Classes Of Glycosaminoglycans Research Articles

Molecular mechanisms and potential applications of chondroitin sulphate in managing post-traumatic osteoarthritis.

Post-traumatic osteoarthritis (PTOA), a disorder of the synovium, subchondral bone, and cartilage that affects the entire joint, constitutes approximately 12% of all cases of symptomatic osteoarthritis. This review summarizes the pathogenetic mechanisms that underlie the positive influence of chondroitin sulphates (CSs) on PTOA as means of preventive and therapeutic treatment. Mechanisms of PTOA development involve chondrocytes undergoing various forms of cell death (apoptosis, pyroptosis, necroptosis, ferroptosis and/or necrosis). Chondroitin sulphates are a class of glycosaminoglycans that improve the structure and function of cartilage and subchondral bone, which is associated with their ability to decrease the activation of NF-κB and p38 MAPK, and up-regulate Nrf2. Standardized small fish extract (SSFE) is an example of the drugs that can attenuate NF-κB-mediated systemic inflammation, potentially helping to reduce joint inflammation and cartilage degradation, improve joint function, and alleviate pain and disability in patients with these conditions.

Sulfation Effects of Chondroitin Sulfate to Bind a Chemokine in Aqueous Medium: Conformational Heterogeneity and Dynamics from Molecular Simulation.

The sulfation patterns and degree of sulfation of chondroitin sulfate (CS), an important class of glycosaminoglycans (GAG), and their interactions with chemokines are accountable for various diseases. To realize the underlying mechanism of such complex biological phenomena at a molecular level and their application in rational drug design, a study on conformations and dynamics of CSs is necessary. To explore this, in this study, we performed a series of atomistic molecular dynamics (MD) simulations with different sulfated variants of octadecasaccharide CS, like CS-C, CS-E, and CS-T, in their free forms and when bound to the protein chemokine CXCL8 dimer in an aqueous medium. The calculated binding free energy of CSs with the CXCL8 dimer is favorable, and the degree of sulfation favors the complexation process further with prominent hydrophobic and hydrogen-bonded interactions. We find that the recognition is associated with the configurational entropy loss of the CS molecules as calculated from the Gaussian mixture approach, which supports that the degree of sulfation regulates the process. Cluster analysis through the k-means algorithm and end-to-end distance measurement revealed that although the free CS molecules adopted linear conformations, the nonlinear conformations during binding with protein were noted. Adaptation of nonlinear forms in the bound forms is noteworthy for the less-sulfated CS-C and CS-E. Apart from favorable 4C1 conformations, the occasional appearance of skew-boat forms from the free-energy map of ring pucker for the GlcUA unit was observed, which remains unaffected by the sulfation. We find that during recognition, the average relaxation time of intra-CS and inter-CS-CXCL8 hydrogen bonds (HBs) is about a magnitude lesser than that of CS-water HBs, most prominent on the involvement of higher sulfated CS-T analogues. The translational motion of surrounded water molecules in CSs exhibited sublinear diffusion, and the degree of sublinearity increases around the heavily sulfated molecules due to the hindrance created by them as well as the presence of the chemokine and exhibited markedly slow heterogeneous diffusion.

Predicting Structural Motifs of Glycosaminoglycans using Cryogenic Infrared Spectroscopy and Random Forest.

In recent years, glycosaminoglycans (GAGs) have emerged into the focus of biochemical and biomedical research due to their importance in a variety of physiological processes. These molecules show great diversity, which makes their analysis highly challenging. A promising tool for identifying the structural motifs and conformation of shorter GAG chains is cryogenic gas-phase infrared (IR) spectroscopy. In this work, the cryogenic gas-phase IR spectra of mass-selected heparan sulfate (HS) di-, tetra-, and hexasaccharide ions were recorded to extract vibrational features that are characteristic to structural motifs. The data were augmented with chondroitin sulfate (CS) disaccharide spectra to assemble a training library for random forest (RF) classifiers. These were used to discriminate between GAG classes (CS or HS) and different sulfate positions (2-O-, 4-O-, 6-O-, and N-sulfation). With optimized data preprocessing and RF modeling, a prediction accuracy of >97% was achieved for HS tetra- and hexasaccharides based on a training set of only 21 spectra. These results exemplify the importance of combining gas-phase cryogenic IR ion spectroscopy with machine learning to improve the future analytical workflow for GAG sequencing and that of other biomolecules, such as metabolites.

Odorant Receptor OR2C1 Is an Essential Modulator of Boar Sperm Capacitation by Binding with Heparin

Heparin, a class of glycosaminoglycans (GAGs), is widely used to induce sperm capacitation and fertilization. How heparin induces sperm capacitation remains unclear. Olfactory receptors (ORs) which are G protein-coupled receptors, have been proposed to be involved in sperm capacitation. However, the interaction between ORs and odor molecules and the molecular mechanism of ORs mediating sperm capacitation are still unclear. The present study aimed to explore the underlying interaction and mechanism between heparin and ORs in carrying out the boar sperm capacitation. The results showed that olfactory receptor 2C1 (OR2C1) is a compulsory unit which regulates the sperm capacitation by recognizing and binding with heparin, as determined by Dual-Glo Luciferase Assay and molecular docking. In addition, molecular dynamics (MD) simulation indicated that OR2C1 binds with heparin via a hydrophobic cavity comprises of Arg3, Ala6, Thr7, Asn171, Arg172, Arg173, and Pro287. Furthermore, we demonstrated that knocking down OR2C1 significantly inhibits sperm capacitation. In conclusion, we highlighted a novel olfactory receptor, OR2C1, in boar sperm and disclosed the potential binding of heparin to Pro287, a conserved residue in the transmembrane helices region 7 (TMH7). Our findings will benefit the further understanding of ORs involved in sperm capacitation and fertilization.

Glycosaminoglycan Analysis: Purification, Structural Profiling, and GAG-Protein Interactions.

Glycosaminoglycans (GAGs) are long, linear polysaccharides that are ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These complex carbohydrates are composed of alternating glucosamine and uronic acids that can be heterogeneously N- and O-sulfated. The arrangement and orientation of the sulfated sugar residues specify the location of distinct ligand binding sites on the cell surface, and their capacity to bind ligands impacts cell growth and development, the ability to form tissues and organs, and normal physiology. The heterogeneous nature of GAGs and their inherent structural diversity across different tissues, cell types, and disease states creates challenges to characterizing their structure and function. Here, we describe detailed methods to investigate GAG-protein interactions in vitro and evaluate the structural composition of two classes of sulfated GAGs, heparan sulfate and chondroitin/dermatan sulfate, using liquid chromatography, mass spectrometry, and radiolabeling techniques. Overall, these methods facilitate the evaluation of GAG structure and function to uncover the unique roles these molecules play in cell biology and human disease.

Heparin and Heparin-Based Drug Delivery Systems: Pleiotropic Molecular Effects at Multiple Drug Resistance of Osteosarcoma and Immune Cells.

One of the main problems of modern health care is the growing number of oncological diseases both in the elderly and young population. Inadequately effective chemotherapy, which remains the main method of cancer control, is largely associated with the emergence of multidrug resistance in tumor cells. The search for new solutions to overcome the resistance of malignant cells to pharmacological agents is being actively pursued. Another serious problem is immunosuppression caused both by the tumor cells themselves and by antitumor drugs. Of great interest in this context is heparin, a biomolecule belonging to the class of glycosaminoglycans and possessing a broad spectrum of biological activity, including immunomodulatory and antitumor properties. In the context of the rapid development of the new field of “osteoimmunology,” which focuses on the collaboration of bone and immune cells, heparin and delivery systems based on it may be of intriguing importance for the oncotherapy of malignant bone tumors. Osteosarcoma is a rare but highly aggressive, chemoresistant malignant tumor that affects young adults and is characterized by constant recurrence and metastasis. This review describes the direct and immune-mediated regulatory effects of heparin and drug delivery systems based on it on the molecular mechanisms of (multiple) drug resistance in (onco) pathological conditions of bone tissue, especially osteosarcoma.

A Semi-Quantitative Assay to Measure Glycosaminoglycan Degradation by the Urinary Microbiota.

Glycosaminoglycans (GAGs) are linear polysaccharides and are among the primary components of mucosal surfaces in mammalian systems. The GAG layer lining the mucosal surface of the urinary tract is thought to play a critical role in urinary tract homeostasis and provide a barrier against urinary tract infection (UTI). This key component of the host-microbe interface may serve as a scaffolding site or a nutrient source for the urinary microbiota or invading pathogens, but its exact role in UTI pathogenesis is unclear. Although members of the gut microbiota have been shown to degrade GAGs, the utilization and degradation of GAGs by the urinary microbiota or uropathogens had not been investigated. In this study, we developed an in vitro plate-based assay to measure GAG degradation and utilization and used this assay to screen a library of 37 urinary bacterial isolates representing both urinary microbiota and uropathogenic species. This novel assay is more rapid, inexpensive, and quantitative compared to previously developed assays, and can measure three of the major classes of human GAGs. Our findings demonstrate that this assay captures the well-characterized ability of Streptococcus agalactiae to degrade hyaluronic acid and partially degrade chondroitin sulfate. Additionally, we present the first known report of chondroitin sulfate degradation by Proteus mirabilis, an important uropathogen and a causative agent of acute, recurrent, and catheter-associated urinary tract infections (CAUTI). In contrast, we observed that uropathogenic Escherichia coli (UPEC) and members of the urinary microbiota, including lactobacilli, were unable to degrade GAGs.

Assays for Evaluation of Substrates for and Inhibitors of β-1,4-Galactosyltransferase 7.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by β4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl β-D-xylopyranoside. We have cloned and expressed β4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of β4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of β4GalT7.

Chondroitin Sulfate Disaccharides in the Gas Phase: Differentiation and Conformational Constraints.

Glycosaminoglycans (GAGs) are a family of complex carbohydrates vital to all mammalian organisms and involved in numerous biological processes. Chondroitin and dermatan sulfate, an important class of GAGs, are linear macromolecules consisting of disaccharide building blocks of N-acetylgalactosamine and two different uronic acids. The varying degree and the site of sulfation render their characterization challenging. Here, we combine mass spectrometry with cryogenic infrared spectroscopy in the wavenumber range from 1000 to 1800 cm–1. Fingerprint spectra were recorded for a comprehensive set of disaccharides bearing all known motifs of sulfation. In addition, state-of-the-art quantum chemical calculations were performed to aid the understanding of the differences in the experimental fingerprint spectra. The results show that the degree and position of charged sulfate groups define the size of the conformational landscape in the gas phase. The detailed understanding of cryogenic infrared spectroscopy for acidic and often highly sulfated glycans may pave the way to utilize the technique in fragment-based sequencing approaches.

Glycosaminoglycans.

Glycosaminoglycans (GAGs) are important constituents of human glycome. They are negatively charged unbranched polysaccharides that are usually covalently attached to proteins, forming glycan-protein conjugates, called proteoglycans. Glycosaminoglycans play critical roles in numerous biological processes throughout individual development and are also involved in the pathological processes of various diseases. Based on their remarkable bioactivities and their universal involvement in disease progression, GAGs are applied as therapeutics or are being targeted or used in treating diseases. In this chapter, we introduce the characteristics of the four classes of GAGs that constitute the glycosaminoglycan family. The pathological roles of glycosaminoglycans in major diseases including innate disease, infectious disease, and cancer are discussed. The application of GAGs and their mimetics as therapeutics is introduced, as well as those therapeutic methods developed based on GAGs' role in pathogenesis. In addition, we provide a brief and overall lookback at the history of GAG research and sort out some critical techniques that facilitated GAG and glycomics studies.

Expression optimization and molecular modification of heparin C5 epimerase

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.

Heparan sulfate maintains adult midgut homeostasis in Drosophila.

Tissue homeostasis is controlled by the differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)-like cells and mis-differentiated progeny. HS-deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which, in turn, promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in the inactivation of decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, our data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. Our data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.

Structural analysis of glycosaminoglycans from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a traditional animal-based Chinese medicine. Chondroitin sulfate (CS), dermatan sulfate (DS) and hyaluronic acid (HA) are structurally complex classes of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their possible structural characteristics in CCA are not clear. In this study, GAG fractions containing CS/DS and HA were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). The result showed that CS/DS/HA disaccharides were detected in the three lower salt fractions from anion-exchange chromatography. The sulfation patterns and densities of CS/DS chains in these fractions differed greatly, while HA chains varied in their chain lengths. The quantitative analysis first revealed that the amount of GAGs in CCA varied significantly in total and in each fraction. This novel structural information could help clarify the possible involvement of these polysaccharides in the biological activities of CCA.

Chemoenzymatic Synthesis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) are a family of structurally complex heteropolysaccharides composed of alternating hexosamine and uronic acid or galatose residue that include hyaluronan, chondroitin sulfate and dermatan sulfate, heparin and heparan sulfate, and keratan sulfate. GAGs display a range of critical biological functions, including regulating cell-cell interactions and cell proliferation, inhibiting enzymes, and activating growth factor receptors during various metabolic processes. Indeed, heparin is a widely used GAG-based anticoagulant drug. Unfortunately, naturally derived GAGs are highly heterogeneous, limiting studies of their structure-activity relationships and even resulting in safety concerns. For example, the heparin contamination crisis in 2007 reportedly killed more than a hundred people in the United States. Unfortunately, the chemical synthesis of GAGs, or their oligosaccharides, based on repetitive steps of protection, activation, coupling, and deprotection, is incredibly challenging. Recent advances in chemoenzymatic synthesis integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions, mimicking the biosynthetic pathway of GAGs, and represent a promising strategy to solve many of these synthetic challenges. In this critical Account, we examine the recent progress made, in our laboratory and by others, in the chemoenzymatic synthesis of GAGs, focusing on heparan sulfate and heparin, a class of GAGs with profound physiological and pharmacological importance. A major challenge for the penetration of the heparin market by homogeneous heparin products is their cost-effective large-scale synthesis. In the past decade, we and our collaborators have systematically explored the key factors that impact this process, including better enzyme expression, improved biocatalysts using protein engineering and immobilization, low cost production of enzyme cofactors, optimization of the order of enzymatic transformations, as well as development of efficient technologies, such as using ultraviolet absorbing or fluorous tags, to detect and purify synthetic intermediates. These improvements have successfully resulted in multigram-scale synthesis of low-molecular-weight heparins (LMWHs), with some showing excellent anticoagulant activity and even resulting in more effective protamine reversal than commercial, animal-sourced LMWH drugs. Sophisticated structural analysis is another challenge for marketing heparins, since impurities and contaminants can be present that are difficult to distinguish from heparin drug products. The availability of the diverse library of structurally defined heparin oligosaccharides has facilitated the systematic analytical studies undertaken by our group, resulting in important information for characterizing diverse heparin products, safeguarding their quality. Recently, a series of chemically modified nucleotide sugars have been investigated in our laboratory and have been accepted by synthases to obtain novel GAGs and GAG oligosaccharides. These includefluoride and azido regioselectivelyfunctionalizedsugarsand stable isotope-enriched GAGs and GAG oligosaccharides, critical for better understanding the biological roles of these important biopolymers. We speculate that the repertoire of unnatural acceptors and nucleotide sugar donors will soon be expanded to afford many new GAG analogues with new biological and pharmacological properties including improved specificity and metabolic stability.

Heparan sulfate negatively regulates intestinal stem cell proliferation in Drosophila adult midgut.

ABSTRACTTissue homeostasis is maintained by differentiated progeny of residential stem cells. Both extrinsic signals and intrinsic factors play critical roles in the proliferation and differentiation of adult intestinal stem cells (ISCs). However, how extrinsic signals are transduced into ISCs still remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, negatively regulates progenitor proliferation and differentiation to maintain midgut homeostasis under physiological conditions. Interestingly, HS depletion in progenitors results in inactivation of Decapentaplegic (Dpp) signaling. Dpp signal inactivation in progenitors resembles HS-deficient intestines. Ectopic Dpp signaling completely rescued the defects caused by HS depletion. Taken together, these data demonstrate that HS is required for Dpp signaling to maintain midgut homeostasis. Our results provide insight into the regulatory mechanisms of how extrinsic signals are transduced into stem cells to regulate their proliferation and differentiation.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

The Protective Influence of Chondroitin Sulfate, a Component of Human Milk, on Intestinal Bacterial Invasion and Translocation.

Human milk is known to be protective against necrotizing enterocolitis, a devastating intestinal inflammatory disease affecting the preterm population. Although the pathogenesis of necrotizing enterocolitis is yet to be solidified, intestinal integrity dysfunction, bacterial invasion and/or translocation, and inflammation may play important roles. Glycosaminoglycans, compounds naturally prevalent in both human milk and the intestine, are thought to be anti-inflammatory and capable of altering bacterial interactions within the gut. In this study, we aimed to evaluate the potential of chondroitin sulfate, the most prominent class of glycosaminoglycans in human milk, to protect against bacterial infection in an intestinal in vitro model. T84 cell monolayers were treated with chondroitin sulfate and cell viability was assessed across a number of doses. Monolayers were then pretreated with chondroitin sulfate and subsequently challenged with E. coli invasion and translocation to evaluate any protective role of the compound against infection. Tight junction barrier function was assessed by transepithelial electrical resistance, and cytokine levels were evaluated. Chondroitin sulfate at any dose up to 750 μg/ml was not associated with any statistically significant decrease in cell viability. Additionally, chondroitin sulfate at 750 μg/ml was associated with a 75% decrease in both bacterial invasion and translocation compared to control. These data suggest chondroitin sulfate may protect against bacterial infection through a reduction in both invasion and translocation, importantly without attendant reduction in cell viability.

Analysis of Heparan sulfate/heparin from Colla corii asini by liquid chromatography-electrospray ion trap mass spectrometry.

Colla corii asini (CCA) made from donkey-hide has been widely used as a health-care food and an ingredient of traditional Chinese medicine. Heparan sulfate (HS)/heparin is a structurally complex class of glycosaminoglycans (GAGs) that have been implicated in a wide range of biological activities. However, their presence within CCA, and their possible structural characteristics, were previously unknown. In this study, GAG fractions containing HS/heparin were isolated from CCA and their disaccharide compositions were analyzed by high sensitivity liquid chromatography-ion trap/time-of-flight mass spectrometry (LC-MS-ITTOF). This revealed that, in addition to the eight commonly seen HS disaccharides, the four rare N-unsubstituted disaccharides were also detected in significant quantities. The disaccharide compositions varied significantly between HS/heparin fractions indicating chains with differing domain structures. This novel structural information may lead to a better understanding of the biological activities (i.e. anticoagulation and antitumor action) of CCA.

Keratan sulfate (KS)-proteoglycans and neuronal regulation in health and disease: the importance of KS-glycodynamics and interactive capability with neuroregulatory ligands.

