Glycosaminoglycan interaction networks and databases

Glycosaminoglycans are complex polysaccharides exhibiting a large structural and conformational diversity. These key biological players organize the extracellular matrix, contribute to cell–matrix interactions, and regulate cell signaling. Natural and synthetic libraries of glycosaminoglycans have been spotted on microarrays to find glycosaminoglycan partners and determine the size and the chemical groups promoting protein binding. Advances in glycosaminoglycan sequencing allow the characterization of glycosaminoglycan sequences interacting with proteins, and glycosaminoglycan-mediated pull-down proteomics can identify glycosaminoglycan-binding proteins at a proteome scale in various biological samples. The analysis of the glycosaminoglycan interaction networks generated using these data gives insights into the molecular and cellular mechanisms underlying glycosaminoglycan functions. These interactomes can also be used to design inhibitors targeting specific GAG interactions for therapeutic purpose.     

Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III

A heparan sulfate glycosaminoglycan chain, biotinylated at its reducing-end, was bound to a streptavidin-coated biochip. Surface plasmon resonance spectroscopy showed a low affinity interaction with antithrombin III (ATIII) when it was flowed over a surface containing heparan sulfate. ATIII bound tightly with high affinity when the same surface was enzymatically modified to using 3-O-sulfotransferase isoform 1 (3-OST-1) in the presence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The 3-OST-1 enzyme is involved in heparan sulfate biosynthesis and introduces a critical 3-O-sulfo group into this glycosaminoglycan affording the appropriate pentasaccharide sequence capable of high affinity binding to ATIII. This experiment demonstrates the specific structural modification of a glycosaminoglycan bound to a biochip using a biosynthetic enzyme, suggesting a new approach to rapid screening glycosaminoglycan-protein interactions.     

Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways:     

The synthesis of a novel thio-linked disaccharide of chondroitin as a potential inhibitor of polysaccharide lyases

A thio-linked disaccharide based on the structure of the glycosaminoglycan chondroitin was synthesized as a potential inhibitor of chondroitin AC lyase from Flavobacterium heparinum for structural analysis of the active site. Instead it was found to be a slow substrate, thereby demonstrating that lyases, in contrast to glycosidases, can cleave thioglycoside links between sugars.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. I. Soluble complexes.

The interaction of acridine orange with dermatan and chondrotin sulfates results in the formation of complexes containing bound dye molecules ordered into dissymmetric arrays. Complexes containing an excess of available disaccharide residues compared to dye are completely soluble, and exhibit biphasic circular dichroism bands. Analysis of the dependence of the extrinsic circular dichrosim on dye aggregation indicates the presence of extended dye stacks bound to the glycosaminoglycan. Complexes formed in solutions containing an excess of dye are only partially soluble, and exhibit circular dichroism spectra having band shifts and intensity changes relative to the soluble complexes. The latter complexes show a sharp drop in induced circular dichroism with temperature, due to a cooperative change in the structure of the complex. The structural order of the dye–glycosaminoglycan complex may differ from the intrinsic structure of the glycosaminoglycan itself in dilute solution.     

Crystal structure of N-glycosylated human glypican-1 core protein: structure of two loops evolutionarily conserved in vertebrate glypican-1

Glypicans are a family of cell-surface proteoglycans that regulate Wnt, hedgehog, bone morphogenetic protein, and fibroblast growth factor signaling. Loss-of-function mutations in glypican core proteins and in glycosaminoglycan-synthesizing enzymes have revealed that glypican core proteins and their glycosaminoglycan chains are important in shaping animal development. Glypican core proteins consist of a stable α-helical domain containing 14 conserved Cys residues followed by a glycosaminoglycan attachment domain that becomes exclusively substituted with heparan sulfate (HS) and presumably adopts a random coil conformation. Removal of the α-helical domain results in almost exclusive addition of the glycosaminoglycan chondroitin sulfate, suggesting that factors in the α-helical domain promote assembly of HS. Glypican-1 is involved in brain development and is one of six members of the vertebrate family of glypicans. We expressed and crystallized N-glycosylated human glypican-1 lacking HS and N-glycosylated glypican-1 lacking the HS attachment domain. The crystal structure of glypican-1 was solved using crystals of selenomethionine-labeled glypican-1 core protein lacking the HS domain. No additional electron density was observed for crystals of glypican-1 containing the HS attachment domain, and CD spectra of the two protein species were highly similar. The crystal structure of N-glycosylated human glypican-1 core protein at 2.5 Å, the first crystal structure of a vertebrate glypican, reveals the complete disulfide bond arrangement of the conserved Cys residues, and it also extends the structural knowledge of glypicans for one α-helix and two long loops. Importantly, the loops are evolutionarily conserved in vertebrate glypican-1, and one of them is involved in glycosaminoglycan class determination.     

