by immunological assays that human umbilical cord blood-derived mesenchymal stromal cells display O-GlcNAc, the product of EOGT, and that O-GlcNAc is further elongated with galactose to form O-linked N-acetyllactosamine. We suggest that these novel glycans are involved in the fine tuning of Notch receptor signaling pathways in stem cells.

We have recently developed an in vitro culture model enabling the large-scale expansion of switched-memory B lymphocytes, producing a polyclonal human IgG repertoire. Given the importance of glycosylation for the functions of immunoglobulins, we analyzed the N-glycosylation profiles of the immunoglobulin G (IgG) in this model. Switched-memory B cells were cultured for 38 days and, using liquid chromatography-mass spectrometry, we analyzed IgGs' glycosylation profiles which were then compared to the glycosylation patterns of commercial intravenous immunoglobulin (IVIG). We observed a reproducible proliferation rate, high viability through the cultures as well as a good maintenance of the switched-memory B cells repertoire. The glycosylation pattern analyses revealed a variety of the typical biantennary N-glycan structures with diverse terminal monosaccharides. While many similarities were detected in comparison to the glycosylation profile of IVIG, in vitro-produced polyclonal IgGs were bearing higher levels of bisecting GlcNAc known to affect the effector functions of therapeutic antibodies. This data highlights the need for monitoring of the glycoform distribution in antibodies produced in vitro., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology.

There is an increasing interest in the modification of cell surface glycosylation to improve the properties of therapeutic cells. For example, glycosylation affects the biodistribution of mesenchymal stromal cells (MSCs). Metabolic glycoengineering is an efficient way to modify the cell surface. The mammalian biosynthetic machinery tolerates the unnatural sialic acid precursor, N-propanoylmannosamine (ManNProp), and incorporates it into cell surface glycoconjugates. We show here by mass spectrometric analysis of cell surface N-glycans that about half of N-acetylneuraminic acid was replaced by N-propanoylneuraminic acid in the N-glycans of human umbilical cord blood-derived MSCs supplemented with ManNProp. In addition, the N-glycan profile was altered. ManNProp-supplemented cells had more multiply fucosylated N-glycan species than control cells. The fucosylated epitopes were shown in tandem mass spectrometric analysis to be Lewis x or blood group H epitopes, but not sialyl Lewis x (sLex). The amounts of tri- and tetra-antennary and polylactosamine-containing N-glycans also increased in ManNProp supplementation. In accordance with previous studies of other cell types, increased expression of the sLex epitope in ManNProp-supplemented MSCs was demonstrated by flow cytometry. In light of the N-glycan analysis, the sLex epitope in these cells is likely to be carried by O-glycans or glycolipids. sLex has been shown to target MSCs to bone marrow, which may be desirable in therapeutic applications. The present results represent the first structural analysis of an N-glycome of ManNProp-supplemented cells and demonstrate the feasibility of modifying cell surface glycosylation of therapeutic cells by this type of metabolic glycoengineering.

Reversed-phase liquid chromatography on the nanoscale coupled to electrospray tandem mass spectrometry was used to analyse a mixture of four commercial glycan standards, and the method was further adapted to N-glycans enzymatically released from alpha-1-acid glycoprotein and immunoglobulin gamma. Glycans were permethylated to enable their separation by reversed-phase chromatography and to facilitate interpretation of fragmentation data. Prior to derivatization of glycans by permethylation, they were reduced to cancel anomerism because, although feasible, it was not desired to separate α- and β-anomers. The effect of supplementing chromatographic solvent with sodium hydroxide to guide adduct formation was investigated. Raising the temperature in which the separation was performed improved chromatographic resolution and affected retention times as expected. It was shown by using the tetrasaccharides sialyl Lewis X and sialyl Lewis A that reversed-phase chromatography could achieve the separation of methylated isobaric glycan analytes. Isobaric glycans were detected among the N-glycans of immunoglobulin gamma and further analysed by tandem mass spectrometry.

