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l-Iduronic acid is a key constituent of heparin and heparan sulfate polysaccharides due to its unique conformational plasticity, which facilitates the binding of polysaccharides to proteins. At the same time, this is the synthetically most challenging unit of heparinoid oligosaccharides; therefore, there is a high demand for its replacement with a more easily accessible sugar unit. In the case of idraparinux, an excellent anticoagulant heparinoid pentasaccharide, we demonstrated that l-iduronic acid can be replaced by an easier-to-produce l-sugar while maintaining its essential biological activity. From the inexpensive d-mannose, through a highly functionalized phenylthio mannoside, the l-gulose donor was prepared by C-5 epimerization in 10 steps with excellent yield. This unit was incorporated into the pentasaccharide by α-selective glycosylation and oxidized to l-guluronic acid. The complete synthesis required only 36 steps, with 21 steps for the longest linear route. The guluronate containing pentasaccharide inhibited coagulation factor Xa by 50% relative to the parent compound, representing an excellent anticoagulant activity. To the best of our knowledge, this is the first biologically active heparinoid anticoagulant which contains a different sugar unit instead of l-iduronic acid.

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.

Hunter syndrome, or mucopolysaccharidosis type II (MPS II), is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), which is responsible for degrading heparan and dermatan sulfate. The IDS gene is located on chromosome Xq28; pathological variants in this gene mostly consist of missense mutations and small and larger deletions, which produce different phenotypes. However, there is only one record in our population concerning the molecular mechanism of this disease; a genotype-phenotype description is not available.There were included 24 unrelated male patients; clinical features were recorded at a database, fluorometric IDS enzyme activity testing was done for each individual, followed by Sanger sequencing to identify mutations.The mutational spectrum was found in 16 out of 24 Mexican patients with MPS II, and its range of phenotypes was described. The most frequent variants were of the missense type. The most affected exons were exon 3 (c.275TG, c.284_287del, c.325TC), exon 8 (c.1035GC, c.550GA), exon 9 (c.1403GC, c.1229_1229del), and exon 7 (c.979AC; this variant has not been previously reported). Exon 5 (c.438CT, a non-pathogenic variant) was the least frequent. It was also found that the most severely affected patients were those with large deletions (2 out of 24) [rsaIDS: IDSP1 (P164)x0, FMR1, AFF2 (P164)x2] involving genes and pseudogenes. We found 2 patients with a synonymous mutation in exon 4.Our results confirmed reports in the literature, since the most frequent variants were reported in exons 3 and 8. However, this result varies from one previous report in our population, which mentions large deletions and rearrangements as the most frequent alterations, since complex rearrangements were not found. According to what has been previously found, the most severely affected patients are those in which a whole gene has been deleted.

Heparan sulfate (HS) is a linear and complex polysaccharide that modulates the biological activities through protein recognition and interaction. Evidence indicates that protein-binding properties of HS are largely dependent on distinctive sulfation and epimerization patterns that are modified by a series of Golgi-localized enzymes. In particular, the glucuronyl C5-epimerase (Hsepi) converts D-glucuronic acid (GlcA) residues to L-iduronic acid (IdoA) and 2-O-sulfotransferase (2OST) catalyzes sulfation at C2 position of IdoA and rarely GlcA residues. Mice lacking both Hsepi and 2OST display multiple development defects, indicating the importance of IdoA in HS. Here, to gain greater insights of HS structure–function relationships, as well as a better understanding of the regulatory mechanisms of Hsepi and 2OST, the fine structure and cellular signaling functions of HS were investigated after restoration of Hsepi in the mutant mouse embryonic fibroblast (MEF) cells. Introduction of Hsepi into the Hsepi mutant MEF cells led to robustly increased proportion of IdoA residues, which rescued the cell signaling in response to fibroblast growth factor 2. However, we found that Hsepi knockout had no influence on either cellular transport or enzymatic activity of 2OST in the MEF cells, which is not in accord with the findings suggesting that the enzymatic activity and cellular transport of 2OST and Hsepi might be differently regulated.

Glycosaminoglycans (GAGs) are complex linear polysaccharides, which are covalently attached to core proteins (except for hyaluronan) to form proteoglycans. They play key roles in the organization of the extracellular matrix, and at the cell surface where they contribute to the regulation of cell signaling and of cell adhesion. To explore the mechanisms and pathways underlying their functions, we have generated an expanded dataset of 4,290 interactions corresponding to 3,464 unique GAG-binding proteins, four times more than the first version of the GAG interactome (Vallet, Clerc, and Ricard-Blum. J Histochem Cytochem 69: 93–104, 2021). The increased size of the GAG network is mostly due to the addition of GAG-binding proteins captured from cell lysates and biological fluids by affinity chromatography and identified by mass spectrometry. We review here the interaction repertoire of natural GAGs and of synthetic sulfated hyaluronan, the specificity and molecular functions of GAG-binding proteins, and the biological processes and pathways they are involved in. This dataset is also used to investigate the differences between proteins binding to iduronic acid-containing GAGs (dermatan sulfate and heparin/heparan sulfate) and those interacting with GAGs lacking iduronic acid (chondroitin sulfate, hyaluronan, and keratan sulfate).

Two-thirds of patients with mucopolysaccharidosis II (MPS II; Hunter syndrome) have cognitive impairment. This phase 2/3, randomized, controlled, open-label, multicenter study (NCT02055118) investigated the effects of intrathecally administered idursulfase-IT on cognitive function in patients with MPS II. Children older than 3 years with MPS II and mild-to-moderate cognitive impairment (assessed by Differential Ability Scales-II [DAS-II], General Conceptual Ability [GCA] score) who had tolerated intravenous idursulfase for at least 4 months were randomly assigned (2:1) to monthly idursulfase-IT 10 mg (n = 34) via an intrathecal drug delivery device (IDDD; or by lumbar puncture) or no idursulfase-IT treatment (n = 15) for 52 weeks. All patients continued to receive weekly intravenous idursulfase 0.5 mg/kg as standard of care. Of 49 randomized patients, 47 completed the study (two patients receiving idursulfase-IT discontinued). The primary endpoint (change from baseline in DAS-II GCA score at week 52 in a linear mixed-effects model for repeated measures analysis) was not met: although there was a smaller decrease in DAS-II GCA scores with idursulfase-IT than with no idursulfase-IT at week 52, this was not significant (least-squares mean treatment difference [95% confidence interval], 3.0 [-7.3, 13.3]; p = 0.5669). Changes from baseline in Vineland Adaptive Behavioral Scales-II Adaptive Behavior Composite scores at week 52 (key secondary endpoint) were similar in the idursulfase-IT (n = 31) and no idursulfase-IT (n = 14) groups. There were trends towards a potential positive effect of idursulfase-IT across DAS-II composite, cluster, and subtest scores, notably in patients younger than 6 years at baseline. In a post hoc analysis, there was a significant (p = 0.0174), clinically meaningful difference in change from baseline in DAS-II GCA scores at week 52 with idursulfase-IT (n = 13) versus no idursulfase-IT (n = 6) among those younger than 6 years with missense iduronate-2-sulfatase gene variants. Overall, idursulfase-IT reduced cerebrospinal glycosaminoglycan levels from baseline by 72.0% at week 52. Idursulfase-IT was generally well tolerated. These data suggest potential benefits of idursulfase-IT in the treatment of cognitive impairment in some patients with neuronopathic MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Enzyme replacement therapy with weekly infused intravenous (IV) idursulfase is effective in treating somatic symptoms of mucopolysaccharidosis II (MPS II; Hunter syndrome). A formulation of idursulfase for intrathecal administration (idursulfase-IT) is under investigation for the treatment of neuronopathic MPS II. Here, we report 36-month data from the open-label extension (NCT02412787) of a phase 2/3, randomized, controlled study (HGT-HIT-094; NCT02055118) that assessed the safety and efficacy of monthly idursulfase-IT 10 mg in addition to weekly IV idursulfase on cognitive function in children older than 3 years with MPS II and mild-to-moderate cognitive impairment. Participants were also enrolled in this extension from a linked non-randomized sub-study of children younger than 3 years at the start of idursulfase-IT therapy. The extension safety population comprised 56 patients who received idursulfase-IT 10 mg once a month (or age-adjusted dose for sub-study patients) plus IV idursulfase (0.5 mg/kg) once a week. Idursulfase-IT was generally well tolerated over the cumulative treatment period of up to 36 months. Overall, 25.0% of patients had at least one adverse event (AE) related to idursulfase-IT; most treatment-emergent AEs were mild in severity. Of serious AEs (reported by 76.8% patients), none were considered related to idursulfase-IT treatment. There were no deaths or discontinuations owing to AEs. Secondary efficacy analyses (in patients younger than 6 years at phase 2/3 study baseline; n = 40) indicated a trend for improved Differential Ability Scale-II (DAS-II) General Conceptual Ability (GCA) scores in the early idursulfase-IT versus delayed idursulfase-IT group (treatment difference over 36 months from phase 2/3 study baseline: least-squares mean, 6.8 [90% confidence interval: -2.1, 15.8; p = 0.2064]). Post hoc analyses of DAS-II GCA scores by genotype revealed a clinically meaningful treatment effect in patients younger than 6 years with missense variants of the iduronate-2-sulfatase gene (IDS) (least-squares mean [standard error] treatment difference over 36 months, 12.3 [7.24]). These long-term data further suggest the benefits of idursulfase-IT in the treatment of neurocognitive dysfunction in some patients with MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Heparin binds to and activates antithrombin (AT) through a specific pentasaccharide sequence, in which a trisaccharide subsite, containing glucuronic acid (GlcA), has been considered as the initiator in the recognition of the polysaccharide by the protein. Recently it was suggested that sulfated iduronic acid (IdoA2S) could replace this "canonical" GlcA. Indeed, a heparin octasaccharidic sequence obtained by chemoenzymatic synthesis, in which GlcA is replaced with IdoA2S, has been found to similarly bind to and activate antithrombin. By using saturation-transfer-difference (STD) NMR, NOEs, transferred NOEs (tr-NOEs) NMR and molecular dynamics, we show that, upon binding to AT, this IdoA2S unit develops comparable interactions with AT as GlcA. Interestingly, two IdoA2S units, both present in a 1 C4 -2 S0 equilibrium in the unbound saccharide, shift to full 2 S0 and full 1 C4 upon binding to antithrombin, providing the best illustration of the critical role of iduronic acid conformational flexibility in biological systems.

Ulvan, a sulfated polysaccharide extracted from the green seaweed genus Ulva, has bioactive properties including an immunomodulating capacity. The immunomodulatory capacity of ulvan from Ulva ohnoi, however, has not been assessed in detail. We depolymerised purified ulvan from U. ohnoi to obtain a range of molecular weight fractions (Mw 7, 9, 13, 21, 209 kDa), which were characterised by constituent sugar analysis, SEC-MALLS, and NMR. Ulvan fractions contained 48.8–54.7 mol% rhamnose, 32.5–35.9 mol% glucuronic acid, 4.5–7.3 mol% iduronic acid, and 3.3–5.6 mol% xylose. 1H and 13C NMR was consistent with hydrolysis occurring at the anomeric centre without further modification to the oligosaccharide structure. The in vitro immunomodulatory effect of ulvan fractions was quantified by measuring levels of inflammatory-mediating signalling molecules released from LPS-stimulated RAW264.7 murine macrophages. All ulvan fractions showed no toxicity on RAW264.7 cells at concentrations below 100 μg mL−1 over 48 h. Secreted interleukin-10 and prostaglandin E2 demonstrated an anti-inflammatory effect by higher molecular weight ulvan fractions at 100 μg mL−1. To a lesser extent, these fractions also enhanced the LPS-induced inflammation through minor increases of IL-1β and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

The chemoenzymatic synthesis of heparin, through a multienzyme process, represents a critical challenge in providing a safe and effective substitute for this animal-sourced anticoagulant drug. D-glucuronyl C5-epimerase (C5-epi) is an enzyme acting on a heparin precursor, N-sulfoheparosan, catalyzing the reversible epimerization of D-glucuronic acid (GlcA) to L-iduronic acid (IdoA). The absence of reliable assays for C5-epi has limited elucidation of the enzymatic reaction and kinetic mechanisms. Real time and offline assays are described that rely on 1D 1H NMR to study the activity of C5-epi. Apparent steady-state kinetic parameters for both the forward and the pseudo-reverse reactions of C5-epi are determined for the first time using polysaccharide substrates directly relevant to the chemoenzymatic synthesis and biosynthesis of heparin. The forward reaction shows unusual sigmoidal kinetic behavior, and the pseudo-reverse reaction displays nonsaturating kinetic behavior. The atypical sigmoidal behavior of the forward reaction was probed using a range of buffer additives. Surprisingly, the addition of 25 mM each of CaCl2 and MgCl2 resulted in a forward reaction exhibiting more conventional Michaelis–Menten kinetics. The addition of 2-O-sulfotransferase, the next enzyme involved in heparin synthesis, in the absence of 3′-phosphoadenosine 5′-phosphosulfate, also resulted in C5-epi exhibiting a more conventional Michaelis–Menten kinetic behavior in the forward reaction accompanied by a significant increase in apparent Vmax. This study provides critical information for understanding the reaction kinetics of C5-epi, which may result in improved methods for the chemoenzymatic synthesis of bioengineered heparin.

Recently, the activity in heparan sulfate (HS) has led to the discovery of many drug molecules that have the potential to impact both medical science and human health. However, structural diversity and synthetic challenges impede the progress of HS research. Here we show that synthetic HS mimics can be engineered to produce many of the functions of native HS. HS mimics were synthesized from an L-Iduronic acid (IdoA) scaffold, which was homooligomerized by stereoselective α-1,4-glycosylation and regioselective sulfation. The exhaustive NMR analysis of these HS mimics, using both NOE and vicinal 3JH-H coupling constants, confirmed that sulfation at the O-4 enhances the 1C4 geometry population at the corresponding ring. Interestingly, the 1C4 conformer becomes almost exclusive upon additional sulfation at O-2. Microarray and SPR analysis of HS mimics with different growth factors established that oligosaccharide length, sulfation code, and exclusive IdoA conformation synergistically affect the specificity and activity of growth factors. Particularly, 4-O-sulfated IdoA disaccharide had a strong binding affinity to vascular endothelial growth factor (VEGF165) and therefore, modulated endothelial cell proliferation, migration, and angiogenesis. These results establish the potential of sulfated IdoA oligosaccharides to be used as structurally well-defined HS mimics as they provide several functions of native HS.

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.

Enoxaparin, widely used antithrombotic drug, is a polydisperse glycosaminoglycan with highly microheterogeneous structure dictated by both parent heparin heterogeneity and depolymerization conditions. While the process-related modifications of internal and terminal sequences of enoxaparin have been extensively studied, very little is known about the authentic non-reducing ends (NRE). In the present study a multi-step isolation and thorough structural elucidation by NMR and LC/MS allowed to identify 16 saturated tetramers along with 23 unsaturated ones in the complex enoxaparin tetrasaccharide fraction. Altogether the elucidated structures represent a unique enoxaparin signature, whereas the composition of saturated tetramers provides a structural readout strictly related to the biosynthesis of parent heparin NRE. In particular, both glucuronic and iduronic acids were detected at the NRE of macromolecular heparin. The tetrasaccharides bearing glucosamine at the NRE are most likely associated with the heparanase hydrolytic action. High sulfation degree and 3-O-sulfation are characteristic for both types of NRE.

Heparan sulfate (HS) and dermatan sulfate (DS) are l-iduronic acid containing glycosaminoglycans (GAGs) which are implicated in a number of biological processes and conditions including cancer and viral infection. Chemical synthesis of HS and DS is required to generate structurally defined oligosaccharides for a biological study. Herein, we present a new synthetic approach to HS and DS oligosaccharides using chemoselective glycosylation which relies on a disarmed [2.2.2] l-ido lactone motif. The strategy provides a general approach for iterative-reducing end chain extension, using only shelf-stable thioglycoside building blocks, exploiting a conformational switch to control reactivity, and thus requires no anomeric manipulation steps between glycosylations.

A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N-acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA2R-(α1,4)-D-GlcNAc-(α1-, in which R was −OH (˜80%) or –OSO3− (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC50, 0.10 μM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity.

Low molecular weight heparins (LMWHs) are currently used as an anticoagulant agent since unfractionated heparin (UFH) can cause serious adverse drug reactions. LMWHs are commercially prepared using different methods such as nitrous acid cleavage and β-elimination under strong reaction conditions or with harsh chemicals, which may cause the saccharide units within the polysaccharide backbone to be decomposed and noticeably modified. This study demonstrates an effective method for depolymerizing heparin via the production of large amounts of free radicals from H2O2/ascorbic acid and ultrasonic power; this results in highly pure products because ascorbic acid can decompose during the reaction, which is different from the previously reported H2O2/Cu2+ method. The reaction conditions-including concentration of ascorbic acid, reaction temperature and intensity of ultrasonic power-were investigated and optimized. We found that the degradation behavior of heparin in this combined physicochemical process conformed to first-order reaction kinetics. The chemical composition and structures of different LMWHs were analyzed. The results showed the primary structure and sulfate esters were well preserved after the depolymerization, the major repeat units are (1-4)-linked glucosamine and iduronic acid. The further in vitro assays indicated that the LMWHs produced by H2O2/ascorbic acid with ultrasonic power have an anti-metastatic effect in A549 cells, which suggested the LMWHs rapidly prepared in this physicochemical way have a potential for anti-tumor metastatic function.

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.

Glucuronyl C5-epimerase (Hsepi) catalyzes the conversion of glucuronic acid to iduronic acid in the process of heparan sulfate biosynthesis. Targeted interruption of the gene, Glce, in mice resulted in neonatal lethality with varied defects in organ development. To understand the underlying molecular mechanisms of the phenotypes, we used mouse embryonic fibroblasts (MEF) as a model to examine selected signaling pathways. Our earlier studies found reduced activities of FGF-2, GDNF, but increased activity of sonic hedgehog in the mutant cells. In this study, we focused on the bone morphogenetic protein (BMP) signaling pathway. Western blotting detected substantially elevated endogenous Smad1/5/8 phosphorylation in the Hsepi mutant (KO) MEF cells, which is reverted by re-expression of the enzyme in the KO cells. The mutant cells displayed an enhanced proliferation and elevated alkaline phosphatase activitywhen cultured in osteogenic medium. Analysis of the genes involved in the BMP signaling pathway revealed upregulation of a number of BMP ligands, but reduced expression of several Smads and BMP antagonist (Grem1) in the KO MEF cells. The high level of Smad1/5/8 phosphorylation was also found in primary calvarial cells isolated from the KO mice. The results suggest that Hsepi expression modulates BMP signaling activity, which, at least partially, is associated with defected molecular structure of heparan sulfate expressed in the cells.

Mucopolysaccharidosis type II (Hunter Syndrome) is a rare X-linked inherited lysosomal storage disorder presenting a wide genetic heterogeneity. It is due to pathogenic variants in the IDS gene, causing the deficit of the lysosomal hydrolase iduronate 2-sulfatase, degrading the glycosaminoglycans (GAGs) heparan- and dermatan-sulfate. Based on the presence/absence of neurocognitive signs, commonly two forms are recognized, the severe and the attenuate ones. Here we describe a line of induced pluripotent stem cells, generated from dermal fibroblasts, carrying the mutation c.479C>T, and obtained from a patient showing an attenuated phenotype. The line will be useful to study the disease neuropathogenesis.

Clinically approved anti-coagulant Fondaparinux is safe since it has zero contamination problems often associated with animal based heparins. Fondaparinux is a synthetic pentasaccharide based on the antithrombin-binding domain of Heparin sulfate and contains glucosamine, glucuronic acid and iduronic acid in its sequence. Here, we show the formal synthesis of Fondaparinux pentasaccharide by performing all glycosidations in a catalytic fashion for the first time to the best of our knowledge. Designer monosaccharides were synthesized avoiding harsh reaction conditions or reagents. Further, those were subjected to reciprocal donor-acceptor selectivity studies to guide [Au]/[Ag]-catalytic glycosidations for assembling the pentasaccharide in a highly convergent [3 + 2] or [3 + 1 + 1] manner. Catalytic and mild activation during glycosidations that produce desired glycosides exclusively, scalable route to the synthesis of unnatural and expensive iduronic acid, minimal number of steps and facile purifications, shared use of functionalized building blocks and excellent process efficiency are the salient features. Despite strong demand for their clinical use, the synthesis of heparin oligosaccharides as anticoagulants remains challenging. Here, a mild and scalable formal synthesis of Fondaparinux pentasaccharide is presented through [Au]/[Ag]-catalyzed glycosidations.

Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva.

Of all posttranslational modifications known, glycosaminoglycans (GAGs) remain one of the most challenging to study, and despite the recent years of advancement in MS technologies and bioinformatics, detailed knowledge about the complete structures of GAGs as part of proteoglycans (PGs) is limited. To address this issue, we have developed a protocol to study PG-derived GAGs. Chondroitin/dermatan sulfate conjugates from the rat insulinoma cell line, INS-1832/13, known to produce primarily the PG chromogranin-A, were enriched by anion-exchange chromatography after pronase digestion. Following benzonase and hyaluronidase digestions, included in the sample preparation due to the apparent interference from oligonucleotides and hyaluronic acid in the analysis, the GAGs were orthogonally depolymerized and analyzed using nano-flow reversed-phase LC-MS/MS in negative mode. To facilitate the data interpretation, we applied an automated LC-MS peak detection and intensity measurement via the Proteome Discoverer software. This approach effectively provided a detailed structural description of the nonreducing end, internal, and linkage region domains of the CS/DS of chromogranin-A. The copolymeric CS/DS GAGs constituted primarily consecutive glucuronic-acid-containing disaccharide units, or CS motifs, of which the N-acetylgalactosamine residues were 4-O-sulfated, interspersed by single iduronic-acid-containing disaccharide units. Our data suggest a certain heterogeneity of the GAGs due to the identification of not only CS/DS GAGs but also of GAGs entirely of CS character. The presented protocol allows for the detailed characterization of PG-derived GAGs, which may greatly increase the knowledge about GAG structures in general and eventually lead to better understanding of how GAG structures are related to biological functions., Graphical Abstract, Highlights • Protocol developed to structurally characterize glycosaminoglycans of proteoglycans. • Comprehensive characterization of cellular glycosaminoglycan structures. • Relative quantification of nonreducing end, internal, and linkage region domains. • Overall chondroitin/dermatan sulfate glycosaminoglycan structures of chromogranin-A., In Brief Glycosaminoglycans (GAGs) remain one of the most challenging posttranslational modifications to study, much due to their structural complexity and heterogeneity, and new methods for analysis are therefore required. We have developed a protocol for enrichment and structural characterization of GAGs of proteoglycans using nLC-MS/MS. We provide detailed information on the nonreducing end, internal, and linkage region GAG domains and use the data to determine an overall GAG structure of chromogranin-A of rat INS-1832/13 cells.

Crude anionic polysaccharides extracted from the Pacific starfish Lysastrosoma anthosticta were separated by anion-exchange chromatography into fractions LA-F1 and LA-F2. The main fraction LA-F1 was solvolytically desulfated giving rise to preparation LA-F1-DS with a structure of dermatan core [→3)-β-d-GalNAc-(1→4)-α-l-IdoA-(1→]n. Reduction of LA-F1 afforded preparation LA-F1-RED composed mainly of the repeating disaccharide units →3)-β-d-GalNAc4R-(1→4)-α-l-Ido2S3S-(1→, where R was SO3- or H. Analysis of the NMR spectra of the parent fraction LA-F1 led to determine the main component as the oversulfated dermatan sulfate LA-Derm bearing sulfate groups at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor fraction LA-F2 contained a mixture of LA-Derm and heparinoid LA-Hep, the latter being composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→ and →4)-α-d-GlcNS3S-(1→4)-α-l-IdoA2S3S-(1→. The presence of 2,3-di-O-sulfated iduronic acid residues is very unusual both for natural dermatan sulfate and heparinoid. Preparations LA-F1, LA-F2 and LA-F1-RED demonstrated significant anticoagulant effect in vitro.

Glycosaminoglycans (GAGs) are a family of anionic carbohydrates that play an essential role in the physiology and pathology of all eukaryotic life forms. Experimental determination of GAG-protein complexes is challenging due to their difficult isolation from biological sources, natural heterogeneity, and conformational flexibility-including possible ring puckering of sulfated iduronic acid from 1C4 to 2SO conformation. To overcome these challenges, we present GlycoTorch Vina (GTV), a molecular docking tool based on the carbohydrate docking program VinaCarb (VC). Our program is unique in that it contains parameters to model 2SO sugars while also supporting glycosidic linkages specific to GAGs. We discuss how crystallographic models of carbohydrates can be biased by the choice of refinement software and structural dictionaries. To overcome these variations, we carefully curated 12 of the best available GAG and GAG-like crystal structures (ranging from tetra- to octasaccharides or longer) obtained from the PDB-REDO server and refined using the same protocol. Both GTV and VC produced pose predictions with a mean root-mean-square deviation (RMSD) of 3.1 A from the native crystal structure-a statistically significant improvement when compared to AutoDock Vina (4.5 A) and the commercial software Glide (5.9 A). Examples of how real-space correlation coefficients can be used to better assess the accuracy of docking pose predictions are given. Comparisons between statistical distributions of empirical "salt bridge" interactions, relevant to GAGs, were compared to density functional theory (DFT) studies of model salt bridges, and water-mediated salt bridges; however, there was generally a poor agreement between these data. Water bridges appear to play an important, yet poorly understood, role in the structures of GAG-protein complexes. To aid in the rapid prototyping of future pose scoring functions, we include a module that allows users to include their own torsional and nonbonded parameters.

(1)H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the (1)C(4):(2)S(0) forms of the FPX residue IdoA(2S) is altered from ≈ 35:65 (free FPX) to ≈ 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

Ulvan is a sulfated polysaccharide from green algae with potent antitumor, antiviral, and immunomodulatory activities. However, no chemical synthesis of ulvan saccharides has been reported to date. In this paper, we performed the first efficient synthesis of the unique sulfated tetrasaccharide motif of type B ulvanobiuronic acid 3-sulfate. Based on the gold(i)-catalyzed glycosylation with glycosyl ynenoates as donors, efficient construction of the challenging α-(1 → 4)-glycosidic bonds between iduronic acid and rhamnose building blocks was achieved to afford the tetrasaccharide skeleton in a stereospecific manner. The synthetic sulfated tetrasaccharide was found to significantly improve the phagocytic activity of macrophage RAW264.7 cells.

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.肝素和硫酸乙酰肝素是一类应用于临床抗凝血的糖胺聚糖。肝素葡萄糖醛酸C5 异构酶(Heparosan-N-sulfate-glucuronate 5-epimerase，C5，EC 5.1.3.17) 是肝素和硫酸乙酰肝素合成过程中重要的修饰酶，催化N-硫酸化肝素前体 (N-sulfoheparosan) 的D-葡萄糖醛酸 (D-GlcA) 上5 号位羧基翻转生成L-艾杜糖醛酸(L-iduronic acid，L-IdoA)。文中以大肠杆菌Escherichia coli 为宿主对斑马鱼来源的肝素葡萄糖醛酸C5 异构酶基因Glce 进行重组表达优化与分子改造。比较了3 种不同的表达载体pET20b(+)、pET28a(+) 和pCold Ⅲ对C5 表达的差异情况，其中以嗜冷启动型载体pCold Ⅲ表达酶活最高，达到(1 873.61±5.42) U/L。为了进一步提高C5 的可溶表达量，在N 端融合促溶标签SET2 后，可溶蛋白表达量比对照提高了50%，酶活达到 (2 409.25±6.43) U/L。在此基础上，通过理性设计对底物结合口袋进行定点突变，获得最优突变体 (V153R) 的酶活和比酶活分别为(5 804.32±5.63) U/L 和(145.14±2.33) U/mg，是原始酶的2.41 倍和2.28 倍。肝素C5 异构酶改造与表达优化为酶法催化合成肝素奠定了基础。.

Tetra-tert-butyl-3-chloro-1-hydroxydistannoxane has been found to selectively cleave with high efficiency primary acetates on complex oligosaccharides containing esterified l-iduronic acid and bearing an anomeric acetate. This tin based catalyst was found much more effective than magnesium methoxide to carry out selective deacetylation.

Heparan sulfate (HS) is a class of linear, sulfated, anionic polysaccharides, called glycosaminoglycans (GAGs), which present on the mammalian cell surfaces and extracellular matrix. HS GAGs display a wide range of critical biological functions, particularly in cell signaling. HS is composed of repeating units of 1 → 4 glucosidically linked uronic acid and glucosamine residues. Heparin, a pharmacologically important version of HS, having higher sulfation and a higher content of iduronic acid than HS, is a widely used clinical anticoagulant. However, due to their heterogeneity and complex structure, HS and heparin are very challenging to analyze, limiting biological studies and even resulting in safety concerns in their therapeutic application. Therefore, reliable methods of structural analysis of HS and heparin are critically needed. In addition to the structural analysis of heparin, its concentration in blood needs to be closely monitored to avoid complications such as thrombocytopenia or hemorrhage caused by heparin overdose. This review summarizes the progress in biotechnological approaches in the structural characterization of HS and heparin over the past decade and includes the development of the ultrasensitive approaches for detection and measurement in biological samples.

Ulvan, a kind of polyanionic heteropolysaccharide consisting of 3-sulfated rhamnose, uronic acids (iduronic acid and glucuronic acid) and xylose, has been widely applied in food and cosmetic industries. In addition, ulvan can be converted into fermentable monosaccharides through the cascade system of carbohydrate-active enzymes. Ulvan lyases can degrade ulvan into ulvan oligosaccharides, which is the first step in the fully degradation of ulvan. Various ulvan lyases have been cloned and characterized from marine bacteria and grouped into five polysaccharide lyase (PL) families, namely: PL24, PL25, PL28, PL37 and PL40 families. The elucidation of the biochemical characterization, action pattern and catalytic mechanism of ulvan lyase would definitely enhance our understanding of the deep utilization of marine bioresource and marine carbon cycling. In this review, we summarized the recent progresses about the source and biochemical characteristics of ulvan lyase. Additionally, the structural characteristics and catalytic mechanisms have been introduced in detail. This comprehensive information should be helpful regarding the application of ulvan lyases.

Low-molecular-weight heparin and ultra-low-molecular-weight heparin are widely used as anticoagulant and antithrombotic drugs. An atmospheric pressure dielectric-barrier discharge procedure was developed to depolymerize heparin to low- or ultra-low-molecular-weight fragments. Dielectric-barrier discharge treatment of heparin resulted in several well-separated and uniform fragments. The structural character of digested fragments below 3,000 Da was elucidated. Expected digestion products such as sulfated disaccharide, sulfated tetrasaccharide, and pentasaccharides with alternatingly-linked N-acetyl glucosamine and iduronic acid were detected. The anticoagulant potential of the digested heparin was tested. The results indicated that dielectric-barrier discharge treatment can produce various molecular weight (MW) heparin fragments and maintain anticoagulant potency.

Ulvan is a complex water-soluble sulfated polysaccharide in the cell wall of green algae belonging to genus Ulva. It is composed of l-rhamnose-3-sulfate (Rha3S), glucuronic acid (GluA), iduronic acid (IduA), and d-xylose (Xyl) distributed in three repetition moieties. The first step of a bacterial ulvan degradation is the cleavage of the β-glycosidic bond between Rha3S and GluA/IduA through a β-elimination mechanism by a ulvan lyase to produce oligo-ulvans with unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronate (Δ) at the non-reducing end. We have identified an ulvan associated polysaccharide utilization locus (PUL) residing between two ulvan lyase genes belonging to families of polysaccharide lyase 24 (PL24) and PL25 in the genome of a ulvan-utilizing bacterium Glaciecola KUL10 strain. The PUL contains many genes responsible for oligo-ulvan degradation. Among them, we demonstrated that both KUL10_26540 and KUL10_26770 had an unsaturated β-glucuronyl hydrolase activity to produce Rha3S and oligosaccharides, such as Rha3S-GluA-Rha3S, Rha3S-IduA-Rha3S and, Rha3S-Xyl-Rha3S, by releasing 5-dehydro-4-deoxy-d-glucuronate. KUL10_26540 showed much higher activity than KUL10_26770 and was more active on disaccharide than tetrasaccharide. We also found a rhamnosidase activity on four KUL10 gene products, although they could not react on the sulfated rhamnose.

Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.

Several l-idose and l-iduronic acid glycosyl donors (mostly thioglycosides but also halides and trichloroacetimidates) with acyl protecting groups at the C-2 position were prepared and evaluated in glycosylation reactions with simple acceptors. In glycosaminoglycan oligosaccharide syntheses in the literature, the presence of C-2 acyl protecting groups in l-ido-configured glycosyl donors generally results in exclusive formation of 1,2-trans glycosidic linkages, a finding that has typically been attributed to neighbouring-group participation. However, glycosylations of simple alcohols with l-ido-configured donors (particularly thioglycosides), reported here, generally displayed incomplete stereocontrol and gave mixtures of the 1,2-trans and 1,2-cis products, suggesting that neighbouring-group participation has lesser importance in these reactions. Glycosyl donors and reaction conditions were identified that gave improved, but not exclusive, selectivity for the desired α-l-anomer (1,2-trans) as the major product. Interestingly, glycosylations under the same reaction conditions with more complex monosaccharide acceptors gave exclusively the expected 1,2-trans products. The role of neighbouring-group participation in these glycosylations was explored with density functional theory (DFT) calculations, which revealed that the non-stereoselective addition of the acceptor alcohol to the intermediate oxocarbenium ion is competitive with the stereospecific addition of the acceptor to the acyloxonium ion intermediate.

Ulvan, extracted from the marine green algae belonging to the genus Ulva, is mainly composed of four monosaccharides consisting of rhamnose, xylose, glucuronic acid and iduronic acid. It has great potential in the fields of food, pharmaceuticals and chemistry. It can be degraded by ulvan lyase with a β-elimination mechanism, which cleaves the β-glycosidic bond between sulfated rhamnose and glucuronic acid/iduronic acid, thereby producing an Ulva oligosaccharide with unsaturated bond. These oligosaccharides have several pleasing functions, including antioxidant, antiviral, antilipidic, antibacterial, and other effects. The latest CAZy data shows that ulvan lyases are composed of four families of lyases: PL24, PL25, PL28, and PL40. The structural features of some enzymes have been fully demonstrated, which is highly helpful in understanding their catalytic mechanisms. This review summarizes key sources, classifications, and key research advances in the biochemical properties of enzymes. In addition, its structural features and catalytic mechanism have been organized. Finally, we briefly summarized the potential of various active functional oligosaccharides produced by degrading ulvan in future applications. This detailed information can give us a better understanding of ulvan.

Green algae belonging to the genus Ulvales are known to produce ulvan which is one of the main polysaccharide components of their cell wall. Ulvan is composed of 3O-sulfate-rhamnose (Rha3S), glucuronic acid (GlcA), iduronic acid (IduA) and xylose (Xyl) distributed in three disaccharide repetition moieties: [→ 4)- β -D-GlcA-(1 → 4)- α -L-Rha3S-(1 →], [→ 4)- α -L-IdoA-(1 → 4)- α -L-Rha3S(1 →] and [→ 4)- β -D-Xyl-(1 → 4)- α -L-Rha3S(1 →]. The ability of bacteria to degrade complex algal polysaccharides such as ulvan is usually encoded in clusters of genes referred to as polysaccharide utilization loci (PUL). Full saccharification of ulvan is expected to require an ulvan lyase, which cleaves the β -(1 → 4)-glycosidic bond between Rha3S and GluA or IduA through a β -elimination mechanism. In addition, enzymes with β -glucuronyl hydrolase, rhamnosidase, xylosidase and sulfatase activity are also expected. Recently, the genomes of several ulvan degrading bacteria were sequenced, which led to the identification of a new family of polysaccharide lyases family 24 (PL24). In this work, we have continued to mine the genomic data of one of the sequenced strains, Alteromonas sp. LOR. Here we report the identification of an ulvan associated PUL residing between open reading frames ( lor_19 – lor_34 ). This PUL contains a TonB dependent receptor, along with an experimentally verified rhamnosidase, a β -glucuronyl hydrolase and predicted sulfatases. Interestingly, we also identified in the PUL a new ulvan lyase (LOR_29) which showed no homology to previously reported ulvan lyases making it a founding member of yet another new family of polysaccharide lyases (PL25). Finally, this enzyme prompted us to mine other genomes where we identified additional potential ulvan PULs harboring this gene in other bacterial species. Taken together our report provides further insight into ulvan degradation mechanisms in bacteria and reveals a new family of polysaccharide lyases.

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B3 ´ –CO2, which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2-O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2-O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA.

For a more insightful investigation into the specificity of bacterial heparinase I, a series of structurally well-defined heparin oligosaccharides was synthesized using a highly efficient chemoenzymatic strategy. Apart from the primary cleavage site, five glycosidic linkages of oligosaccharides with varying modifications to obtain secondary cleavage sites were degraded by a high concentration of heparinase I. The reactivity of linkages toward heparinase I was not entirely dependent on the 2-O-sulfated iduronic acid being cleaved or the neighboring 6-O-sulfated glucosamine residues, but it was dependent on higher degrees of sulfation of oligosaccharides and indispensable N-substituted glucosamine adjacent to the cleavable linkage. Moreover, the enzyme demonstrated less preferential cleavage toward glycosidic linkages containing glucuronic acid than those containing iduronic acid of the counterpart oligosaccharides. Biolayer interferometry revealed differences in reactivity that are not completely consistent with different affinities of substrates to enzyme. Our study presented accurate information on the cleavage promiscuity of heparinase I that is crucial for heparin depolymerization.

Objectives To assess the outcome of population-based newborn screening for mucopolysaccharidosis type II (MPS II) during the first year of screening in Illinois. Study design Tandem mass spectrometry was used to measure iduronate-2-sulfatase (I2S) activity in dried blood spot specimens obtained from 162 000 infant samples sent to the Newborn Screening Laboratory of the Illinois Department of Public Health in Chicago. Results One case of MPS II and 14 infants with pseudodeficiency for I2S were identified. Conclusions Newborn screening for MPS II by measurement of I2S enzyme activity was successfully integrated into the statewide newborn screening program in Illinois.

Pathogens are able to exploit specific glycosaminoglycans (GAGs), especially iduronic acid (IdoA)-containing GAGs, to invade the host. By analyzing Escherichia coli proteome chip data, we identified the interactomes of three IdoA-containing GAGs: heparin, heparin sulfate (HS), and chondroitin sulfate B (CSB). Using non-IdoA-containing GAG, chondroitin sulfate C, as a negative control, 157 proteins specifically binding with IdoA-containing GAGs were revealed in the present study. These proteins showed functional enrichment in protein synthesis and metabolism. Fifteen proteins which commonly interacts with three IdoA-containing GAGs were further examined. The regular expression for motif showed these common IdoA interactome shared a conserved sequence. Among them, we identified a second flagellar system outer membrane protein, MbhA. The MbhA has Kd values of 8.9 × 10−8 M, 5.3 × 10−7 M, and 1.79 × 10−7 M to interact with heparin, HS, and CSB, respectively. Using flow cytometry, we confirmed that the MbhA protein can bind to human epithelial cells HCT-8. Overexpression of mbhA increased the percentage of invasion in E. coli which lacks a second flagellar system. Moreover, pre-blocking of HCT-8 cells with MbhA inhibited the bacterial invasion, implying the importance of the direct interaction of MbhA and the host cell surface on bacterial invasion. Significance We analyzed the Escherichia coli proteomic data to elucidate the interactomes of three different IdoA-containing GAGs (heparin, HS, and CSB) because these IdoA-containing GAGs can mediate bacterial invasion to the host. Through proteomic and systematic analysis, a second flagellar system outer membrane protein, MbhA, was also identified in the present study. Affinity assay confirmed that MbhA can bind to three IdoA-containing GAGs heparin, HS, and CSB. The result of flow cytometry also showed MbhA can interact with human epithelial cells HCT-8. Results of bacteria invasion assay showed overexpression of mbhA promoted the bacterial invasion. Moreover, pre-blocking of HCT-8 cells with MbhA also reduced the percentage of bacterial invasion. These findings correspond well that MbhA is one of invasion factors.

Musculocontractural Ehlers-Danlos syndrome (MC-EDS) is a rare recessive disorder that is characterized by connective tissue fragility, distinct craniofacial features and congenital malformations. MC-EDS patients have defects in the enzymes dermatan sulfate epimerase-1 and dermatan 4-O-sulfotransferase-1, which are involved in the biosynthesis of iduronic acid in the chondroitin sulfate/dermatan sulfate (CS/DS) chains of proteoglycans (PGs). While the connective tissue defect is a result of disturbed collagen fibril assembly based on a decreased iduronic acid content of interacting CS/DS-PGs, the cause of the developmental malformations in MC-EDS is not well understood. This review focuses on a new role of CS/DS-PGs in the development of multipotent and highly migratory neural crest (NC) cells in the Xenopus embryo model of MC-EDS. Single iduronic acid residues in CS/DS-PGs are involved in the formation of NC-derived craniofacial structures by facilitating the migration and adhesion of NC cells to fibronectin. Our results suggest a defect in NC development as cause of the craniofacial and other congenital anomalies in MC-EDS patients, which might contribute to an improved diagnosis and etiology-based therapy. (Less)

Currently porcine intestine is the only approved source for producing pharmaceutical heparin in most countries. Enoxaparin, prepared by benzylation and alkaline depolymerization from porcine intestine heparin, is prevalent in the anticoagulant drug market. It is predicted that porcine intestine heparin-derived enoxaparin (PIE) will encounter shortage, and expanding its production from heparins obtained from other animal tissues may, therefore, be inevitable. Bovine lung heparin is a potential alternative source for producing enoxaparin. Critical processing parameters for producing bovine lung heparin-derived enoxaparin (BLE) are discussed. Three batches of BLEs were prepared and their detailed structures were compared with PIEs using modern analytical techniques, including disaccharide composition, intact chain mapping by liquid chromatography-mass spectrometry and 2-dimensional nuclear magnetic resonance spectroscopy. The results suggested that the differences between PIEs and BLEs mainly result from N-acetylation differences derived from the parent heparins. In addition, bioactivities of BLEs were about 70% of PIEs based on anti-factor IIa and Xa chromogenic assays. We conclude that BLE has the potential to be developed as an analogue of PIE, although some challenges still remain.

Heparan sulfate (HS) is widely distributed in mammalian tissues in the form of HS proteoglycans, which play essential roles in various physiological and pathological processes. In contrast to the template-guided processes involved in the synthesis of DNA and proteins, HS biosynthesis is not believed to involve a template. However, it appears that the final structure of HS chains was strictly regulated. Herein, we report research based hypothesis that two major steps, namely "coding" and "decoding" steps, are involved in the biosynthesis of HS, which strictly regulate its chemical structure and biological activity. The "coding" process in this context is based on the distribution of sulfate moieties on the amino groups of the glucosamine residues in the HS chains. The sulfation of these amine groups is catalyzed by N-deacetylase/N-sulfotransferase, which has four isozymes. The composition and distribution of sulfate groups and iduronic acid residues on the glycan chains of HS are determined by several other modification enzymes, which can recognize these coding sequences (i.e., the "decoding" process). The degree and pattern of the sulfation and epimerization in the HS chains determines the extent of their interactions with several different protein factors, which further influences their biological activity.

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice.

Diabetic foot ulcer (DFU) is a common high-risk complication in patients with diabetes mellitus, but current drugs and therapies in management of this disease cannot meet the urgent clinical needs. In this study, a snail glycosaminoglycan (SGAG) from the cultured China white jade snail was purified and structurally clarified. This snail glycosaminoglycan is a regular sulfated polysaccharide, composed of iduronic acid (IdoA) and N-acetyl-glucosamine (GlcNAc) with the repeating sequence of →4)-α-GlcNAc (1→4)-α-IdoA2S (1→. The biological assays showed that SGAG had no anticoagulant activity for lacking specific heparin pentasaccharide sequence. The pharmacological experiments suggested that SGAG markedly accelerated the healing of full-thickness wounds in diabetic mice skin. Histologic and immunohistochemical analysis revealed that SGAG treatment alleviated the inflammation and dermal edema, and promoted angiogenesis. This is the first report applying the snail glycosaminoglycan to favor diabetic wound healing.

The cell wall polysaccharide ulvan was isolated from two species of the seaweed Ulva collected along the Swedish west coast. Acidic extraction was benchmarked against hot water extraction with enzymatic purification and against commercial ulvan. Extracted ulvan contained 11–18 % g/g of ash, some protein (up to 1.3 % g N/g) but minimal colored impurities. The ulvans had high molecular weights (660,000–760,000 g/mol) and were composed of 77–79 % g/g carbohydrates, mainly rhamnose, xylose, glucose, glucuronic acid, and iduronic acid. The extraction protocol and the ulvan source strongly impact the molecular weight and the chemical composition. Acidic extraction caused almost complete desulfation of the isolated ulvan while the other method preserved a significant degree of SO3 substituents. Elemental analysis of ash remaining after thermal degradation showed presence of common mineral elements such as Na, Ca, Mg, Al, and K, but none of the heavy metals Pb, Hg, or As.

Dermatan sulfate (DS) is a glycosaminoglycan (GAG) that is produced through the epimerization of the glucuronic acid on chondroitin sulfate into iduronic acid (IduA) by dermatan sulfate epimerase (DS-epi) 1 and 2. Proteoglycans (PGs) play essential physiological and pathological roles during cellular development, proliferation, differentiation, and cancer metastasis. DS proteoglycans play vital roles during the process of tumorigenesis, due to the increased flexibility of the polysaccharide chain in the presence of IduA residues, which facilitate specific interactions with proteins, such as growth factors, cytokines, and angiogenic factors. Furthermore, DS-epi is highly expressed in many tumors, especially in esophageal squamous cell carcinoma. This study aimed to investigate the expression of DS-epi1 in multiple breast cancer cell lines, including MCF7 (luminal A), MDA-MB-231 (triple-negative) and SKBR3 (human epidermal growth factor receptor 2-positive), and its involvement in cancer progression. A SKBR3 variant, SKBR3m, presented the most erratic cell growth pattern when compared with those for MCF7 and MDA-MB-231. Moreover, SKBR3m cells demonstrated the highest level of DS-epi1 gene expression and higher 35S-DS content. However, at the protein level, MCF7 cells displayed the highest protein level for DS-epi1, whereas MDA-MB-231 cells had the lowest level. DS-epi1 was found in vesicles and in the perinuclear compartment only in SKBR3m cells, suggesting localization in the Golgi apparatus in these cells, in contrast with the cytoplasmic localization observed in MCF7 and MDA-MB-231 cells. The cytoplasm location of DS-epi1 likely compromised the formation of DS chains, but the core protein was detected using a decorin antibody. Golgi-specific labeling confirmed the localization of DS-epi1 in SKBR3m cells at the Golgi apparatus, indicating that the location of the enzyme was a determinant for the synthesis of DS in this cell line, suggesting that DS may play a decisive role in the tumor growth observed in this breast cancer cell line.

Self-assembling aliphatic heparin derivatives were shown to inhibit the immune system by antagonizing Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD2). In the present study, glycol split heparin– d -erythro-sphingosine conjugates (NAHNP) and its regioselectively desulfated derivatives with shortened aliphatic chains were investigated regarding their biophysical properties in the interaction with TLR4/MD2. Two-dimensional nuclear Overhauser effect spectroscopy studies showed that upon glycol splitting, the heparin backbone gains extra adaptability that facilitates binding to proteins. However, unlike native heparin or glycol split non-anticoagulant heparin (NAH), hydrophobic derivatization of NAH forces sulfated iduronic acid residues to change configuration from a 2S0 skew-boat to a 1C4 chair form. Whereas neither heparin nor NAH had any appreciable effect, NAHNP significantly inhibited lipopolysaccharide-induced activation of the NF-κB transcription factor. We showed that NAHNP binds to TLR4/MD2 with an affinity of 62.3 nM. In line with computational studies, biosensor-based structure-kinetic relationship studies demonstrated that 6-O-sulfo groups of d -glucosamine residue were essential in binding to arginines of both TLR4 and MD2 domains of the receptor complex. The desulfation of 6-O-sulfo groups decreases the association kinetics from 4.2 × 104 M−1 s−1 to 3.8 × 103 M−1 s−1, which results in a decreased affinity of 800 nM. Two aliphatic chains of NAHNP bound to the MD2 pocket similarly to lipopolysaccharide. A decrease in chain length resulted in a loss of inhibitory activity on NF-κB transcription and binding affinity to TLR4/MD2. In conclusion, the present study characterizes the immunosuppressive effect of aliphatic heparin derivatives and provides a promising strategy to develop selective immunosuppressants for acute and chronic inflammatory disorders.

Highly sulfated domains of heparan sulfate (HS), also known as HS S-domains, consist of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)-]. The expression of HS S-domains at the cell surface is determined by two mechanisms: tightly regulated biosynthetic machinery and enzymatic remodeling by extracellular endoglucosamine 6-sulfatases, Sulf-1 and Sulf-2. Intracellular or extracellular deposits of misfolded and aggregated proteins are characteristic of protein aggregation diseases. Although proteins can aggregate alone, deposits of protein aggregates in vivo contain a number of proteinaceous and non-protein components. HS S-domains are one non-protein component of these aggregated deposits. HS S-domains are considered to be critical for signal transduction of several growth factors and several disease conditions, such as tumor progression, but their roles in protein aggregation diseases are not yet fully understood. This review summarizes the current understanding of the possible roles of HS S-domains and Sulfs in the formation and cytotoxicity of protein aggregates.