Theoretical investigations on the functionalization of carbon nanotubes

In this paper, our recent work concerning theoretical studies on the functionalization of carbon nanotubes (CNTs) is reviewed. In particular, two different aspects of the functionalization process are taken into account. On the one hand, the chemical functionalization of the sidewall is exploited as a way to develop nanostructured gas sensing devices. On the other hand, we investigated the possibility of functionalizing the sidewall with transition metal complexes, thus extending the concepts of organometallic chemistry to CNTs. Calculations were performed by applying statical and dynamical (Car-Parrinello) density functional theory methods, as well as hybrid (quantum mechanics/molecular mechanics) schemes. The structural and electronic peculiarities of the CNT model under study, due, for example to the presence of defects, were found to play a crucial role in the modelization of the functionalization process. In most cases, the use of realistic models was essential to achieve a full agreement with experiments.     

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.     

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multimolecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell-binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell-surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.     

Mechanical model for a collagen fibril pair in extracellular matrix

In this paper, we model the mechanics of a collagen pair in the connective tissue extracellular matrix that exists in abundance throughout animals, including the human body. This connective tissue comprises repeated units of two main structures, namely collagens as well as axial, parallel and regular anionic glycosaminoglycan between collagens. The collagen fibril can be modeled by Hooke’s law whereas anionic glycosaminoglycan behaves more like a rubber-band rod and as such can be better modeled by the worm-like chain model. While both computer simulations and continuum mechanics models have been investigated for the behavior of this connective tissue typically, authors either assume a simple form of the molecular potential energy or entirely ignore the microscopic structure of the connective tissue. Here, we apply basic physical methodologies and simple applied mathematical modeling techniques to describe the collagen pair quantitatively. We found that the growth of fibrils was intimately related to the maximum length of the anionic glycosaminoglycan and the relative displacement of two adjacent fibrils, which in return was closely related to the effectiveness of anionic glycosaminoglycan in transmitting forces between fibrils. These reveal the importance of the anionic glycosaminoglycan in maintaining the structural shape of the connective tissue extracellular matrix and eventually the shape modulus of human tissues. We also found that some macroscopic properties, like the maximum molecular energy and the breaking fraction of the collagen, were also related to the microscopic characteristics of the anionic glycosaminoglycan.     

Two-dimensional boron nitride structures functionalization: first principles studies

Density functional theory calculations have been performed to investigate two-dimensional hexagonal boron nitride (2D hBN) structures functionalization with organic molecules. 2x2, 4x4 and 6x6 periodic 2D hBN layers have been considered to interact with acetylene. To deal with the exchange-correlation energy the generalized gradient approximation (GGA) is invoked. The electron-ion interaction is treated with the pseudopotential method. The GGA with the Perdew-Burke-Ernzerhoff (PBE) functionals together with van der Waals interactions are considered to deal with the composed systems. To investigate the functionalization two main configurations have been explored; in one case the molecule interacts with the boron atom and in the other with the nitrogen atom. Results of the adsorption energies indicate chemisorption in both cases. The total density of states (DOS) displays an energy gap in both cases. The projected DOS indicate that the B-p and N-p orbitals are those that make the most important contribution in the valence band and the H-s and C-p orbitals provide an important contribution in the conduction band to the DOS. Provided that the interactions of the acetylene with the 2D layer modify the structural and electronic properties of the hBN the possibility of structural functionalization using organic molecules may be concluded.     