Despite recent technical advances in glycan analysis, the rapidly growing field of glycomics still lacks methods that are high throughput and robust, and yet allow detailed and reliable identification of different glycans. LC-MS-MS(2) methods have a large potential for glycan analysis as they enable separation and identification of different glycans, including structural isomers. The major drawback is the complexity of the data with different charge states and adduct combinations. In practice, manual data analysis, still largely used for MALDI-TOF data, is no more achievable for LC-MS-MS(2) data. To solve the problem, we developed a glycan analysis software GlycanID for the analysis of LC-MS-MS(2) data to identify and profile glycan compositions in combination with existing proteomic software. IgG was used as an example of an individual glycoprotein and extracted cell surface proteins of human fibroblasts as a more complex sample to demonstrate the power of the novel data analysis approach. N-glycans were isolated from the samples and analyzed as permethylated sugar alditols by LC-MS-MS(2), permitting semiquantitative glycan profiling. The data analysis consisted of five steps: 1) extraction of LC-MS features and MS(2) spectra, 2) mapping potential glycans based on feature distribution, 3) matching the feature masses with a glycan composition database and de novo generated compositions, 4) scoring MS(2) spectra with theoretical glycan fragments, and 5) composing the glycan profile for the identified glycan compositions. The resulting N-glycan profile of IgG revealed 28 glycan compositions and was in good correlation with the published IgG profile. More than 50 glycan compositions were reliably identified from the cell surface N-glycan profile of human fibroblasts. Use of the GlycanID software made relatively rapid analysis of complex glycan LC-MS-MS(2) data feasible. The results demonstrate that the complexity of glycan LC-MS-MS(2) data can be used as an asset to increase the reliability of the identifications.

Multipotent mesenchymal stem cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. However, there is a lack of methods to quickly and efficiently isolate, characterize, and ex vivo expand desired cell populations for therapeutic purposes. Single markers to identify cell populations have not been characterized; instead, all characterizations rely on panels of functional and phenotypical properties. Glycan epitopes can be used for identifying and isolating specific cell types from heterogeneous populations, on the basis of their cell-type specific expression and prominent cell surface localization. We have now studied in detail the cell surface expression of the blood group i epitope (linear poly-N-acetyllactosamine chain) in umbilical cord blood (UCB)-derived MSCs. We used flow cytometry and mass spectrometric glycan analysis and discovered that linear poly-N-acetyllactosamine structures are expressed in UCB-derived MSCs, but not in cells differentiated from them. We further verified the findings by mass spectrometric glycan analysis. Gene expression analysis indicated that the stem-cell specific expression of the i antigen is determined by β3-N-acetylglucosaminyltransferase 5. The i antigen is a ligand for the galectin family of soluble lectins. We found concomitant cell surface expression of galectin-3, which has been reported to mediate the immunosuppressive effects exerted by MSCs. The i antigen may serve as an endogenous ligand for this immunosuppressive agent in the MSC microenvironment. Based on these findings, we suggest that linear poly-N-acetyllactosamine could be used as a novel UCB-MSC marker either alone or within an array of MSC markers.

Intestinal microbiota plays an important role in human health, and its composition is determined by several factors, such as diet and host genotype. However, thus far it has remained unknown which host genes are determinants for the microbiota composition. We studied the diversity and abundance of dominant bacteria and bifidobacteria from the faecal samples of 71 healthy individuals. In this cohort, 14 were non-secretor individuals and the remainders were secretors. The secretor status is defined by the expression of the ABH and Lewis histo-blood group antigens in the intestinal mucus and other secretions. It is determined by fucosyltransferase 2 enzyme, encoded by the FUT2 gene. Non-functional enzyme resulting from a nonsense mutation in the FUT2 gene leads to the non-secretor phenotype. PCR-DGGE and qPCR methods were applied for the intestinal microbiota analysis. Principal component analysis of bifidobacterial DGGE profiles showed that the samples of non-secretor individuals formed a separate cluster within the secretor samples. Moreover, bifidobacterial diversity (p<0.0001), richness (p<0.0003), and abundance (p<0.05) were significantly reduced in the samples from the non-secretor individuals as compared with those from the secretor individuals. The non-secretor individuals lacked, or were rarely colonized by, several genotypes related to B. bifidum, B. adolescentis and B. catenulatum/pseudocatenulatum. In contrast to bifidobacteria, several bacterial genotypes were more common and the richness (p<0.04) of dominant bacteria as detected by PCR-DGGE was higher in the non-secretor individuals than in the secretor individuals. We showed that the diversity and composition of the human bifidobacterial population is strongly associated with the histo-blood group ABH secretor/non-secretor status, which consequently appears to be one of the host genetic determinants for the composition of the intestinal microbiota. This association can be explained by the difference between the secretor and non-secretor individuals in their expression of ABH and Lewis glycan epitopes in the mucosa.

