Human umbilical cord blood-derived mesenchymal stromal cells display a novel interaction between P-selectin and galectin-1.

Human multipotent mesenchymal stromal/stem cells (MSCs) have been shown to exert immunomodulatory properties that have great potential in therapies for various inflammatory and autoimmune disorders. However, intravenous delivery of these cells is followed by massive cell entrapment in the lungs and insufficient homing to target tissues or organs. In targeting to tissues, MSCs and other therapeutic cells employ similar mechanisms as leucocytes, including a cascade of rolling and adhesion steps mediated by selectins, integrins and their ligands. However, the mechanisms of MSCs homing are not well understood. We discovered that P-selectin (CD62P) binds to umbilical cord blood (UCB)-derived MSCs independently of the previously known sialyl Lewis x (sLex)-containing ligands such as P-selectin glycoprotein ligand-1 (PSGL-1, CD162). By biochemical assays, we identified galectin-1 as a novel ligand for P-selectin. Galectin-1 has previously been shown to be a key mediator of the immunosuppressive effects of human MSCs. We conclude that this novel interaction is likely to play a major role in the immunomodulatory targeting of human UCB-derived MSCs.

Cloning and expression of Helicobacter pylori GDP-l-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae.

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.

Core 2 beta1,6-N-acetylglucosaminyltransferases and alpha1,3-fucosyltransferases regulate the synthesis of O-glycans on selectin ligands on oral cavity carcinoma cells.

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.

Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography.

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.

Functional expression of Escherichia coli enzymes synthesizing GDP-L-fucose from inherent GDP-D-mannose in Saccharomyces cerevisiae.

Fucosylation of glycans on glycoproteins and -lipids requires the enzymatic activity of relevant fucosyltransferases and GDP-L-fucose as the donor. Due to the biological importance of fucosylated glycans, a readily accessible source of GDP-L-fucose would be required. Here we describe the construction of a stable recombinant S.cerevisiae strain expressing the E.coli genes gmd and wcaG encoding the two enzymes, GDP-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase/4-reductase (GMER(FX)) respectively, needed to convert GDP-mannose to GDP-fucose via the de novo pathway. Taking advantage of the rich inherent cytosolic GDP-mannose pool in S.cerevisiae cells we could easily produce 0.2 mg/l of GDP-L-fucose with this recombinant yeast strain without addition of any external GDP-mannose. The GDP-L-fucose product could be used as the fucose donor for alpha1,3fucosyltransferase to synthesize sialyl Lewis x (sLex), a glycan crucial for the selectin-dependent leukocyte traffic.

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose.

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.

High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

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).

A time-resolved immunofluorometric assay of sialyl Lewis x-degrading alpha 2,3-sialidase activity.

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.

In vitro experimental studies of sialyl Lewis x and sialyl Lewis a on endothelial and carcinoma cells: crucial glycans on 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.

Enzymatic transfer of a beta1,6-linked N-acetylglucosamine to the alpha-galactose of globo-N-tetraose: in vitro synthesis of a novel hybrid pentasaccharide of lacto-globo type.

A novel saccharide was synthesized by incubating globo-N-tetraose, GalNAc beta1-3Gal alpha1-4Gal beta1-4Glc, and UDP[3H]GlcNAc with hog gastric mucosal microsomes, known to contain beta1,6-N-acetylglucosaminyltransferase activity of a broad acceptor specificity. Chromatography and MALDI-TOF mass spectrometry of the product, as well as the amount of incorporated radioactivity indicated that one [3H]GlcNAc residue was transferred to the acceptor saccharide. One- and two-dimensional 1H NMR-spectroscopic analysis of the product and ESI-CID mass spectrometry of the pentasaccharide in permethylated form established its structure as GalNAc beta1-3([3H]GlcNAc beta1-6)Gal alpha1-4Gal beta1-4Glc. The new enzyme activity possesses substrate specificity features common to a purified beta1,6-GlcNAc-transferase from bovine tracheal epithelium, which forms branches at the subterminal beta1,3-substituted galactose and accepts both GlcNAc- and Gal-configuration at the terminal residue of the acceptor (Ropp et al. (1991) J. Biol. Chem., 266, 23863-23871). The new beta1,6-GlcNAc-branch was readily galactosylated by bovine milk beta1,4-galactosyltransferase, revealing a pathway to novel hybrid type glycans with N-acetyllactosamine chains on globotype saccharides. This pathway may lead to the rare IP blood-group antigen and to globoside-like molecules mediating cell adhesion.

A time-resolved immunofluorometric method for the measurement of sialyl Lewis x-synthesizing alpha1,3-fucosyltransferase activity.

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.

1H and 13C NMR analysis of the pentasaccharide Gal beta (1-->4)GlcNAc beta (1-->3)-[GlcNAc beta (1-->6)]Gal beta (1-->4)GlcNAc synthesized by the mid-chain beta-(1-->6)-D-N-acetylglucosaminyltransferase of rat serum.

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.

Enzymatic synthesis of site-specifically (alpha 1-3)-fucosylated polylactosamines containing either a sialyl Lewis (x), a VIM-2, or a sialylated and internally difucosylated sequence.

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.

Enzyme-assisted synthesis of a bivalent high-affinity dodecasaccharide inhibitor of mouse gamete adhesion. The length of the chains carrying distal alpha 1,3-bonded galactose residues is critical.

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.