Standardization of Staining in Giycosaminoglycan Histochemistry: Alcian Blue, its Analogues, and Diamine Methods

Glycosaminoglycans are identified in tissue sections by various histochemical techniques including staining with alcian blue and its analogues, such as cuprolinic blue and cupromeronic blue, or with high and low iron diamine methods. The variation in staining results is particularly confusing in the case of alcian blue, where not only are several different brands of alcian blue available but also several different staining protocols are used. If the results obtained by these techniques are compared, they often do not match. We have developed a dot blot technique for quality control of glycosaminoglycan histochemistry to standardize the staining protocols. This staining technique enables his-tochemists to test particular batches of alcian blue or its analogues for selective glycosaminoglycan staining, thus improving control of histochemical results. The results obtained using the dot blot assay indicate that it is necessary to test each batch of dye individually to obtain valid results in glycosaminoglycan histochemistry.     

Anticoagulant motifs of marine sulfated glycans

Sulfated polysaccharides, like the glycosaminoglycan (GAG) heparin, are known to exhibit anticoagulant properties when certain structural features are present. The structural requirement for this action is well-established for heparin, in which a pentasaccharide motif plays a key role for keeping the high-affinity interaction to antithrombin. Over the last years of this glycomic era, several novel anticoagulant sulfated glycans have been described. Those from marine sources have been awakening special attention mainly because of their impressive anticoagulant effects together with structural uniqueness. The commonest of these glycans are the sulfated fucans (SFs), the sulfated galactans (SGs), and the marine invertebrate GAGs like the fucosylated chondroitin sulfate and ascidian dermatan sulfate. Since these marine sulfated glycans do not bear within their polymeric chains the specific pentasaccharide motif of heparin, other structural features must be necessary to trigger the anticoagulant effect. The objective of this report is to present the anticoagulant motifs of the marine SFs, SGs and GAGs.     

Covalent hybridization of CNT by thymine and uracil: A computational study

We have investigated the electronic and structural properties of covalent functionalization of the tip of (5,0) carbon nanotube (CNT) by di-keto and keto-enol forms of thymine (T) and uracil (U) nucleobases. Density functional theory (DFT) calculations have been performed to optimize the investigated structures and to calculate the properties such as dipole moment, bond length, band gap, total energy, binding energy and quadrupole coupling constant. The results indicated that, due to the functionalization of CNT by T and U, the hybrids exhibit new properties in which they are similar in both types of CNT-T and CNT-U hybrids.     

Semi-synthetic heparin derivatives: chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups

The glycosaminoglycan heparin is a polyanionic polysaccharide most recognized for its anticoagulant activity. Heparin binds to cationic regions in hundreds of prokaryotic and eukaryotic proteins, termed heparin-binding proteins. The endogenous ligand for many of these heparin-binding proteins is a structurally similar glycosaminoglycan, heparan sulfate (HS). Chemical and biosynthetic modifications of heparin and HS have been employed to discern specific sequences and charge-substitution patterns required for these polysaccharides to bind specific proteins, with the goal of understanding structural requirements for protein binding well enough to elucidate the function of the saccharide-protein interactions and/or to develop new or improved heparin-based pharmaceuticals. The most common modifications to heparin structure have been alteration of sulfate substitution patterns, carboxyl reduction, replacement N-sulfo groups with N-acetyl groups, and chain fragmentation. However, an accumulation of reports over the past 50 years describe semi-synthetic heparin derivatives obtained by incorporating aliphatic, aryl, and heteroaryl moieties into the heparin structure. A primary goal in many of these reports has been to identify heparin-derived structures as new or improved heparin-based therapeutics. Presented here is a perspective on the introduction of non-anionic structural motifs into heparin structure, with a focus on such modifications as a strategy to generate novel reduced-charge heparin-based bind-and-block antagonists of HS-protein interactions. The chemical methods employed to synthesize such derivatives, as well as other unique heparin conjugates, are reviewed.     

