Oligosaccharide Structures Research Articles

Synthesis of a Lewis b hexasaccharide thioglycoside donor and its use towards an extended mucin core Tn heptasaccharide structure and a photoreactive biotinylated serine linked hexasaccharide.

Investigation into Heliobacter pylori binding to Lewis b (Leb) antigens through the blood group antigen binding adhesion protein (BabA) requires structurally well-defined tools. A Leb hexasaccharide thioglycoside donor was chemically prepared through a linear approach starting from D-lactose. This donor can be used to attach reducing end linkers providing a range of options for conjugation techniques or to further extend the oligosaccharide structure. To evaluate its efficiency as a donor, it was coupled to a 6-OH GalNAc acceptor, producing an extended Leb-containing Tn mucin core structure in 84% yield, and to L-serine in 72% yield. The latter compound was subsequently functionalized with a photolabile diazirine linker and biotin, creating a Leb hexasaccharide structure-function tool suitable for lectin tagging interaction studies. This donor opens a wide range of possibilities for conjugation of Leb structures to produce a variety of chemical biology tools to assist in the study of these interactions.

Human milk oligosaccharides and non-digestible carbohydrates reduce pathogen adhesion to intestinal epithelial cells by decoy effects or by attenuating bacterial virulence

This work investigated the effects of different chemical structures of human milk oligosaccharides (hMOs) and non-digestible carbohydrates (NDCs) on pathogen adhesion by serving as decoy receptors. Pre-exposure of pathogens to inulins and low degree of methylation (DM) pectin prevented binding to gut epithelial Caco2-cells, but effects were dependent on the molecules’ chemistry, pathogen strain and growth phase. Pre-exposure to 3-fucosyllactose increased E. coli WA321 adhesion (28%, p < 0.05), and DM69 pectin increased E. coli ET8 (15 fold, p < 0.05) and E. coli WA321 (50%, p < 0.05) adhesion. Transcriptomics analysis revealed that DM69 pectin upregulated flagella and cell membrane associated genes. However, the top 10 downregulated genes were associated with lowering of bacteria virulence. DM69 pectin increased pathogen adhesion but bacterial virulence was attenuated illustrating different mechanisms may lower pathogen adhesion. Our study illustrates that both hMOs and NDCs can reduce adhesion or attenuate virulence of pathogens but that these effects are chemistry dependent.

The Flexibility of Oligosaccharides Unveiled Through Residual Dipolar Coupling Analysis.

The intrinsic flexibility of glycans complicates the study of their structures and dynamics, which are often important for their biological function. NMR has provided insights into the conformational, dynamic and recognition features of glycans, but suffers from severe chemical shift degeneracy. We employed labelled glycans to explore the conformational behaviour of a β(1-6)-Glc hexasaccharide model through residual dipolar couplings (RDCs). RDC delivered information on the relative orientation of specific residues along the glycan chain and provided experimental clues for the existence of certain geometries. The use of two different aligning media demonstrated the adaptability of flexible oligosaccharide structures to different environments.

The Potential Utility of Prebiotics to Modulate Alzheimer's Disease: A Review of the Evidence.

The gut microbiome has recently emerged as a critical modulator of brain function, with the so-called gut-brain axis having multiple links with a variety of neurodegenerative and mental health conditions, including Alzheimer’s Disease (AD). Various approaches for modulating the gut microbiome toward compositional and functional states that are consistent with improved cognitive health outcomes have been documented, including probiotics and prebiotics. While probiotics are live microorganisms that directly confer beneficial health effects, prebiotics are oligosaccharide and polysaccharide structures that can beneficially modulate the gut microbiome by enhancing the growth, survival, and/or function of gut microbes that in turn have beneficial effects on the human host. In this review, we discuss evidence showing the potential link between gut microbiome composition and AD onset or development, provide an overview of prebiotic types and their roles in altering gut microbial composition, discuss the effectiveness of prebiotics in regulating gut microbiome composition and microbially derived metabolites, and discuss the current evidence linking prebiotics with health outcomes related to AD in both animal models and human trials. Though there is a paucity of human clinical trials demonstrating the effectiveness of prebiotics in altering gut microbiome-mediated health outcomes in AD, current evidence highlights the potential of various prebiotic approaches for beneficially altering the gut microbiota or gut physiology by promoting the production of butyrate, indoles, and secondary bile acid profiles that further regulate gut immunity and mucosal homeostasis, which are associated with beneficial effects on the central immune system and brain functionality.

