Complex Oligosaccharides Research Articles

Biosynthesis of the IFN-γ binding protein of ectromelia virus, the causative agent of mousepox

Ectromelia virus (ECTV), the causative agent of mousepox, expresses an extracellular interferon-γ binding protein (IFN-γBP) with homology to the ligand binding domains of the IFN-γ high affinity receptor (IFN-γR1). Unlike the cellular receptor, the IFN-γBP binds IFN-γ from several species. The IFN-γBP is synthesized early after infection, accumulating in the extracellular milieu as dimers composed of two protein species with M r of 34.6 or 33.0 kDa. Homodimers are covalently linked by an interchain disulphide bond at position 216. The IFN-γBP has complex N-linked oligosaccharides at positions 41 and 149 as determined by site-directed mutagenesis and glycosidase treatment. Glycosylation at position 41 is required for secretion from mammalian cells and may play a role in the activity of the IFN-γBP. Glycosylation at position 149 is not required for secretion, and the lack of glycosylation at this site does not diminish ligand binding as measured by surface plasmon resonance (SPR) and ELISA.

Selectin Antagonists

Asthma and COPD are chronic inflammatory conditions that affect hundreds of millions of patients worldwide. New therapeutics are desperately needed, especially those that target the underlying causes and prevent disease progression. Although asthma and COPD have distinct etiologies, both are associated with reduced airflow caused by excess infiltration of inflammatory cells into healthy lung tissues. As selectin-mediated adhesion of leukocytes to the vascular endothelium is a key early event in the initiation of the inflammatory response, selectin inhibition is thought to be a good target for therapeutic intervention.Three known selectins are expressed in distinct subsets of cells: P-selectin is presented on the surface of activated platelets and endothelial cells, L-selectin is constitutively expressed on leukocytes, and E-selectin synthesis is upregulated in activated endothelial cells. They mediate cell-cell adhesion in the shear flow of the bloodstream via specialized interactions with clusters of oligosaccharides presented on cell surface glycopeptide ligands. The role of selectin-ligand interactions in the inflammatory response has been demonstrated in various animal models, prompting considerable attention from the pharmaceutical industry.Drug discovery efforts have yielded many different classes of selectin inhibitors, including soluble protein ligands, antibodies, oligosaccharides and small molecules. Although many selectin inhibitors have shown activity in preclinical models, clinical progress of selectin-directed therapies has been slow. Early approaches employed carbohydrate-based inhibitors to mimic the natural ligand sialyl Lewis X; however, these compounds proved challenging to develop. Cytel’s CY 1503, a complex oligosaccharide, progressed to phase II/III trials for reperfusion injury, but further development was halted when it failed to demonstrate clinical efficacy. Two protein-based selectin inhibitors have reached phase II development. These included Wyeth’s recombinant soluble P-selectin ligand, TSI (PSGL-1), which was discontinued after disappointing results in myocardial infarction trials and Protein Design Labs’ humanized anti-L-selectin monoclonal antibody, which is currently in development for trauma. Bimosiamose, discovered by Encysive Pharmaceutical and presently being developed by Revotar Biopharmaceuticals, is an 863 g/mol molecular weight dimer with minimal carbohydrate content and is, to date, the leading selectin inhibitor in clinical development. This compound has shown promise in a phase Ha ‘proof of concept’ trial in patients with asthma, reducing airway recruitment of eosinophils after intravenous administration. Further clinical development of an inhaled formulation is underway.Despite a significant need for new therapeutics, selectin inhibitors have not yet been explored for the treatment of COPD. Bimosiamose represents an important proof of principle, and hopefully continued success will spark renewed interest in selectin-directed therapeutics for respiratory diseases.

RecombinantClostridium perfringensalpha-N-Acetylgalactosaminidase Blood Group A2Degrading Activity

The ABO blood group antigens (A, B and H antigens) are composed of complex oligosaccharides linked to membrane proteins and lipids [[1], [2]] and abundant on red blood cell membranes. Each antigen ...

Generation of a Novel Engineered Murine Apyrase in Pichia Pastoris. Enzymatic Profile and Inhibition of Human Platelet Reactivity.

To study thrombogenesis in wild-type and transgenic mice we engineered a recombinant, soluble form of murine ENTPDase1/CD39 for use as a therapeutic agent. ENTPDase1 contains N- and C-terminal intracellular and transmembrane regions flanking an extracellular domain containing the 5 enzymatically active apyrase-conserved regions. The soluble portion of murine CD39 was amplified using PCR with 5′-XhoI and 3′-XbaI linked primers and cloned into the XhoI/XbaI-digested yeast expression vector pGAPZα. The vector, which features the constitutive GAP promoter and α factor secretion signal, was modified by elimination of the Ste13 cleavage sites and by introduction of a 5′-6His tag separated from the murine solCD39 coding sequence by an enterokinase cleavage site. Stably expressing clones were selected. Aliquots of 30-fold concentrated conditioned medium, obtained following 72 h culture of a yeast clone, were assayed for ADPase and ATPase activity and for the ability to inhibit platelet reactivity. Nucleotidase activity was monitored by native gel electrophoresis and staining using ATP metabolism to phosphate and conversion of Ca-phosphate into PbS, as well as by radio-TLC analyses. Platelet aggregation was quantitated using ADP and collagen as stimuli in heparinized and citrated human platelet-rich plasma (PRP). SolCD39 produced in mammalian cells (CHO, or COS-1) yields an active enzyme that is heavily N-glycosylated. Similarly, Pichia pastoris-produced (murine) solCD39 yields a comparably active protein that is also heavily glycosylated, albeit with the yeast-specific high mannose-type N-glycans. Its ADPase to ATPase activity ratio was ~1.2. Yeast-derived murine solCD39 was treated with PNGase F, an amidase that cleaves between the innermost N-acetyl glucosamine of complex oligosaccharides and asparagine residues of glycoproteins. This removed all saccharides from solCD39, as shown by PAS staining, and by mobility shifts by Western blot analysis and Pb staining in native gels. Interestingly, the deglycosylated protein retained ~60% of both enzymatic and biological activities as compared to the parent molecule. The figures shows platelet aggregation to 5 μM ADP in citrated PRP for control (no murine solCD39), isolated solCD39, and equal quantites of solCD39 that was deglycosylated, as well as subjected to the deglycosylation reaction in the absence of PNGase F. These data are similar for the present murine yeast-produced molecule and COS-produced human solCD39. Our data indicate the possibility of high scale production of solCD39 in yeast, with excellent yield, stability and antithrombotic activity. [Display omitted]

A selective Sc(OTf) 3-catalyzed trialkylsilyl ether to acetyl ester exchange reaction with β- l-idopyranoside and 3,4- O-isopropylidene-β- d-galactopyranoside derivatives

The selective silylation of monosaccharide building blocks is useful for preparing complex oligosaccharides. We now report that the diol, methyl (dimethylthexylsilyl 3- O-pivaloyl-β- l-idopyranosyl)uronate, can be selectively silylated at the O-2 position by trialkylsilyl triflates. After protection of O-4, the O-2 silyl group can be selectively replaced by acetate by taking advantage of a trialkylsilyl–acetate exchange reaction catalyzed by Sc(OTf) 3 in the presence of acetic anhydride. The high O-2 selectivity is shown for triethylsilyl (TES), tert-butyldimethylsilyl (TBS), and triisopropylsilyl (TIPS). The selective cleavage reaction only worked well for TES and TBS derivatives. A selection of silyl triflates and silyl chlorides were used as silylating reagents with ethyl 3,4- O-isopropylidene-1-thio-β- d-galactopyranoside. In most cases, silylation afforded 2,6-di- O-silylated products in high yields. Studies on the cleavage reaction showed that only the primary silylated protecting groups were replaced by acetyl groups. This reaction worked with a variety of silyl protecting groups but not the tert-butyldiphenylsilyl (TBDPS) protecting group. Unfortunately, the 1-thioethyl group was also sensitive to the Sc(OTf) 3, leading in these conditions to α/β mixtures of the 1-acetates, which compromised the synthetic utility of this reaction for these compounds. The sequence presented here is a useful synthetic route to differentially protected l-iduronic acid building blocks.

