Sulfated Oligosaccharides Research Articles

Insights into structure, affinity, specificity, and function of GAG-protein interactions through the chemoenzymatic preparation of defined sulfated oligohyaluronans.

High amounts of glycosaminoglycans (GAG) such as hyaluronan (HA) occur in connective tissues. There is nowadays increasing evidence that a "sulfation code" exists which mediates numerous GAG functions. High molecular weight and inhomogeneity of GAG, however, aggravated detailed studies. Thus, synthetic oligosaccharides were urgently required. We will review here chemoenzymatic and analytic strategies to provide defined sulfated and anomerically modified GAG oligosaccharides of the HA type. Representative studies of protein/GAG interactions by (bio)chemical and biophysical methods are reported yielding novel insights into GAG-protein binding. Finally, the biological conclusions and in vivo applications of defined sulfated GAG oligosaccharides will be discussed.

Heparan Sulfate Proteoglycans as Attachment Factor for SARS-CoV-2.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is causing an unprecedented global pandemic demanding the urgent development of therapeutic strategies. Microarray binding experiments, using an extensive heparan sulfate (HS) oligosaccharide library, showed that the receptor binding domain (RBD) of the spike of SARS-CoV-2 can bind HS in a length- and sequence-dependent manner. A hexasaccharide composed of IdoA2S-GlcNS6S repeating units was identified as the minimal binding epitope. Surface plasmon resonance showed the SARS-CoV-2 spike protein binds with a much higher affinity to heparin (KD = 55 nM) compared to the RBD (KD = 1 μM) alone. It was also found that heparin does not interfere in angiotensin-converting enzyme 2 (ACE2) binding or proteolytic processing of the spike. However, exogenous administered heparin or a highly sulfated HS oligosaccharide inhibited RBD binding to cells. Furthermore, an enzymatic removal of HS proteoglycan from physiological relevant tissue resulted in a loss of RBD binding. The data support a model in which HS functions as the point of initial attachment allowing the virus to travel through the glycocalyx by low-affinity high-avidity interactions to reach the cell membrane, where it can engage with ACE2 for cell entry. Microarray binding experiments showed that ACE2 and HS can simultaneously engage with the RBD, and it is likely no dissociation between HS and RBD is required for binding to ACE2. The results highlight the potential of using HS oligosaccharides as a starting material for therapeutic agent development.

Ultra-high-performance liquid chromatography charge transfer dissociation mass spectrometry (UHPLC-CTD-MS) as a tool for analyzing the structural heterogeneity in carrageenan oligosaccharides.

Ultra-high-performance liquid chromatography (UHPLC) with charge transfer dissociation mass spectrometry (CTD-MS) is presented for the analysis of a mixture of complex sulfated oligosaccharides. The mixture contained kappa (κ), iota (ι), and lambda (λ) carrageenans that contain anhydro bridges, different degrees of sulfation ranging from one to three per dimer, different positioning of the sulfate groups along the backbone, and varying degrees of polymerization (DP) between 4 and 12. Optimization studies using standard mixtures of carrageenans helped establish the optimal conditions for online UHPLC-CTD-MS/MS analysis. Optimization included (1) UHPLC conditions; (2) ion source conditions, such as the capillary voltage, drying gas and nebulizing gas temperature, and flow rate; and (3) CTD-MS conditions, including data-dependent CTD-MS. The UHPLC-CTD results were contrasted with UHPLC-CID results of the same mixture on the same instrument. Whereas CID tends to produce B/Y and C/Z ions with many neutral losses, CTD produced more abundant A/X ions and less abundant neutral losses, which enabled more confident structural detail. The results demonstrate that He-CTD is compatible with the timescale of UHPLC and provides more structural information about carrageenans compared to state-of-the-art methods like UHPLC-CID analysis.

A Rapid and Mild Sulfation Strategy Reveals Conformational Preferences in Therapeutically Relevant Sulfated Xylooligosaccharides.

Although sulfated xylooligosaccharides are promising therapeutic leads for a multitude of afflictions, the structural complexity and heterogeneity of commercially deployed forms (e. g. Pentosan polysulfate 1) complicates their path to further clinical development. We describe herein the synthesis of the largest homogeneous persulfated xylooligomers prepared to date, comprising up to eight xylose residues, as standards for biological studies. Near quantitative sulfation was accomplished using a remarkably mild and operationally simple protocol which avoids the need for high temperatures and a large excess of the sulfating reagent. Moreover, the sulfated xylooligomer standards so obtained enabled definitive identification of a pyridinium contaminant in a sample of a commercially prepared Pentosan drug and provided significant insights into the conformational preferences of the constituent persulfated monosaccharide residues. As the spatial distribution of sulfates is a key determinant of the binding of sulfated oligosaccharides to endogenous targets, these findings have broad implications for the advancement of Pentosan-based treatments.

Influence of saccharide modifications on heparin lyase III substrate specificities.

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

Effect of sulfated modification on rheological and physiological properties of oat β-glucan oligosaccharides prepared by acid or oxidative degradation

Two kinds of oat β-glucan oligosaccharides (βG-H and βG-O) were obtained by acid and oxidative degradation methods, respectively. Further, six kinds of sulfated oat β-glucan oligosaccharides (βG-H-S1, βG-H-S2, βG-H-S3, βG-O-S1, βG-O-S2, and βG-O-S3) with different degree of substitution were prepared by controlling the reaction time. The structure analysis showed that the molecular weights (Mw) and degree of substitution (DS) of sulfated oat β-glucan oligosaccharides increased with the prolonged reaction time. After sulfated modification, the apparent viscosity of oat β-glucan oligosaccharides increased and their viscoelasticity was changed. Sulfated modification endowed βG-H with enhanced fat binding capacity and bile acid binding capacity, and the above-mentioned properties increased with the increasing Mw and DS. Meanwhile, sulfated modification endowed βG-O with enhanced fat binding capacity and attenuated bile acid binding capacity, and the capacities of βG-O-S decreased with the increasing Mw and DS. Sulfated modification enhanced hypoglycemic effect of oat β-glucan oligosaccharides, it showed ability to decrease the glucose availability and inhibit α-glucosidase activity. The results revealed that the physiological properties of sulfated β-glucan oligosaccharides depend on their Mw, DS, and degradation method of β-glucan oligosaccharides.

Free radical scavenging activity of type II collagen peptides and chondroitin sulfate oligosaccharides from by-products of mottled skate processing

The by-products of the skate processing industry have bioactive properties but are mostly discarded. In the present study, the properties of skate cartilage, a major by-product of skate processing that is rich in chondroitin sulfate (CS) and type II collagen were investigated. Enzymatically-hydrolyzed type II collagen peptides (CP) and thermally hydrolyzed CS oligosaccharides (CSo) from skate cartilage were prepared and their antioxidant activities were studied. High free radical scavenging activity was observed in the <3 kDa MW fraction of the papain hydrolyzed CP (Pa-CP). CSo also had high free radical scavenging activity, but the activity was mainly due to the intermediate and final products of the non-enzymatic browning reaction resulting from high-temperature hydrolysis. These products may have included flavonoid-like compounds. Furthermore, Pa-CP and CSo were not cytotoxic. Thus, Pa-CP and CSo derived from skate by-products may be beneficial antioxidant ingredients. The present study may contribute to the development of functional health foods and achieving zero-waste skate processing.

Characterization of rat and mouse acidic milk oligosaccharides based on hydrophilic interaction chromatography coupled with electrospray tandem mass spectrometry

Oligosaccharides are one of the most important components in mammalian milk. Milk oligosaccharides can promote colonization of gut microbiota and protect newborns from infections. The diversity and structures of MOs differ among mammalian species. MOs in human and farm animals have been well-documented. However, the knowledge on MOs in rat and mouse have been very limited even though they are the most-widely used models for studies of human physiology and disease. Herein, we use a high-sensitivity online solid-phase extraction and HILIC coupled with electrospray tandem mass spectrometry to analyze the acidic MOs in rat and mouse. Among the fifteen MOs identified, twelve were reported for the first time in rat and mouse together with two novel sulphated oligosaccharides. The complete list of acidic oligosaccharides present in rat and mouse milk is the baseline information of these animals and should contribute to biological/biomedical studies using rats and mice as models.

Characterization of defined sulfated heparin-like oligosaccharides by electrospray ionization ion trap mass spectrometry.

Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.

Preparation of Possible P Selectin Inhibitor from Bovine Thyroglobulin, (Di-hydrido) Sulfo Hydrate 1, 5 Anhydro L-fucitol

P selectin is known to mediate several disease states through the binding of epitopes on the surface of endothelial cells. These diseases include cancer, cancer metastasis and inflammation. Sulfo Lewis a is a sulfated oligosaccharide could bind P selectin. A possible novel inhibitor of P-selectin binding may attenuate these diseases. One possible inhibitor is prepared from bovine thyroglobulin in one step, with appropriate work-up, from a readily abundant source, bovine thyroid gland, via novel chemistry. That molecule is (di-hydrido) sulfo hydrate 1, 5 anhydro L-fucitol. This chemistry is known to occur for the preparation of 1, 5 anhydro oligosaccharides from K casein and bovine milk. Another example of this chemistry has also been prepared; the di-(hydrido) di-phosphate di-hydrate 2, 5 anhydro mannitol (glucitol) from an ethanol extract of banana fruit. The molecule, here, would be the first report of H^- nucleophile attack of a non-phosphorylated glycoside. In addition to components of the O-linked oligosaccharide, to originate from bovine thyroglobulin requiring a tyrosine sulfate for binding. This work provides methods for preparing (di-hydrido) sulfo 1, 5 anhydro L-fucitol, as a possible inhibitor of P-selectin binding, in addition to those reported. These include; the di (di-hydrido) trisaccharide di-(hydrido) di-phosphate di-hydrate serinyl (di-hydrido) sulfo tyrosine dipeptide as well as the tri (di-hydrido) sulfo tri-hydrate 1, 5 anhydro trisaccharide alditol. Both of the latter two originate from bovine thyroglobulin and have been or will, possibly, be reported.

Discovery of rare sulfated N-unsubstituted glucosamine based heparan sulfate analogs selectively activating chemokines.

Achieving selective inhibition of chemokines with structurally well-defined heparan sulfate (HS) oligosaccharides can provide important insights into cancer cell migration and metastasis. However, HS is highly heterogeneous in chemical composition, which limits its therapeutic use. Here, we report the rational design and synthesis of N-unsubstituted (NU) and N-acetylated (NA) heparan sulfate tetrasaccharides that selectively inhibit structurally homologous chemokines. HS analogs were produced by divergent synthesis, where fully protected HS tetrasaccharide precursor was subjected to selective deprotection and regioselectively O-sulfated, and O-phosphorylated to obtain 13 novel HS tetrasaccharides. HS microarray and SPR analysis with a wide range of chemokines revealed the structural significance of sulfation patterns and NU domain in chemokine activities for the first time. Particularly, HT-3,6S-NH revealed selective recognition by CCL2 chemokine. Further systematic interrogation of the role of HT-3,6S-NH in cancer demonstrated an effective blockade of CCL2 and its receptor CCR2 interactions, thereby impairing cancer cell proliferation, migration and invasion, a step towards designing novel drug molecules.

Strategies in Synthesis of Heparin/Heparan Sulfate Oligosaccharides: 2000-Present.

Heparin and heparan sulfate are members of the glycosaminoglycan family that are involved in a multitude of biological processes. The great interests in the anticoagulant properties of heparin have stimulated major advances in synthetic strategies toward clinically effective analogues, as demonstrated importantly by the approval of the fully synthetic pentasaccharide fragment, termed fondaparinux (Arixtra®), of the heparin macromolecule for treatment of deep-vein thrombosis. Given the highly complex nature of heparin and heparan sulfate, the chemical synthesis of their components is a challenging endeavor. In the past decade, multiple approaches have been developed to improve the overall synthetic efficiency. New strategies have emerged that can generate libraries of oligosaccharide components of heparin and heparan sulfate. This article discusses recent developments in the assembly of heparin and heparan sulfate oligosaccharides and the associated challenges in their synthesis.

Sulfation of O-glycans on Mucin-type Proteins From Serous Ovarian Epithelial Tumors

Despite sulfated O-linked glycans being abundant on ovarian cancer (OC) glycoproteins, their regulation during cancer development and involvement in cancer pathogenesis remain unexplored. We characterized O-glycans carrying sulfation on galactose residues and compared their expression with defined sulfotransferases regulated during OC development. Desialylated sulfated oligosaccharides were released from acidic glycoproteins in the cyst fluid from one patient with a benign serous cyst and one patient with serous OC. Oligosaccharides characterized by LC-MSn were identified as core 1 and core 2 O-glycans up to the size of decamers and with 1 to 4 sulfates linked to GlcNAc residues and to C-3 and/or C-6 of Gal. To study the specificity of the potential ovarian sulfotransferases involved, Gal3ST2 (Gal-3S)-, Gal3ST4 (Gal-3S)-, and CHST1 (Gal-6S)-encoding expression plasmids were transfected individually into CHO cells also expressing the P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b (PSGL-1/mIg G2b) fusion protein and the human core 2 transferase (GCNT1). Characterization of the PSGL-1/mIg G2b O-glycans showed that Gal3ST2 preferentially sulfated Gal on the C-6 branch of core 2 structures and Gal3ST4 preferred Gal on the C-3 branch independently if core-1 or -2. CHST1 sulfated Gal residues on both the C-3 (core 1/2) and C-6 branches of core 2 structures. Using serous ovarian tissue micro array, Gal3ST2 was found to be decreased in tissue classified as malignant compared with tissues classified as benign or borderline, with the lowest expression in poorly differentiated malignant tissue. Neither Gal3ST4 nor CHST1 was differentially expressed in benign, borderline, or malignant tissue, and there was no correlation between expression level and differentiation stage. The data displays a complex sulfation pattern of O-glycans on OC glycoproteins and that aggressiveness of the cancer is associated with a decreased expression of the Gal3ST2 transferase.

Rational designing of glyco-nanovehicles to target cellular heterogeneity.

The aberrant expression of endocytic epidermal growth factor receptors (EGFRs) in cancer cells has emerged as a key target for therapeutic intervention. Here, we describe for the first time a state-of-the-art design for a heparan sulfate (HS) oligosaccharide-based nanovehicle to target EGFR-overexpressed cancer cells in cellular heterogeneity. An ELISA plate IC50 inhibition assay and surface plasma resonance (SPR) binding assay of structurally well-defined HS oligosaccharides showed that 6-O-sulfation (6-O-S) and 6-O-phosphorylation (6-O-P) of HS tetrasaccharides significantly enhanced EGFR cognate growth factor binding. The conjugation of these HS ligands to multivalent fluorescent gold nanoparticles (AuNPs) enabled the specific and efficient targeting of EGFR-overexpressed cancer cells. In addition, this heparinoid-nanovehicle exhibited selective homing to NPs in cancer cells in three-dimensional (3D) coculture spheroids, thus providing a novel target for cancer therapy and diagnostics in the tumor microenvironment (TME).

Anti-HIV Mechanism of Sulfated Poly and Oligosaccharides

Anti-HIV Mechanism of Sulfated Poly and Oligosaccharides

Qualitative mucin disorders in patients with primary Sjögren's syndrome: a literature review.

Background It is a common opinion that Primary Sjögren Syndrome (pSS) damages the exocrine glands and determines the reduction of secreted saliva, some studies show that there are qualitative anomalies of the mucins produced in saliva, including MUC7, MUC5B, MUC1. The purpose of this study is to trace all the information useful to establish whether there is a qualitative or quantitative defect of the mucins in the pSS. Material and Methods We reviewed the literature by looking for publications relevant to the topic in electronic databases. Sixteen articles met the search criteria. The studies were divided into two categories, those that studied the rheological characteristics of the saliva and those that studied the structural and / or metabolism modifications of the muciparous cells in the salivary glands. Results in Patients with pSS, xerostomia and the reduction of salivary spinnbarkeit are only partially related to the reduction of the unstimulated salivary flow. In pSS, pathological alterations of mucins’ chemical-physical properties prevail as a cause of the clinical characteristics. Moreover, in pSS there are structural and metabolism changes in salivary glands’ muciparous cells. Conclusions There is much evidence that supports the presence of qualitative alterations in the saliva’s rheological properties in Patients with pSS, and these are the main cause, more than the reduction of the unstimulated salivary flow, of the disease clinical characteristics - dry mouth and complications in the oral cavity. Therefore we propose to add to the classification criteria of pSS also a qualitative test of salivary glycoproteins. Key words:Primary Sjögren's syndrome, mucin, MUC7, MUC5B, MUC1, sulphate oligosaccharides.

Cloning and characterization of two chondroitin sulfate ABC lyases from Edwardsiella tarda LMG2793

Chondroitinase ABC can be used to prepare chondroitin sulfate (CS) oligosaccharides efficiently and environmentally. It also promotes nerve recovery through enzymatic degradation of glycosaminoglycan chains in damaged nerve tissue. In this study, two new chondroitin sulfate ABC lyases were expressed and characterized from Edwardsiella tarda LMG2793, with molecular weight of 116.8 kDa and 115.9 kDa, respectively. Two lyases ChABC I and ChABC II belonged to the polysaccharide lyase (PL) family 8. ChABC I and ChABC II showed enzyme activity towards chondroitin sulfate A (CS-A), CS-B, CS-C and CS-D, but had no activity towards hyaluronan (HA). The optimal temperature for ChABC I to exhibit the highest activity against CS-A was 40 °C and the optimal pH was 7.0. ChABC II showed the highest activity to CS-A at optimal temperature of 40 °C and pH of 9.0. ChABC I and ChABC II were stable at 37 °C and remained about 90 % of activity after incubation at 37 °C for 3 h. Many metal ions had no effect on the activity of ChABC I and ChABC II. These properties were beneficial to their further basic research and application. ChABC I was an endo-type enzyme while ChABC II was an exo-type enzyme. A group of amino acids were selected for further study by evaluating the sequence homology with other CS degradation lyases. Mutagenesis studies speculated that the catalytic residues in ChABC I were His522, Tyr529 and Arg581. The catalytic residues of ChABC II were His498, Tyr505 and Arg558. This work will contribute to the structural and functional characterization of biomedically relevant CS and promote the application of CS lyase in further basic research and therapeutics.