Compared to the other classes of glycosaminoglycans (GAGs), that is, chondroitin/dermatan sulfate, heparin/heparan sulfate and hyaluronan, keratan sulfate (KS), have the least known of its interactive properties. In the human body, the cornea and the brain are the two most abundant tissue sources of KS. Embryonic KS is synthesized as a linear poly-N-acetyllactosamine chain of d-galactose-GlcNAc repeat disaccharides which become progressively sulfated with development, sulfation of GlcNAc is more predominant than galactose. KS contains multi-sulfated high-charge density, monosulfated and non-sulfated poly-N-acetyllactosamine regions and thus is a heterogeneous molecule in terms of chain length and charge distribution. A recent proteomics study on corneal KS demonstrated its interactivity with members of the Slit-Robbo and Ephrin-Ephrin receptor families and proteins which regulate Rho GTPase signaling and actin polymerization/depolymerization in neural development and differentiation. KS decorates a number of peripheral nervous system/CNS proteoglycan (PG) core proteins. The astrocyte KS-PG abakan defines functional margins of the brain and is up-regulated following trauma. The chondroitin sulfate/KS PG aggrecan forms perineuronal nets which aredynamic neuroprotective structures with anti-oxidant properties and roles in neural differentiation, development and synaptic plasticity. Brain phosphacan a chondroitin sulfate, KS, HNK-1 PG have roles in neural development and repair. The intracellular microtubule and synaptic vesicle KS-PGs MAP1B and SV2 have roles in metabolite transport, storage, and export of neurotransmitters and cytoskeletal assembly. MAP1B has binding sites for tubulin and actin through which it promotes cytoskeletal development in growth cones and is highly expressed during neurite extension. The interactive capability of KS with neuroregulatory ligands indicate varied roles for KS-PGs in development and regenerative neural processes.

RNA Aptamers with Specificity for Heparosan and Chondroitin Glycosaminoglycans.

In this study, two respective groups of RNA aptamers have been selected against two main classes of glycosaminoglycans (GAGs), heparosan, and chondroitin, as they have proven difficult to specifically detect in biological samples. GAGs are linear, anionic, polydisperse polysaccharides found ubiquitously in nature, yet their detection remains problematic. GAGs comprised repeating disaccharide units, consisting of uronic acid and hexosamine residues that are often also sulfated at various positions. Monoclonal antibodies are frequently used in biology and medicine to recognize various biological analytes with high affinity and specificity. However, GAGs are conserved across the whole animal phylogenic tree and are nonimmunogenic in hosts traditionally used for natural antibody generation. Thus, it has been challenging to obtain high affinity, selective antibodies that recognize various GAGs. In the absence of anti-GAG antibodies, glycobiologists have relied on the use of specific enzymes to convert GAGs to oligosaccharides for analysis by mass spectrometry. Unfortunately, while these methods are sensitive, they can be labor-intensive and cannot be used for in situ detection of intact GAGs in cells and tissues. Aptamers are single-stranded oligonucleotide (DNA or RNA) ligands capable of high selectivity and high affinity detection of biological analytes. Aptamers can be developed in vitro by the systematic evolution of ligands by exponential enrichment (SELEX) to recognize nonimmunogenic targets, including neutral carbohydrates. This study utilizes the SELEX method to generate RNA aptamers, which specifically bind to the unmodified GAGs, heparosan, and chondroitin. Binding confirmation and cross-screening with other GAGs were performed using confocal microscopy to afford three specific GAGs to each target. Affinity constant of each RNA aptamer was obtained by fluorescent output after interaction with the respective GAG target immobilized on plates; the KD values were determined to be 0.71–1.0 μM for all aptamers. Upon the success of chemical modification (to stabilize RNA aptamers in actual biological systems) and fluorescent tagging (to only visualize RNA aptamers) of these aptamers, they would be able to serve as a specific detection reagent of these important GAGs in biological samples.