Structural characterization of N-glycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry.

The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique described to date has proven to be a convenient and rapid method for identification and characterizations of proteins. However, the general MALDI-TOF MS analysis of complex carbohydrates and glycopeptides still entails special consideration of ionization and the fragmentation characteristics of labile carbohydrate moieties. In this study, an efficient and practical method we termed the matrix-dependent selective fragmentation (MDSF) technique of MALDI-TOF/TOF MS, which allows highly sensitive and reliable fragmentation of oligosaccharides and N-glycopeptides. Results from application of the MDSF technique to TOF/TOF MS analysis demonstrated that in comparison to the conventional postsource decay up to 170 times more sensitive product ion peaks could be obtained. It was also suggested that MDSF generates desired structural information based on the controlled cleavage of the singly charged precursor ion with different electronic excited states made by this method. Ideal product ion peaks observed by MDSF in TOF/TOF MS facilitated structural characterization of complex oligosaccharide derivatives including unstable Neu5Ac and Fuc residues and N-glycopeptides.

The H,K-ATPase β Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting

The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.

Conformations of cell surface oligosaccharides and recognition by lectins from pathogens

Pseudomonas aeruginosa is essentially a saprophytic bacterium that can turn into an aggressive pathogen when in contact with damaged and inflamed tissues such as burns and lungs of cystic fibrosis (CF) patients. In all of these cases, the host cell surfaces present altered glycosylations. The most studied example is that of CF where point mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, results in altered ion movements and also influences the N‐glycosylation of the CFTR protein and other cell surface glycoproteins. Increased fucosylation and decreased sialylation with higher levels of Lewis x and Lewis a epitopes correlate to P. aeruginosa‐specific binding. In addition, both the mucus covering the airway epithelia of CF patients and their salivary mucins display higher levels of sialylated and sulfated Lewis oligosaccharides that attract P. aeruginosa.The galactose‐ and fucose‐binding (PA‐IL and PA‐IIL) lectins of P. aeruginosa contribute to the virulence of this pathogenic bacterium, which is a major cause of morbidity and mortality in CF patients. Database searching in newly sequenced genomes indicates that the presence of these lectins is characteristics of high environmental adaptability of some opportunistic bacteria. The crystal structures of PA‐IL and PA‐IIL lectins have been solved in complex with the corresponding monosaccharides. Molecular modelling studies were used in order to predict the binding mode of PA‐IIL with complex oligosaccharides. Combination of theoretical studies with binding assays give clear indication that the histo‐blood group epitope Lewis a could be the biological ligand present in the lung of CF patients.

Covalent display of oligosaccharide arrays in microtiter plates.

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).

Solid‐Phase Synthesis of Complex Oligosaccharides Using Azidoglucose as a Glycosyl Acceptor

AbstractThe solid phase synthesis of oligosaccharides 1−3 was performed on Merrifield resin. These syntheses are based on the hydroxymethylbenzyl benzoate spacer‐linker system and, for regioselective chain extension, the use of O‐glycosyl trichloroacetimidates as glycosyl donors containing temporary O‐Fmoc protection. The important lactosamine‐containing oligosaccharides 1−3 are accessible in excellent yields by using a 2‐azidoglucose residue as the glucosamine building block adjacent to the spacer‐linker system. Only standard amounts of the glycosyl donor are employed in this solid phase oligosaccharide synthesis and we did not have to resort to any capping procedures. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004)

Recombinant anti‐hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core‐xylose and core‐α(1,3)‐fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.

Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor

In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [3H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.

Influence of N-linked glycans on intracellular transport of hepatitis C virus E1 chimeric glycoprotein and its role in pseudotype virus infectivity

We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine → glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.

Expression of a truncated secreted form of the mGluR3 subtype of metabotropic glutamate receptor

In this study, 10 truncated constructs encompassing all or part of the extracellular ligand binding domain of the mGluR3 subtype of metabotropic glutamate receptor were generated, expressed in human embryonic kidney cells, and tested for secretion and binding of the high affinity agonist [3H]DCG-IV. The effect of inserting epitope tags into the N or C termini on cell secretion and radioligand binding was also examined. Secretion into the cell culture media was observed for 8 of the 10 truncated receptors and all secreted forms displayed high affinity agonist binding. The highest level of binding was observed in the C-terminal polyhistidine-tagged receptor truncated at serine 507. Reduction and enzymatic deglycosylation of the serine 507 truncated receptor using endoglycosidase H and PNGase F showed that the secreted receptor was a disulfide-linked dimer containing complex oligosaccharides. Pharmacological characterization demonstrated that the truncated receptor showed the same rank order of potency of agonist binding, a relatively small 2-fold decrease in agonist affinity, and a larger 10-fold decrease in affinity for the antagonist LY341495 compared to the full-length membrane-bound receptor. These results define the essential requirements for ligand binding to the extracellular domain of mGluR3 and highlight parameters important for the optimization of receptor expression in mammalian cells.

Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation.

Misfolded or incompletely assembled multisubunit glycoproteins undergo endoplasmic reticulum-associated degradation (ERAD) regulated in large measure by their N-linked polymannose oligosaccharides. In this quality control system lectin interaction with Glc(3)Man(9)GlcNAc(2) glycans after trimming with endoplasmic reticulum (ER) alpha-glucosidases and alpha-mannosidases sorts out persistently unfolded glycoproteins for N-deglycosylation and proteolytic degradation. Monoglucosylated (Glc(1)Man(9)GlcNAc(2)) glycoproteins take part in the calnexin/calreticulin glucosylation-deglucosylation cycle, while the Man(8)GlcNAc(2) isomer B product of ER mannosidase I interacts with EDEM. Proteasomal degradation requires retrotranslocation into the cytosol through a Sec61 channel and deglycosylation by peptide: N-glycosidase (PNGase); in alternate models both PNGase and proteasomes may be either free in the cytosol or ER membrane-imbedded/attached. Numerous proteins appear to undergo nonproteasomal degradation in which deglycosylation and proteolysis take place in the ER lumen. The released free oligosaccharides (OS) are transported to the cytosol as OS-GlcNAc(2) along with similar components produced by the hydrolytic action of the oligosaccharyltransferase, where they together with OS from the proteasomal pathway are trimmed to Man(5)GlcNAc(1) by the action of cytosolic endo-beta- N-acetylglucosaminidase and alpha-mannosidase before entering the lysosomes. Some misfolded glycoproteins can recycle between the ER, intermediate and Golgi compartments, where they are further processed before ERAD. Moreover, properly folded glycoproteins with mannose-trimmed glycans can be deglucosylated in the Golgi by endomannosidase, thereby releasing calreticulin and permitting formation of complex OS. A number of regulatory controls have been described, including the glucosidase-glucosyltransferase shuttle, which controls the level of Glc(3)Man(9)GlcNAc(2)-P-P-Dol, and the unfolded protein response, which enhances synthesis of components of the quality control system.

Carbohydrate chips for studying high-throughput carbohydrate-protein interactions.

Carbohydrate-protein interactions play important biological roles in living organisms. For the most part, biophysical and biochemical methods have been used for studying these biomolecular interactions. Less attention has been given to the development of high-throughput methods to elucidate recognition events between carbohydrates and proteins. In the current effort to develop a novel high-throughput tool for monitoring carbohydrate-protein interactions, we prepared carbohydrate microarrays by immobilizing maleimide-linked carbohydrates on thiol-derivatized glass slides and carried out lectin binding experiments by using these microarrays. The results showed that carbohydrates with different structural features selectively bound to the corresponding lectins with relative binding affinities that correlated with those obtained from solution-based assays. In addition, binding affinities of lectins to carbohydrates were also quantitatively analyzed by determining IC(50) values of soluble carbohydrates with the carbohydrate microarrays. To fabricate carbohydrate chips that contained more diverse carbohydrate probes, solution-phase parallel and enzymatic glycosylations were performed. Three model disaccharides were in parallel synthesized in solution-phase and used as carbohydrate probes for the fabrication of carbohydrate chips. Three enzymatic glycosylations on glass slides were consecutively performed to generate carbohydrate microarrays that contained the complex oligosaccharide, sialyl Le(x). Overall, these works demonstrated that carbohydrate chips could be efficiently prepared by covalent immobilization of maleimide-linked carbohydrates on the thiol-coated glass slides and applied for the high-throughput analyses of carbohydrate-protein interactions.

Oligosaccharide—Peptide Ligation of Glycosyl Thiolates with Dehydropeptides: Synthesis of S‐Linked Mucin‐Related Glycopeptide Conjugates.

A chemoselective strategy for oligosaccharide-peptide ligation is described in which alpha-thio analogues of mucin-related glycoconjugates can be readily accessed through site-selective conjugate addition of complex oligosaccharide thiolates to dehydroalanine-containing peptides. The efficiency of the ligation is highlighted by the rapid convergent assembly of thio-isosteres of the four tumor-associated carbohydrate antigens, T(N), T, ST(N), and 2,6-ST, as a pair of diastereoisomers at the newly formed cysteine stereocenter. The process proceeds in high yield and with complete retention of the alpha-anomeric configuration.

Structural Characterization of TSC-36/Flik: ANALYSIS OF TWO CHARGE ISOFORMS

Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties.

Regulation of ganglioside biosynthesis in the nervous system

Regulation of ganglioside biosynthesis in the nervous system

Conformational analysis of complex oligosaccharides: the CICADA approach to the uromodulin O-glycans

Uromodulin is the pregnancy-associated Tamm–Horsfall glycoprotein, with the enhanced ability to inhibit T-cell proliferation. Pregnancy-associated structural changes mainly occur in the O-glycosylation of this glycoprotein. These include up to 12 glycan structures, made up of an unusual core type 2 sequence terminated with one, two, or three sialyl Lewis x sequences; this type of O-glycans could serve as E- and P-selectin ligands. The present work focuses on the most complex one; a tetradecamer made up of a type 2 core carrying three sialyl Lewis x branches. Five different monosaccharides are assembled by 14 glycosidic linkages. The conformational behavior of the constituting disaccharide segments was evaluated using the flexible residue procedure of the MM3 molecular mechanics procedure. For each disaccharide, the adiabatic energy surface, along with the local energy minima were established. All these results were used for the generation, prior to complete optimization of the tetradecamer. This was followed by a complete exploration of conformational hyperspace throughout the use of the single coordinate method as implemented in the CICADA program. Despite the potential flexibility of the tetradecasaccharide, only four conformational families occur, accounting for more than 95% of the total low energy conformations. For each family, the molecular properties (electrostatic, lipophilicity, and hydrogen potential) were studied. The shape of the tetradecasaccharide is best described as a flat ribbon, flanked by three branches having terminal sialyl residues. Two of the branches interact through nonbonded interactions, bringing further energy stabilization, and limiting the conformational flexibility of the sialyl residues. Only one branch maintains the original conformational features of sialyl Lewis x. This O-glycan can be seen as a fascinating example of ‘dendrimeric’ structure, where the spatial arrangement of three S-Le x epitopes may favor its complementary ‘presentations’ for the interactions with E- and P-selectins.