Distinct Mycoplasma pneumoniae Interactions with Sulfated and Sialylated Receptors.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the conducting airways, causing bronchitis and atypical or "walking" pneumonia in humans. M. pneumoniae recognizes sialylated and sulfated oligosaccharide receptors to colonize the respiratory tract, but the contribution of the latter is particularly unclear. We used chamber slides coated with sulfatide (3-O-sulfogalactosylceramide) to provide a baseline for M. pneumoniae binding and gliding motility. As expected, M. pneumoniae bound to surfaces coated with sulfatide in a manner that was dependent on sulfatide concentration and incubation temperature and inhibited by competing dextran sulfate. However, mycoplasmas bound to sulfatide exhibited no gliding motility, regardless of receptor density. M. pneumoniae also bound lactose 3'-sulfate ligated to an inert polymer scaffold, and binding was inhibited by competing dextran sulfate. The major adhesin protein P1 mediates adherence to terminal sialic acids linked α-2,3, but P1-specific antibodies that blocked M. pneumoniae hemadsorption (HA) and binding to the sialylated glycoprotein laminin by 95% failed to inhibit mycoplasma binding to sulfatide, suggesting that P1 does not mediate binding to sulfated galactose. Consistent with this conclusion, the M. pneumoniae HA-negative mutant II-3 failed to bind to sialylated receptors but adhered to sulfatide in a temperature-dependent manner.

Modular Synthesis of Heparan Sulfate Oligosaccharides Having N-Acetyl and N-Sulfate Moieties.

Heparan sulfates are structurally diverse sulfated polysaccharides that reside at the surface of all animal cells where they can interact with a multitude of proteins, thereby modulating a wide range of physiological and disease processes. We describe here a modular synthetic methodology that can provide libraries of heparan sulfate oligosaccharides that have glucosamine residues modified by different patterns of N-acetyl and N-sulfate moieties. It is based on the use of glycosyl donors that are modified at C2 by an azido- or trifluoromethylphenyl-methanimine moiety, which allowed the selective installation of α-glycosides. The amino protecting groups can be selectively unmasked by a reduction or acid treatment, allowing the installation of N-acetyl and N-sulfate moieties, respectively. In combination with the orthogonal hydroxyl protecting groups levulinic (Lev) ester, thexyldimethylsilyl (TDS) ether, allyloxycarbonate (Alloc), and 9-fluorenylmethyl carbonate (Fmoc), different patterns of O-sulfation can be installed. The methodology was applied to prepare four hexasaccharides that differ in the pattern of N- and O-sulfation. These compounds, together with a number of previously prepared HS oligosaccharides, were printed as a glycan microarray to examine the binding selectivities of several HS-binding proteins.

Study on the relationships between molecular weights of chondroitin sulfate oligosaccharides and Aβ-induced oxidative stress and the related mechanisms.

In the present study, we studied anti-Alzheimer's disease (AD) activities of chondroitin sulfate (CS) oligosaccharides with different molecular weights. CS from shark cartilage was degraded by a recombinant CS endolyase, chondroitinase ABC I (CHSase ABC I), and CS disaccharide (DP2), tetrasaccharide (DP4), hexasaccharide (DP6), octasaccharide (DP8), decasaccharide (DP10) and dodecasaccharide (DP12) were obtained by separation with gel filtration. Anti-AD activities of CS oligosaccharides were assessed using Aβ-injured SH-SY5Y cells and BV2 cells. It was shown that CS oligosaccharides could block Aβ-induced oxidative stress, mitochondrial dysfunction and activation of intrinsic apoptotic pathway for SH-SY5Y cells. Furthermore, these activities increased with the increase of molecular weights. For Aβ-injured BV2 cells, CS oligosaccharides inhibited oxidative stress, the production of proinflammatory cytokines and the activation of toll-like receptor pathway, and CS DP2 had the best activity among them. In conclusion, CS oligosaccharides suppressed Aβ-induced oxidative stress and relevant injury in vitro, and these effects had different relationships with the molecular weights of CS oligosaccharides for different cell lines, which might be caused by different mechanisms.