Background: Although ischemia-reperfusion (I/R) injury represents a major problem in posttransplant organ failure, effective treatment is not available. The acute phase protein alpha-1-acid glycoprotein (AGP) has been shown to be protective against experimental I/R injury. The effects of AGP are thought to be mediated by fucose groups expressed on the AGP protein inhibiting neutrophil infiltration. However, the precise mechanism of protection remains to be established. We therefore studied the effects of exogenous human AGP (hAGP) in a mouse model of ischemic acute renal failure., Methods: Mice were subjected to renal I/R and treated with hAGP, fucose-depleted hAGP, or control treated. Also, transgenic mice over-expressing rat AGP or wild-type controls were subjected to renal I/R., Results: Treatment was with hAGP as well as fucose-depleted hAGP protected mice against I/R-induced acute renal failure. Surprisingly, AGP-over-expressing mice were not protected against I/R injury. Both natural and fucose-depleted hAGP inhibited the activation of the complement system, as determined by renal C3 deposition and influx of neutrophils measured by immunohistochemistry and myeloperoxidase-enzyme-linked immunoadsorbent assay. Tubular epithelial cell structure (actin cytoskeleton) and cell-cell interaction (tight-junction architecture) were completely preserved in AGP-treated mice. Also, epithelial caspase activation and apoptotic DNA cleavage were prevented by AGP treatment., Conclusions: Both natural and fucose-depleted hAGP protect against renal I/R injury by preservation of tubular epithelial structure and inhibition of apoptosis and subsequent inflammation. Therefore, hAGP can be regarded as a potential new therapeutic intervention in the treatment of acute renal failure, as seen after transplantation of ischemically injured kidneys.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides.

Helicobacter pylori is a Gram-negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O-antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP-l-fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP-l-fucose synthesis, H. pylori gmd coding for GDP-d-mannose dehydratase (GMD), and gmer coding for GDP-4-keto-6-deoxy-d-mannose-3,5-epimerase/4-reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP-l-fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl-N-acetyllactosamine to sialyl Lewis X.

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

Selectin-dependent cell binding has importance in the extravasation of blood-circulating tumor cells and in the generation of metastases. Cell surface glycoproteins decorated with sialylated, fucosylated epitopes, such as sialyl Lewis(x) (sLe(x)), are ligands for selectins. Not only terminal sLe(x) moieties but also proximal core structures contribute to the formation of binding epitopes for selectins. Core 2 beta1,6-N-acetylglucosaminyltransferases (C2GnT) and alpha1,3-fucosyltransferases (alpha1,3-FucT) have been suggested to be the rate-limiting enzymes in the synthesis of selectin ligands. We analyzed oral cavity epithelial carcinoma cell lines and showed their expression of RNA transcripts for C2GnT and alpha1,3-FucT, identified alpha1,3-FucT enzyme activities, and analyzed the cell surface sLe(x) expression levels. Neither the pattern of expressed enzymes nor the alpha1,3-FucT activity directly predicted the binding capacity of E-selectin. However, only the sLe(x)-expressing cell lines were capable of binding to E-selectin, but not to P-selectin, thus putatively promoting the selectin-mediated metastasis. These findings suggest that C2GnT in combination with alpha1,3-Fuc-T contribute to the selectin-mediated metastasis in oral cavity carcinomas.

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner., (Copyright 2000 Academic Press.)

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by alpha(2,3)sialidase(s).

We have developed an assay for alpha 2,3-sialidase (EC 3.2.1.18) which employs a biotinylated carbohydrate-polyacrylamide conjugate as substrate for the enzyme. The solution-phase sialidase reactions are followed by a selective capture of biotinylated neoglycoconjugates onto a microtitration plate coated with streptavidin. The amount of the reaction product formed is then rapidly and easily quantified using a product-specific primary antibody and europium chelate-labeled secondary antibody. This method combines the advantages of solution-phase enzymatic reaction and suitability for high-throughput screening typical of solid-phase assays. The assay gives a detectable signal with 0.4% of substrate sites desialylated. We have demonstrated the utility of the assay by measuring alpha 2,3-sialidase activity from crude lysates of cultured rat endothelial cells by using biotinylated sialyl Lewis x glycoconjugate as substrate. Endothelial sialidase(s) showed up to 250-fold higher activity toward soluble compared to immobilized substrate. Product formation detected with an anti-Lewis x antibody was linear in the range of 0.1-4 micrograms/ml of protein in endothelial cell lysate. High sensitivity of the assay was achieved by using solution-phase enzyme reaction and time-resolved fluorometric detection. The same assay format used here is easily adapted to detect activities of several different glycosidases or glycosyl-transferases by using appropriate substrates and antibodies.

By using two different reaction pathways, we generated enzymatically three sialylated and site-specifically alpha 1-3-fucosylated polylactosamines. Two of these are isomeric hexasaccharides Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc and Neu5Ac(alpha 2-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-3)Gal(beta 1-4) GlcNAc, containing epitopes that correspond to VIM-2 and sialyl Lewis (x), respectively. The third one, nonasaccharide Neu5Ac(alpha 2-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)] GlcNAc(beta 1-3)Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc, is a sialylated and internally difucosylated derivative of a trimeric N-acetyllactosamine. All three oligosaccharides have one fucose-free N-acetyllactosaminyl unit and can be used as acceptors for recombinant alpha 1-3-fucosyltransferases in determining the biosynthesis pathways leading to polyfucosylated selectin ligands.

Extravasation from the blood of malignant tumour cells that form metastasis and leukocytes that go into tissues require contact between selectins and their sialyl Lewis x and sialyl Lewis a (sLe(x) and sLe(a) respectively) decorated ligands. Endothelial cells have been shown to express sLe(x) epitopes in lymph nodes and at sites of inflammation, and this is crucial for the selectin-dependent leukocyte traffic. Besides the ability to synthesize sLe(x) on sialylated N-acetyllactosamine via the action of alpha(1,3)fucosyltransferase(s), endothelial cells can also degrade sLe(x) to Lewis x through the action of alpha(2,3)sialidase(s). In addition, several epithelial tumors possess the machinery to synthesize sLe(x), which facilitates their adhesion to endothelial E- and P-selectin.

We describe here an assay that employs a highly sensitive nonradioactive method, time-resolved fluorometry, for measuring the activity of the enzyme GDP-Fuc:NeuNAcalpha2-3Galbeta1-4GlcNAc-R (Fuc to GlcNAc) alpha1,3-fucosyltransferase (alpha1,3FT). In this assay, a neoglycoprotein substrate of alpha1,3FT is immobilized on a microtiter plate. Incubation with the fucose donor GDP-fucose and enzyme source converts the acceptor NeuNAcalpha2-3Galbeta1-4GlcNAc-R to the product NeuNAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc-R, which is quantified using a product-specific (antisialyl Lewis x) primary antibody and europium chelate-labeled secondary antibody. In the development of the assay, we used extracts of alpha1,3FT-transfected insect cells as the specific enzyme source. The reaction product formation was proportional to time of incubation (0-2 h) and the extract added (0.1-10 microU of enzyme) and was dependent on the GDP-fucose and glycoconjugate acceptor. We have also demonstrated with different cultured cancer cell lines that this time-resolved immunofluorometric assay allows rapid measurement of alpha1,3FT activity from a large number of crude cell lysate samples. Our results indicated that cell lines which expressed more sialyl Lewis x determinant on their surfaces had higher levels of alpha1,3FT activity. The advantages of this new assay are high sensitivity and a wide linear range of measurement. The assay is expected to be useful in the determination of regulation mechanisms of sialyl Lewis x-synthesizing alpha1,3-fucosyltransferases.

Chemical shifts and coupling constants of completely assigned 1H and 13C NMR spectra at 500 MHz, as well as ROESY and HMBC connectivities were used to establish the structure of the pentasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)[GlcNAc beta (1-->6)]Gal beta (1-->4)GlcNAc, synthesized by the action of the mid-chain beta-(1-->6)-D-N-acetylglucosaminyltransferase of rat serum from UDP-GlcNAc and the linear tetrasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)Gal beta (1-->4)GlcNAc.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.