An improved method for galactosyl oligosaccharide characterization

Due to beneficial effects of galactosyl oligosaccharides (GOS) on digestive and immune health, their characterization has become increasingly important. This is especially so as GOS are synthesized enzymatically and contain oligosaccharides of different sizes and linkages. High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is widely used for GOS characterization. With its high resolving power, it can separate structural isomers. Here we present a significant improvement to currently used methods. Our approach combines high resolution HPAE separation on a CarboPac PA300 column with 4 µm particle size with PAD and Orbitrap mass spectrometry (MS) detections to provide in-depth information on GOS composition. Oligosaccharide resolution, especially in the disaccharide region, is significantly improved and can be routinely achieved. Improvement in technology to remove sodium before MS results in minimal peak dispersion, allowing GOS degrees of polymerization 2 to 6 to be identified based on mass spectra obtained from intact oligosaccharides and confirmed using fragmentation patterns observed in MS/MS data. Combining HPAE with MS led to identification of 28 oligosaccharides in a commercial GOS sample. We attempted to correlate oligosaccharide structure with observed elution behavior. To our knowledge this is first such attempt and can form a basis for a comprehensive structure vs HPAE elution behavior database.

The anti-inflammatory activity of specific-sized hyaluronic acid oligosaccharides

Severe acute inflammatory conditions may cause tissue damage, sepsis, and death. As a critical component of the extracellular matrix, hyaluronic acid (HA) has been reported to possess pro- and anti-inflammatory properties via Toll-like receptors (TLRs). In this study, we prepared different sizes and structures of HA oligosaccharides and derivatives and investigated the effects on inflammation in vitro and in vivo. Our results showed that HA tetra-saccharide was the minimum fragment to enhance inflammation, whereas HA disaccharide competitively blocked TLR4-dependent inflammation. The enzymatic HA disaccharide (ΔHA2) inhibited lipopolysaccharide (LPS)-induced inflammation. Based on structure-activity relationship analysis, we observed that anti-inflammatory activity depended on HAs polymerization degree, acetyl group, and configuration. In addition, we demonstrated that ΔHA2 reduced LPS-induced pro-inflammatory cytokines production in vivo. ΔHA2, a native metabolite of HA polysaccharides, may have a potential role against LPS-mediated inflammatory diseases.

Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10–60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3−. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.

Production of Organic Acid and Short-Chain Fatty Acids (SCFA) from Lactic Acid Bacteria Isolate on Oligosaccharide Media

The growth of microorganisms in food, one of which is lactic acid bacteria (LAB), can produce metabolites beneficial to health. It is essential to study the results of LAB metabolism to improve the quality of a functional food product. This study aimed to evaluate the isolates Lactobacillus acidophilus FNCC 0051 and Lactobacillus rhamnosus R23 to metabolize oligosaccharides as a carbon source so that the final fermentation product can benefit health especially in lowering cholesterol. In vitro testing was carried out on MRS media with or without oligosaccharides, either singly or in a combination consisting of galactooligosaccharides (GOS), fructooligosaccharides (FOS), inulin (IN), inulin hydrolyzate (HI), or their combination as prebiotics by adding 0.3 % oxbile (bile salt) and inoculated with 1% v/v LAB isolate culture and incubated at 37°C for 24 hours. The results showed that the main product of oligosaccharide metabolism by L. acidophilus FNCC 0051 and L. rhamnosus R23 produced several organic acids (lactic acid), including short-chain fatty acids (SCFA) (acetic acid, propionic acid, and butyric acid). The single and combined carbon sources affected the proportion of lactic acid and acetic acid produced by L. acidophilus FNCC0051 (p&lt;0.05). However, they did not affect the proportions of propionic acid and butyric acid. While in L. rhamnosus R23 (p&lt;0.05), the presence of a single carbon source significantly affected the proportions of lactic acid, acetic acid, propionic acid, and butyric acid, while the combination of oligosaccharides affected the proportions of lactic acid and butyric acid produced. SCFA is the main product of prebiotic metabolism, but the characteristics of the acid produced have not been identified. The fermentation pattern is thought to be related to molecular weight, chain length, and oligosaccharide structure. Short-chain molecules, such as FOS generally ferment more rapidly than long-chain molecules such as inulin. The results of this study indicate that both isolates can be used as probiotics in the development of symbiotic products with the addition of oligosaccharides, which have a physiological effect in lowering cholesterol levels.

A general approach to explore prokaryotic protein glycosylation reveals the unique surface layer modulation of an anammox bacterium.

The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes their large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolve a remarkable array of oligosaccharides,which are produced by two seemingly unrelated biosynthetic routes, andwhich modify the same surface-layer protein simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism-the anaerobic oxidation of ammonium-which depends on the acquisition of substrates of opposite charges. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal a remarkable example for the evolution of complex oligosaccharides in bacteria.

The possible association of clusterin fucosylation changes with male fertility disorders

In the seminal plasma (n = 118) and serum (n = 90) clusterin (CLU) the fucosylation and the expression of selected fucosyltransferases (FUTs) were analyzed. Samples from infertile men were divided into groups based on the results of the standard semen analysis: normozoospermic (N), teratozoospermic (T), asthenoteratozoospermic (AT) and oligoasthenoteratozoospermic (OAT). The CLU fucosylation was analyzed using lectin-ELISAs with biotinylated lectins specific to α1,3-, α1,2-linked antennary fucose, and α1,6-linked core fucose (LTA, UEA, and LCA, respectively). The concentrations of FUT3 and FUT4, reflecting the expression of Le oligosaccharide structures, were measured using ELISA tests. The differences in serum CLU and FUT4 concentrations, and in the expression of core fucose and antennary fucose α1,2-linked in CLU glycans between the N group and other groups examined suggest that the disturbances in sperm count, motility, and morphology are not the only cause of male infertility. Lack of similarities between levels of examined parameters in blood serum and seminal plasma may suggest the differences in mechanisms leading to glycoproteins glycosylation. It confirmed the observed differences in concentrations of seminal plasma CLU, FUT3, and FUT4 between the OAT group and N, T, AT groups, indicating that decreased sperm count may be related to these parameters expression. The serum CLU concentrations and expression of core fucose and fucose α1,2-linked in CLU, seem to be good markers differentiating normozoospermic men from those with abnormal sperm parameters, which was not observed for seminal plasma.

Separation and identification of neutral oligosaccharides with prebiotic activities from apple pectin

Plant-derived neutral oligosaccharides and pectic-oligosaccharides (POS) have been suggested as prebiotics based on their degree of polymerization (DP) and structural complexity. In the present study, neutral oligosaccharides without galacturonic acid were produced from commercial apple pectin using an optimized concentration of pectinase and degradation time. The crude oligosaccharide solution was refined by membrane separation using a DEAE-Sepharose CL-6B column, a graphite carbon column and an Xbridge C18 column, which resulted in the neutral oligosaccharides with DP 3–13. The monosaccharide composition of oligosaccharides primarily included glucose, arabinose, galactose and xylose without galacturonic acid. The structure of the oligosaccharides was analyzed using mass spectrometry with an electrospray ionization source by UPLC on an HILIC column and 1H NMR. The MS/MS spectra in positive ion mode and 1H NMR showed a main oligosaccharide structure corresponding to the connection of arabinogalactan with Galβ1→4Galβ1→4Galβ1→4Galβ1→α3Araf. The growth of the selected Lactobacillus. strains was improved by adding refined neutral oligosaccharides with DP = 3–13 compared with commercial inulin, and growth of Escherichia coli had not basically grown. Therefore, neutral oligosaccharides (DP = 3–13) can be considered effective prebiotics for potential application in functional food.

A Multidimensional Mass Spectrometry-Based Workflow for De Novo Structural Elucidation of Oligosaccharides from Polysaccharides.

Carbohydrates play essential roles in a variety of biological processes that are dictated by their structures. However, characterization of carbohydrate structures remains extremely difficult and generally unsolved. In this work, a de novo mass spectrometry-based workflow was developed to isolate and structurally elucidate oligosaccharides to provide sequence, monosaccharide compositions, and glycosidic linkage positions. The approach employs liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based methods in a 3-dimensional concept: one high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-QTOF MS) analysis for oligosaccharide sequencing and two ultra high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ MS) analyses on fractionated oligosaccharides to determine their monosaccharides and linkages compositions. The workflow was validated by applying the procedure to maltooligosaccharide standards. The approach was then used to determine the structures of oligosaccharides derived from polysaccharide standards and whole food products. The integrated LC–MS workflow will reveal the in-depth structures of oligosaccharides.

Oligator: a flexible interface to draw oligosaccharide structures and generate their theoretical tandem mass spectra.

Oligator is software designed to assist scientists in their exploration of MS/MS experiments, especially for oligosaccharides bearing unreferenced chemical substitutions. Through a graphical interface, users have the total flexibility to build a candidate glycan structure and produce the corresponding theoretical MS/MS spectrum in accordance with the usual ion nomenclature. The structural information is saved using standard notations, in text format, which facilitates the capitalization and exchange of data as well as any other processing of the information. Source code and user manual are freely available at https://github.com/vlollier/oligator. Supplementary data are available at Bioinformatics online.

Modernized uniform representation of carbohydrate molecules in the Protein Data Bank.

Since 1971, the Protein Data Bank (PDB) has served as the single global archive for experimentally determined 3D structures of biological macromolecules made freely available to the global community according to the FAIR principles of Findability-Accessibility-Interoperability-Reusability. During the first 50years of continuous PDB operations, standards for data representation have evolved to better represent rich and complex biological phenomena. Carbohydrate molecules present in more than 14,000 PDB structures have recently been reviewed and remediated to conform to a new standardized format. This machine-readable data representation for carbohydrates occurring in the PDB structures and the corresponding reference data improves the findability, accessibility, interoperability and reusability of structural information pertaining to these molecules. The PDB Exchange MacroMolecular Crystallographic Information File data dictionary now supports (i) standardized atom nomenclature that conforms to International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology (IUPAC-IUBMB) recommendations for carbohydrates, (ii) uniform representation of branched entities for oligosaccharides, (iii) commonly used linear descriptors of carbohydrates developed by the glycoscience community and (iv) annotation of glycosylation sites in proteins. For the first time, carbohydrates in PDB structures are consistently represented as collections of standardized monosaccharides, which precisely describe oligosaccharide structures and enable improved carbohydrate visualization, structure validation, robust quantitative and qualitative analyses, search for dendritic structures and classification. The uniform representation of carbohydrate molecules in the PDB described herein will facilitate broader usage of the resource by the glycoscience community and researchers studying glycoproteins.

Multilevel capillary gel electrophoresis characterization of new antibody modalities

Capillary gel electrophoresis-based methods were applied to comprehensively characterize two development phase new modality monoclonal antibodies including a glycoengineered and a bispecific test compound. The samples were subjected to multilevel characterization at the intact (both by SDS-SGE and cIEF) as well as the reduced protein and the released N-glycan levels. SDS capillary gel electrophoresis analysis showed excellent separation of the light and heavy chains of both samples. The bispecific antibody required a special temperature gradient denaturation process and a longer capillary to resolve its two light chain fragments. Separation of PNGase F digested antibodies revealed migration time shifts, suggesting the presence of N-linked glycosylation on the corresponding subunits. For efficient glycan removal, the highly glycosylated glycoengineered monoclonal antibody was trypsin digested prior to the endoglycosidase treatment. The released glycans were profiled by capillary gel electrophoresis after APTS labeling and their oligosaccharide structures were identified by exoglycosidase based carbohydrate sequencing. Finally, capillary isoelectric focusing shed light on the charge heterogeneity of the test compounds, providing important complementary information. A flowchart was established for workflow optimization.

Application of the Antibody-Inducing Activity of Glycosphingolipids to Human Diseases.

Glycosphingolipids (GSLs) are composed of a mono-, di-, or oligosaccharide and a ceramide and function as constituents of cell membranes. Various molecular species of GSLs have been identified in mammalian cells due to differences in the structures of oligosaccharides. The oligosaccharide structure can vary depending on cell lineage, differentiation stage, and pathology; this property can be used as a cell identification marker. Furthermore, GSLs are involved in various aspects of the immune response, such as cytokine production, immune signaling, migration of immune cells, and antibody production. GSLs containing certain structures exhibit strong immunogenicity in immunized animals and promote the production of anti-GSL antibodies. By exploiting this property, it is possible to generate antibodies that recognize the fine oligosaccharide structure of specific GSLs or glycoproteins. In our study using artificially synthesized GSLs (artGSLs), we found that several structural features are correlated with the antibody-inducing activity of GSLs. Based on these findings, we designed artGSLs that efficiently induce the production of antibodies accompanied by class switching and developed several antibodies that recognize not only certain glycan structures of GSLs but also those of glycoproteins. This review comprehensively introduces the immune activities of GSLs and their application as pharmaceuticals.

LC-MS/MS-based non-isotopically paired labeling (NIPL) strategy for the qualification and quantification of monosaccharides

Investigation into monosaccharides is critical for studies of oligosaccharides structure and function in biological processes. However, monosaccharides quantification is still challenge due to their isomeric structure and high hydrophilic properties. Besides, it was difficult to obtain isotopic internal standards (IS) of each monosaccharide in complex matrixes. Herein, we developed a novel strategy for the qualification and quantification of monosaccharides in urine using two structure analogs 1-(4-methylphenyl)-3-methyl-5-pyrazolone (MPMP) and1-phenyl-3-methyl-5-pyrazolone (PMP) as non-isotopically paired labeling (NIPL) reagents by liquid chromatograph-tandem mass spectrometry (LC-MS/MS). The derivatized monosaccharides by NIPL method not only had sufficient retention time differences on reversed-phase column, but also exhibited predominant product ion pairs (m/z 189 &m/z 175) in the multiple reaction monitoring (MRM) mode. In this method, PMP labeled standards were adopted as one-to-one internal standards (ISs). 12 urinary monosaccharides were successfully determined and the linear ranges expanded five orders of magnitude with limit of quantification (LOQ) varied from 0.09 ng mL−1 to 0.36 ng mL−1 as well as the accuracy higher than 98.15% and the relative standard derivation (RSD) lower than 7.92%. With assistance of multivariate analysis, the targeted monosaccharide biomarkers were firstly obtained for the diagnosis of bladder cancer. By the inexpensive NIPL reagents—MPMP/PMP, the developed strategy possessed the specific advantages of low cost, simple operation, high sensitivity and high accuracy for the qualification and quantitation of monosaccharides. As expected, this method will provide an alternative application potential for targeted metabolomics analysis.

Structural characterisation of the oligosaccharide from Moraxella bovoculi type strain 237 (ATCC BAA-1259) lipooligosaccharide

The Gram-negative bacterium Moraxella bovoculi is associated with infectious bovine keratoconjunctivitis (IBK), colloquially known as ‘pink-eye’. IBK is an extremely contagious ocular disease of cattle. We report here the structure of the oligosaccharide derived from the lipooligosaccharide from M. bovoculi type strain 237 (also known as ATCC BAA-1259T). GLC-MS and correlation NMR analysis of the oligosaccharide revealed 5 sugar residues, with a notable central branched 3,4,6-α-D-Glcp. An additional α-D-Manp was present ~30% on the sub-terminal α-D-Manp of the 4-linked branch. This oligosaccharide structure was consistent with other members of the Moraxellaceae where no heptose was present and 5-linked Kdo was directly attached to the central 3,4,6-α-D-Glcp.

Molecular modeling provides insights into the loading of sialic acid-containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture.

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM197. However, not only is there a surprisingly low limit for antigen chain attachment to CRM197, but different serogroup saccharides have consistently different CRM197 loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW > NmC > NmY) and the number of chains attached to CRM197, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.

Deciphering the structural attributes of protein-heparan sulfate interactions using chemo-enzymatic approaches and NMR spectroscopy.

Heparan sulfates (HS) is a polysaccharide found at the cell surface, where it mediates interactions with hundreds of proteins and regulates major pathophysiological processes. HS is highly heterogeneous and structurally complex and examples that define their structure-activity relationships remain limited. Here, in order to characterize a protein-HS interface and define the corresponding saccharide-binding domain, we present a chemo-enzymatic approach that generates 13C-labeled HS-based oligosaccharide structures. Nuclear magnetic resonance (NMR) spectroscopy, which efficiently discriminates between important or redundant chemical groups in the oligosaccharides, is employed to characterize these molecules alone and in interaction with proteins. Using chemokines as model system, docking based on NMR data on both proteins and oligosaccharides enable the identification of the structural determinant involved in the complex. This study shows that both the position of the sulfo groups along the chain and their mode of presentation, rather than their overall number, are key determinant and further points out the usefulness of these 13C-labeled oligosaccharides in obtaining detailed structural information on HS-protein complexes.