Reconstruction of glycosaminoglycan chains in decorin

The glycosaminoglycan chain of decorin from human spinal ligaments was digested using the hydrolysis of bovine testicular hyaluronidase. As a result, decorin with hexasaccharide, octasaccharide, and decasaccharide including the linkage region, GlcA-Gal-Gal-Xyl, was obtained. The obtained decorin as an acceptor and hyaluronic acid as a donor were incubated with bovine testicular hyaluronidase under the condition of transglycosylation reaction. The transglycosylation reaction product had hexasaccharide to triacontasaccharide. Judging from the analysis of glycosaminoglycan chain in the transglycosylation reaction product, it was confirmed that hyaluronic acid chain as a donor was transferred to the retained glycosaminoglycan chain of decorin as an acceptor. Similarly, it was possible to reconstruct the glycosaminoglycan chain in decorin to chondroitin, chondroitin 4-sulfate or chondroitin 6-sulfate. Therefore, we succeeded in synthesizing an artificial family of decorins.     

Effects of molecular size and chemical structure on renal and hepatic removal of exogenously administered chondroitin sulfate in rats

Chondroitin sulfate, a glycosaminoglycan that is widely distributed among mammals, is used as a therapeutic agent in various diseases. Here, we focus on its absorption, excretion and tissue accumulation in rats. The concentration of 35S-chondroitin sulfate (35S-CS) in plasma reaches a peak in the first 5 min after intravenous administration and simultaneously increases in the urine. Approximately 25% of the 35S found in the urine appears as inorganic sulfate, indicating that 35S-CS is partially degraded during its renal filtration. The glycosaminoglycan is retained mainly by the liver and the kidney, where the amount of 35S reaches a plateau in the first 30 min, remains constant up to 2 h and then decreases markedly. Renal filtration and organ accumulation of 35S-CS decreases as the size of the glycosaminoglycan is reduced, especially in the liver. A derivative of 35S-CS that resists hyaluronidase digestion due to reduction of its glucuronic acid carboxyl groups appears at lower concentrations in plasma and in urine when compared with native 35S-CS. This derivative reaches higher levels in the kidney but lower levels in the liver when compared with the native molecule. Overall, our results indicate a balance between renal and hepatic mechanisms for removing chondroitin sulfate from plasma. The renal filtration increases as the molecular weight of the glycosaminoglycan decreases, whereas hepatic removal requires structural integrity and the presence of high-molecular-weight chains.     

Conversion of native lignin to a highly phenolic functional polymer and its separation from lignocellulosics

An original reaction system (the phase separative reaction system) has been designed for derivatizing native lignins to highly phenolic, functional polymers. This system is composed of a phenol derivative and concentrated acid, which are not miscible at room temperature. The key point of the lignin functionalization process, including the phase separative system, is that lignin and carbohdrates, which are totally different in structures and reactivitie, are modified individually in the different phases: lignin is present in the organic phase and carbohydrates in the aqueous phase. Through the process, lignin was modified selectively at Calpha-positions of side chains, the most reactive sites, to give highly phenolic, light-colored, diphenylmethane-type materials which still retained original interunit linkages formed by the dehydrogenative polymerization during the biosynthesis. The carbohydrates were swollen, followed by partial hydrolysis and dissolution in the acid solution, resulting in the perfect decomposition of interpenetrating polymer network structures in the cell wall. Therefore, the functionalization of lignin and the separation of resulting lignin from carbohydrates were quickly achieved at room temperature, independent of wood species. This process would be a powerful tool for estimating strutures and reactivities of lignins as well as the functionalization of lignins, because of the selective structural modifications. (c) 1995 John Wiley & Sons, Inc.     

Advances in analysis of glycosaminoglycans: its application for the assessment of physiological and pathological states of connective tissues

Glycosaminoglycans are a class of biological macromolecules found mainly in connective tissues as constituents of proteoglycans, covalently linked to their core protein. Hyaluronan is the only glycosaminoglycan present under its single form and possesses the ability to aggregate with the class of proteoglycans termed hyalectans. Proteoglycans are localised both at the extracellular and cellular (cell-surface and intracellular) levels and, via either their glycosaminoglycan chains or their core proteins participate in and regulate several cellular events and (patho)physiological processes. Advances in analytical separational techniques, including high-performance liquid chromatography, capillary electrophoresis and fluorophore assisted carbohydrate electrophoresis, make possible to examine alterations of glycosaminoglycans with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this review we present the chromatographic and electromigration procedures developed to analyse and characterise glycosaminoglycans. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.     

Fabrication and selective functionalization of amine-reactive polymer multilayers on topographically patterned microwell cell culture arrays

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. "Reactive" layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4'-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts.