Repeating Disaccharide Units Research Articles

Peptidoglycan synthesis in Tannerella forsythia: Scavenging is the modus operandi

Tannerella forsythia is a Gram-negative oral pathogen strongly associated with periodontitis. This bacterium has an absolute requirement for exogenous N-acetylmuramic acid (MurNAc), an amino sugar that forms the repeating disaccharide unit with amino sugar N-acetylglucosamine (GlcNAc) of the peptidoglycan backbone. In silico genome analysis indicates that T.forsythia lacks the key biosynthetic enzymes needed for the de novo synthesis of MurNAc, and so relies on alternative ways to meet its requirement for peptidoglycan biosynthesis. In the subgingival niche, the bacterium can acquire MurNAc and peptidoglycan fragments (muropeptides) released by the cohabiting bacteria during their cell wall breakdown associated with cell division. Tannerella forsythia is able to also use host sialic acid (Neu5Ac) in lieu of MurNAc or muropeptides for its survival during the biofilm growth. Evidence suggests that the bacterium might be able to shunt sialic acid into a metabolic pathway leading to peptidoglycan synthesis. In this review, we explore the mechanisms by which T. forsythia is able to scavenge MurNAc, muropeptide and sialic acid for its peptidoglycan synthesis, and the impact of these scavenging activities on pathogenesis.

Two structurally similar fucosylated chondroitin sulfates from the holothurian species Stichopus chloronotus and Stichopus horrens

Two fucosylated chondroitin sulfates SC and SH were isolated from the holothurian species Stichopus chloronotus and Stichopus horrens, respectively. The molar ratio of monosaccharides and sulfate (GalNAc:GlcA:Fuc:SO3Na) was suggested as ∼1:1:1:4 for both polysaccharides. Really this theoretical ratio was slightly distorted by the presence of some fucan sulfate in both preparations (about 2% in SH and 10% in SC), which could not be separated probably due to coincidence with the main components in charge density and molecular weight. The 1D and 2D NMR spectroscopic methods were applied for the detailed structural characterization of SC and SH, which were found to have similar structures. The main chain of SC and SH was shown to be composed of the repeating disaccharide units →4)-β-d-GlcA-(1 → 3)-β-d-GalNAc-(1→ sulfated at O-4 or both at O-4 and O-6 of the N-acetyl-galactosaminyl residues. The ratio of mono- and disulfated GalNAc residues was determined as 1:9 for SC and SH. Only one type of branches linked to O-3 of glucuronyl residues, namely fucosyl 2,4-disulfate residues, were found in both polysaccharides. Therefore polysaccharides SC and SH are two new examples of highly regular fucosylated chondroitin sulfates.

Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry

Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6-4.9ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA-GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biology. Graphical abstract Extraction and HILIC UPLC-MS analysis of procainamide-labelled heparan sulphate disaccharides.

Synthetic Xylosides: Probing the Glycosaminoglycan Biosynthetic Machinery for Biomedical Applications.

Glycosaminoglycans (GAGs) are polysaccharides ubiquitously found on cell surfaces and in the extracellular matrix (ECM). They regulate numerous cellular signaling events involved in many developmental and pathophysiological processes. GAGs are composed of complex sequences of repeating disaccharide units, each of which can carry many different modifications. The tremendous structural variations account for their ability to bind many proteins and thus, for their numerous functions. Although the sequence of GAG biosynthetic events and the enzymes involved mostly were deduced a decade ago, the emergence of tissue or cell specific GAGs from a nontemplate driven process remains an enigma. Current knowledge favors the hypothesis that macromolecular assemblies of GAG biosynthetic enzymes termed "GAGOSOMEs" coordinate polymerization and fine structural modifications in the Golgi apparatus. Distinct GAG structures arise from the differential channeling of substrates through the Golgi apparatus to various GAGOSOMEs. As GAGs perform multiple regulatory roles, it is of great interest to develop molecular strategies to selectively interfere with GAG biosynthesis for therapeutic applications. In this Account, we assess our present knowledge on GAG biosynthesis, the manipulation of GAG biosynthesis using synthetic xylosides, and the unrealized potential of these xylosides in various biomedical applications. Synthetic xylosides are small molecules consisting of a xylose attached to an aglycone group, and they compete with endogenous proteins for precursors and biosynthetic enzymes to assemble GAGs. This competition reduces endogenous proteoglycan-bound GAGs while increasing xyloside-bound free GAGs, mostly chondroitin sulfate (CS) and less heparan sulfate (HS), resulting in a variety of biological consequences. To date, hundreds of xylosides have been published and the importance of the aglycone group in determining the structure of the primed GAG chains is well established. However, the structure-activity relationship has long been cryptic. Nonetheless, xylosides have been designed to increase HS priming, modified to inhibit endogenous GAG production without priming, and engineered to be more biologically relevant. Synthetic xylosides hold great promise in many biomedical applications and as therapeutics. They are small, orally bioavailable, easily excreted, and utilize the host cell biosynthetic machinery to assemble GAGs that are likely nonimmunogenic. Various xylosides have been shown, in different biological systems, to have anticoagulant effects, selectively kill tumor cells, abrogate angiogenic and metastatic pathways, promote angiogenesis and neuronal growth, and affect embryonic development. However, most of these studies utilized the commercially available one or two β-D-xylosides and focused on the impact of endogenous proteoglycan-bound GAG inhibition on biological activity. Nevertheless, the manipulation of cell behavior as a result of stabilizing growth factor signaling with xyloside-primed GAGs is also reckonable but underexplored. Recent advances in the use of molecular modeling and docking simulations to understand the structure-activity relationships of xylosides have opened up the possibility of a more rational aglycone design to achieve a desirable biological outcome through selective priming and inhibitory activities. We envision these advances will encourage more researchers to explore these fascinating xylosides, harness the GAG biosynthetic machinery for a wider range of biomedical applications, and accelerate the successful transition of xyloside-based therapeutics from bench to bedside.

Sequential one-pot multienzyme synthesis of hyaluronan and its derivative

Hyaluronan (HA) is a linear polysaccharide composed of repeating disaccharide units. It has been well documented to play an array of biological functions in cancer events. Here, we reported a sequential one-pot multienzyme (OPME) strategy for in vitro synthesis of HA and its derivatives. The strategy, which combined in situ sugar nucleotides generation with HA chain polymerization, could convert cheap monosaccharides into HA polymers without consuming exogenous sugar nucleotide donors. HA polymers (number-average molecular weight ranged from 1.5×104 to 5.5×105Da) with over 70% yields were efficiently synthesized and purified from this one-pot system. More importantly, partial labeled HA derivative was further synthesized by metabolic incorporation of unnatural monosaccharide analogues into the sequential OPME system. Cross-linked HA hydrogel was achieved via copper (I)-catalyzed azide-alkyne cycloaddition and exhibited novel networks consisting of both inter- and intra-connected HA chains, which could facilitate the potential applications of this unique polysaccharide.

Free radical studies of components of the extracellular matrix: contributions to protection of biomolecules and biomaterials from sterilising doses of ionising radiation

The purpose of the current review is show how the principles and techniques of radiation chemistry have enabled the direct reactions of free radicals with biomolecules and biomaterials to be investigated at the molecular level. In particular, the review focusses on the free radical-induced fragmentation of glycosaminoglycans. Glycosaminoglycans are large linear polysaccharides consisting of repeating disaccharide units and are important components of the extracellular matrix (ECM) either in free form (hyaluronan) or as a component of proteoglycans. Oxidative damage of the extracellular matrix components by either enzymatic or non-enzymatic pathways may have implications for the initiation and progression of a range of human diseases. These include arthritis, kidney disease, cardiovascular disease, lung disease, periodontal disease and chronic inflammation. Oxidative damage to hyaluronan by reactive oxidative species and thus the potential mechanism of damage to the ECM and its role in human pathologies is reviewed with particular focus on damage initiated by potential in vivo free radicals such as superoxide, carbonate and hydroxyl radicals. Such knowledge has also allowed radiation protecting systems to be developed so that sterilising doses of radiation can be delivered to sensitive biomolecules such as proteins and glycosaminoglycans, and also to sensitive biomaterials such as tissue allografts.

Abstract 1153: Heparan sulfate proteoglycans as novel target in cancer precise therapy

Abstract Heparan sulfate proteoglycans (HSPGs) have crucial regulatory roles in tumor onset and progression. HSPGs are composed of a core protein and glycan chains characterized by repeated disaccharide units which can be sulfated at different amount and position. HSPG have enormous structural diversity due to the different possible modifications of the single saccharide units within the polysaccharide, such as position, sulphation and acetylation. As a result HSPG can bind and modulate their binding to signaling molecules such as growth factors, morphogens and chemokines1 . HSPG proved to be important in mediating cancer development and progression by enhancing the binding of growth factors, morphogens and cytokines to their cognate receptors, thus activating signaling pathways that give rise to angiogensis, cell growth and proliferation, together with invasion and metastasis 2-3. NT4 is a branched peptides that targets HSPGs. NT4 specifically binds to sulfated glycosaminoglycans on cancer cells and tissues. NT4 can be conjugated to many different cytotoxic units and tracers. NT4 conjugated to paclitaxel produced tumor regression in a breast cancer orthotopic mouse model 4. NT4 conjugated to tracers can discriminate between tumor and healthy tissue in different human cancer specimen5. We will show the ability of NT4 to drive tracers onto tumor lesions by means of Qdots and in vivo imaging, proving their promising features as theranostics. We will also show NT4 ability to interfere with HSPG- modulated activities such as: tumor cell proliferation, migration and invasion of matrix; as well as endothelial cells proliferation, migration and tube formation. NT4-HSPG interactions and consequent modulation of signaling pathways will prove the importance of this versatile tool, NT4, in addressing tumor cells and interfering in their cell-cell and cell-matrix communications. Bibliography 1. Gharbaran R. et al. Tumour Biol. 2016; 37:11573-11588. 2. Purushothaman A et al. Blood. 2010; 115:2449-57. 3. Lee JH et al. J Biol Chem. 2009; 284:27167-75. 4. Brunetti, J. et al. Scientific Reports. DOI:10.1038/srep17736 5. Falciani C. et al. J Med Chem. 2013; 56:5009-18. Citation Format: Chiara Falciani, Jlenia Brunetti, Lorenzo Depau, Alessandro Pini, Giulia Riolo, Elisabetta Mandarini, Luisa Bracci. Heparan sulfate proteoglycans as novel target in cancer precise therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1153. doi:10.1158/1538-7445.AM2017-1153

Pathogenesis and Inhibition of Flaviviruses from a Carbohydrate Perspective.

Flaviviruses are enveloped, positive single stranded ribonucleic acid (RNA) viruses with various routes of transmission. While the type and severity of symptoms caused by pathogenic flaviviruses vary from hemorrhagic fever to fetal abnormalities, their general mechanism of host cell entry is similar. All pathogenic flaviviruses, such as dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, and Zika virus, bind to glycosaminglycans (GAGs) through the putative GAG binding sites within their envelope proteins to gain access to the surface of host cells. GAGs are long, linear, anionic polysaccharides with a repeating disaccharide unit and are involved in many biological processes, such as cellular signaling, cell adhesion, and pathogenesis. Flavivirus envelope proteins are N-glycosylated surface proteins, which interact with C-type lectins, dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) through their glycans. In this review, we discuss both host and viral surface receptors that have the carbohydrate components, focusing on the surface interactions in the early stage of flavivirus entry. GAG-flavivirus envelope protein interactions as well as interactions between flavivirus envelope proteins and DC-SIGN are discussed in detail. This review also examines natural and synthetic inhibitors of flaviviruses that are carbohydrate-based or carbohydrate-targeting. Both advantages and drawbacks of these inhibitors are explored, as are potential strategies to improve their efficacy to ultimately help eradicate flavivirus infections.

A characteristic chondroitin sulfate trisaccharide unit with a sulfated fucose branch exhibits neurite outgrowth-promoting activity: Novel biological roles of fucosylated chondroitin sulfates isolated from the sea cucumber Apostichopus japonicus

Chondroitin sulfate (CS) is a class of sulfated glycosaminoglycan (GAG) chains that consist of repeating disaccharide unit composed of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc). CS chains are found throughout the pericellular and extracellular spaces and contribute to the formation of functional microenvironments for numerous biological events. However, their structure-function relations remain to be fully characterized. Here, a fucosylated CS (FCS) was isolated from the body wall of the sea cucumber Apostichopus japonicus. Its promotional effects on neurite outgrowth were assessed by using isolated polysaccharides and the chemically synthesized FCS trisaccharide β-D-GalNAc(4,6-O-disulfate) (1–4)[α-l-fucose (2,4-O-disulfate) (1–3)]-β-D-GlcA. FCS polysaccharides contained the E-type disaccharide unit GlcA-GalNAc(4,6-O-disulfate) as a CS major backbone structure and carried distinct sulfated fucose branches. Despite their relatively lower abundance of E unit, FCS polysaccharides exhibited neurite outgrowth-promoting activity comparable to squid cartilage-derived CS-E polysaccharides, which are characterized by their predominant E units, suggesting potential roles of the fucose branch in neurite outgrowth. Indeed, the chemically synthesized FCS trisaccharide was as effective as CS-E tetrasaccharide in stimulating neurite elongation in vitro. In conclusion, FCS trisaccharide units with 2,4-O-disulfated fucose branches may provide new insights into understanding the structure-function relations of CS chains.

Chemo-physical Properties and Biomedical Applications of Hyaluronic Acid in Medicine

Hyaluronic acid is a mucopolysaccharide encountered in most body fluids and extracellular matrix. The aim of our review is to summarize current evidence about chemico-physical properties of hyaluronic acid, highlighting biomedical applications of hyaluronan derivatives. It is a glycosaminoglycan made of repeating disaccharide units containing a carboxylate group, four hydroxyl groups and one carboxylate group, with hydrophilic properties. Its particular structure with multiple coils forming an entangled network results in unique pseudoplastic and viscoelastic characteristics. Its viscous and elastic behavior, depending on the applied strain, makes hyaluronan widely applicable in biomedical field. The large amount of functions and applications is determined by the physico-chemical properties, which allows a polymorphism of the hyaluronic acid structures depending on the molecular weight variations, concentration and ionic status. It is currently used in ophthalmology, orthopedics and rheumatology, in plastic surgery, surgery and otolaryngology as well. Already widely used in clinical practice, hyaluronic acid proves to be often the best solution for difficult medical problems. Future developments in nanomedicine and drug delivery linked to hyaluronic acid are emerging.

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.

New insights into the action of bacterial chondroitinase AC I and hyaluronidase on hyaluronic acid

Hyaluronic acid (HA), a glycosaminoglycan, is a linear polysaccharide with negative charge, composed of a repeating disaccharide unit [→4)-β-d-glucopyranosyluronic acid (1→3)-β-d- N-acetyl-d-glucoaminopyranose (1→]n ([→4) GlcA (1→3) GlcNAc 1→]n). It is widely used in different applications based on its physicochemical properties associated with its molecular weight. Enzymatic digestion by polysaccharides lyases is one of the most important ways to decrease the molecular weight of HA. Thus, it is important to understand the action patterns of lyases acting on HA. In this study, the action patterns of two common lyases, Flavobacterial chondroitinase AC I and Streptomyces hyaluronidase, were investigated by analyzing HA oligosaccharide digestion products. HA oligosaccharides having an odd-number of saccharide residues were observed in the products of both lyases, but their distributions were quite different. Chondroitinase AC acted more efficiently at the β 1–4 glycosidic bond linking GlcNAc and GlcA. Oligosaccharides, having an even number of saccharide residues, and with an unsaturated uronic acid (4-deoxy-α-l-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end represent the major product. A minor amount of oligosaccharides having an odd number of saccharide residues resulted from the irregular terminal residues of HA substrate chains. Hyaluronidase showed a more complicated product mixture. Its minimum recognition and digestion domain is HA heptasaccharide and it could cleave both β 1–4 and β 1–3 glycosidic linkages. The HA oligosaccharides, generated with a 2-acetamido-2,3-di-deoxy-β-d-erythro-hex-2-enopyranose (△HexNAc) at the non-reducing end, are believed to be unstable and undergo breakdown immediately after their generation, and the oligosaccharides with △UA residue at the non-reducing end are formed. Thus, oligosaccharides having both an even and odd-number saccharide residues with a △UA residue at their non-reducing ends, represent the major products of hyaluronidase acting on HA.

Comparison of Enzymatic Hydrolysis of Polysaccharides from Eggshells Membranes

Abstract AHyaluronic acid (HA) is part of the extracellular matrix of connective, epithelial and neural tissues, as well as the synovial fluid, skin, and cartilage. It is composed of repeating disaccharide units of D-glucuronic acid and N-acetyl glucosamine. Hyaluronic acid is used in abdominal surgery, ophthalmology, dermatology, rhinology; it is usable for the osteoarthritis treatment. The membranes of eggshell are a natural source of hyaluronic acid, collagen, glycosaminoglycan and collagenous proteins. In paper, we tested the possibility of extraction hyaluronic acid from the eggshell membranes by enzymatic hydrolysis. We identified optimal conditions of hydrolysis with trypsin at reaction temperature of 37 °C and pH 8; with pepsin at 40 °C and pH 3, as well as with papain at 60 °C and pH 7.5. The content of hyaluronic acid in samples was determined spectrophotometrically using the carbazole method. The experimental results showed a yield of ~ 4 -4.5 % hyaluronic acid per 1 g of dry eggshell membranes.

Erratum to: Molecular dissection of placental malaria protein VAR2CSA interaction with a chemo-enzymatically synthesized chondroitin sulfate library.

Placental malaria, a serious infection caused by the parasite Plasmodium falciparum, is characterized by the selective accumulation of infected erythrocytes (IEs) in the placentas of the pregnant women. Placental adherence is mediated by the malarial VAR2CSA protein, which interacts with chondroitin sulfate (CS) proteoglycans present in the placental tissue. CS is a linear acidic polysaccharide composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-galactosamine that are modified by sulfate groups at different positions. Previous reports have shown that placental-adhering IEs were associated with an unusually low sulfated form of chondroitin sulfate A (CSA) and that a partially sulfated dodecasaccharide is the minimal motif for the interaction. However, the fine molecular structure of this CS chain remains unclear. In this study, we have characterized the CS chain that interacts with a recombinant minimal CS-binding region of VAR2CSA (rVAR2) using a CS library of various defined lengths and sulfate compositions. The CS library was chemo-enzymatically synthesized with bacterial chondroitin polymerase and recombinant CS sulfotransferases. We found that C-4 sulfation of the N-acetyl-d-galactosamine residue is critical for supporting rVAR2 binding, whereas no other sulfate modifications showed effects. Interaction of rVAR2 with CS is highly correlated with the degree of C-4 sulfation and CS chain length. We confirmed that the minimum structure binding to rVAR2 is a tri-sulfated CSA dodecasaccharide, and found that a highly sulfated CSA eicosasaccharide is a more potent inhibitor of rVAR2 binding than the dodecasaccharides. These results suggest that CSA derivatives may potentially serve as targets in therapeutic strategies against placental malaria.

Histone deacetylase-mediated regulation of chondroitin 4-O-sulfotransferase-1 (Chst11) gene expression by Wnt/β-catenin signaling

Chondroitin sulfate (CS) proteoglycans are abundant extracellular and cell surface molecules that consist of a protein core to which highly sulfated CS chains are covalently attached. The CS backbone is composed of repeating disaccharide units [-GlcA-GalNAc-]n, and during synthesis the CS chains acquire structural variability due to the action of sulfotransferases. Specific sulfation patterns are recognized by a large variety of proteins, including growth factors, morphogens, and extracellular matrix proteins, and these interactions regulate key events in development and normal physiology. Therefore, it is important to understand how gene expression of CS sulfotransferases is regulated. We previously found that Wnt signaling regulates the sulfation patterns of cell-associated CS chains by suppressing expression of chondroitin 4-O-sulfotaransferase-1 (C4ST-1), a CS biosynthetic enzyme. Here we investigated the mechanism underlying the regulation of C4ST-1 gene expression by Wnt/β-catenin signaling. Although C4ST-1 mRNA of 3′-UTR contains three binding sites for microRNAs (miRNA), these miRNAs played little role in controlling C4ST-1 gene expression. In contrast, the suppression of C4ST-1 gene expression by Wnt/β-catenin signaling can be recovered by treatment with trichostatin A, but not with 5′-aza-2′-deoxycytidine. These results suggest that the Wnt/β-catenin signal pathway controls C4ST-1 gene expression mainly through histone deacetylase.

Quick characterization of uronic acid-containing polysaccharides in 5 shellfishes by oligosaccharide analysis upon acid hydrolysis

Uronic acid-containing polysaccharides (UACPs) including well-known glycosaminoglycans (GAGs) and some non-GAGs exist widely in animal kingdom. Although numerous methods have been established to analyze GAGs, few methods are available for non-GAG UACPs. In the present study, a protocol to identify all kinds of UACPs with repeating disaccharide units of hexosamine and uronic acid was demonstrated, and UACP components in five shellfishes, namely Turritella fortilirata Sowerby (GTF), Batillaria zonalis (GBZ), Nassarius variciferus (GNV), Monodonta labio Linnaeus (GML), and Argopecten irradians Lamarck (BAI) were primarily revealed. After a simple isolation procedure, crude polysaccharides were depolymerized by controlled acid hydrolysis, and then the resulting oligosaccharides were detected by HPLC coupled with mass spectrometer after 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling. According to chromatograms using the triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) mode, chondroitin sulfate (CS) was found in GNV and GML, a non-GAG named abalone gonad sulfated polysaccharide (AGSP) with a backbone of →4)-β-GlcA-(1 → 2)-α-Man-(1→ repeating units in GBZ, and both of AGSP and CS in BAI and GTF. Further characterization of tetrasaccharides and sulfated/acetylated disaccharides by HPLC combined with an ion trap mass spectrometer confirmed the structural identification of CS and AGSP, and indicated CS in GTF and BAI was Type C. These results suggest the 5 mollusks as potential resources for CS and AGSP. And the analysis protocol presented in this study was powerful and effective for quick characterization of UACPs including GAGs as well as non-GAGs in complicated matrix.

Conformational flexibility of PL12 family heparinases: structure and substrate specificity of heparinase III from Bacteroides thetaiotaomicron (BT4657).

Glycosaminoglycans (GAGs) are linear polysaccharides comprisedof disaccharide repeat units, a hexuronic acid, glucuronic acid or iduronic acid, linked to a hexosamine, N-acetylglucosamine (GlcNAc) or N-acetylgalactosamine. GAGs undergo further modification such as epimerization and sulfation. These polysaccharides are abundant in the extracellular matrix and connective tissues. GAGs function in stabilization of the fibrillar extracellular matrix, control of hydration, regulation of tissue, organism development by controlling cell cycle, cell behavior and differentiation. Niche adapted bacteria express enzymes called polysaccharide lyases (PL), which degrade GAGs for their nutrient content. PL have been classified into 24 sequence-related families. Comparison of 3D structures of the prototypic members of these families allowed identification of distant evolutionary relationships between lyases that were unrecognized at the sequence level, and identified occurrences of convergent evolution. We have characterized structurally and enzymatically heparinase III from Bacteroides thetaiotaomicron (BtHepIII; gene BT4657), which is classified within the PL12 family. BtHepIII is a 72.5 kDa protein. Wepresent the X-ray structures of two crystal forms of BtHepIII at resolution 1.8 and 2.4 Å. BtHepIII contains two domains, the N-terminal α-helical domain forming a toroid and the C-terminal β-sheet domain. Comparison with recently determined structures of two other heparinases from the same PL12 family allowed us to identify structural flexibility in the arrangement of the domains indicating open-close movement. Based on comparison with other GAG lyases, we identified Tyr301 as the main catalytic residue and confirmed this by site-directed mutagenesis. We have characterized substrate preference of BtHepIII toward sulfate-poor heparan sulfate substrate.

Biological function of unique sulfated glycosaminoglycans in primitive chordates.

Glycosaminoglycans with unique sulfation patterns have been identified in different species of ascidians (sea squirts), a group of marine invertebrates of the Phylum Chordata, sub-phylum Tunicata (or Urochordata). Oversulfated dermatan sulfate composed of [4-α-L-IdoA-(2-O-SO3)-1→3-β-D-GalNAc(4-OSO3)-1]n repeating disaccharide units is found in the extracellular matrix of several organs, where it seems to interact with collagen fibers. This dermatan sulfate co-localizes with a decorin-like protein, as indicated by immunohistochemical analysis. Low sulfated heparin/heparan sulfate-like glycans composed mainly of [4-α-L-IdoA-(2-OSO3)-1→4-α-D-GlcN(SO3)-1 (6-O-SO3)-1]n and [4-α-L-IdoA-(2-O-SO3)-1→4-α-D-GlcN(SO3)-1]n have also been described in ascidians. These heparin-like glycans occur in intracellular granules of oocyte assessory cells, named test cells, in circulating basophil-like cells in the hemolymph, and at the basement membrane of different ascidian organs. In this review, we present an overview of the structure, distribution, extracellular and intracellular localization of the sulfated glycosaminoglycans in different species and tissues of ascidians. Considering the phylogenetic position of the subphylum Tunicata in the phylum Chordata, a careful analysis of these data can reveal important information about how these glycans evolved from invertebrate to vertebrate animals.

English

Osteoarthritis (OA) is characterized by low grade inflammation leading to progressive degenerative changes in articular cartilage (Morenko et al., 2004). Joint mobility is reduced owing to pain and swelling of affected area. Severity of OA depends on type of inducer that may be chemical, enzymatic or surgical. Cartilage metabolic disturbances lead to destabilization and degeneration. Clinical manifestations of OA include softening, fibrillation, ulceration, sclerosis, bone eburnation, osteophytes and sub-chondral cysts formation (Fernihough et al., 2004). Pain leading to joint immobilization is prominent manifestation of this chronic malady (Khan et al., 2012). Initially, soft swelling can be observed on affected joints which later on subside with the progression of diseases. Crackling sounds from affected joints during walking and standing (Brandt et al., 2009). Mostly, treatment of OA is done focusing the symptom of pain and joint dysfunction (Flood, 2010). Non-steroidal anti-inflammatory drugs (NSAIDS), opiates, corticosteroids and nutraceuticals are conventional therapeutics for OA (Sofat et al., 2011). Glycosaminoglycans (GAGs) are long un-branched polysaccharides of repeating disaccharide units abundantly present in connective tissues, vitreous humor, synovial fluid, mast cells and predominately in cartilages. The major constituents are chondroitin sulphate, dermatan sulphate, heparin/heparansulphate, glucosamine and hyaluronic acid. Among these, glucosamine sulphate (GS) and chondroitin sulphate (CS) are more effective and safe therapeutic agents for OA (Miller and Clegg, 2010). CS and GS stimulate chondrocyte synthesis of collagen (Adams et al., 2013). Concurrent use of CS and GS increase synthesis of cartilage matrix and decrease progression of OA which ultimately reduces signs and symptoms of disease. In present study, efficacy of glycosaminoglycans on clinical signs of osteoarthritis and C-terminal telopeptides of type II collagen (CTX-II) was evaluated in papain induced OA albino rat model.

Selective Targeting and Restrictive Damage for Nonspecific Cells by Pulsed Laser-Activated Hyaluronan-Gold Nanoparticles.

Herein, we describe an approach that immobilizes low-molecular-weight hyaluronic acid (low-MW HA) on the surface of gold nanoparticles (GNPs), which can serve as a cellular probe and photodamage media, to evaluate the selectivity and efficiency of HA-based GNPs (HGNPs) as a mediator of laser-induced photothermal cell damage. In addition, it is known that solid tumors contain a higher content of low-MW HA than normal tissues. Thus, we used low-MW HA rather than high-MW HA used in other studies. In the present study, we conjugated low-MW HA, which is a linear polysaccharide with a disaccharide repeat unit, to prevent a reduction of the ligand-receptor binding efficiency in contrast to the conjugation of protein or peptides, which have unique three-dimensional structures. Three cell lines-MDA-MB-435 S (with CD44), MDA-MB-453 and NIH/3T3 (both are without CD44)-were investigated in the study, and qualitative observations were conducted by dark-field microscopy and laser scanning confocal microscopy (LSCM). In addition, quantitative measurements calculated using inductively coupled plasma emissions were taken for comparison. Our results showed that within the same treatment time, the uptake dosage of HGNPs by the MDA-MB-435 S cells was higher than that by the MDA-MB-453 and NIH 3T3 cells. Meanwhile, HGNPs uptake by the untreated MDA-MB-435 S cells was higher than that of MDA-MB-435 S cells with CD44 blocked by antibodies or silencing CD44 expression. This result implies that receptor-mediated endocytosis can enhance the cellular uptake of HGNPs. In addition, when exposed to a low-power pulsed laser, the former cell morphologies showed a more laser-induced giant plasma membrane vesicles (GPMV) than the latter morphologies. Therefore, this study utilized the specific photothermal property of HA-modified GNPs with laser-induced blebs to create a possible new method for medical applications.