[Show abstract][Hide abstract] ABSTRACT: For heparan sulfate (HS) to bind and regulate the activity of proteins, the polysaccharide must present an appropriate sequence and adopt a suitable conformation. The conformations of heparin derivatives, as models of HS, are altered via a change in the associated cations, and this can drastically modify their FGF signaling activities. Here, we report that changing the cations associated with an N-acetyl-enriched heparin polysaccharide, from sodium to copper(II), converted it from supporting signaling through the fibroblast growth factor receptor (FGF-1-FGFR1c) tyrosine kinase signaling system to being inhibitory in a cell-based BaF3 assay. Nuclear magnetic resonance and synchrotron radiation circular dichroism (SRCD) spectroscopy demonstrated that the polysaccharide conformation differed in the presence of sodium or copper(II) cations. Electron paramagnetic resonance confirmed the environment of the copper(II) ion on the N-acetyl-enriched polysaccharide was distinct from that previously observed with intact heparin, which supported signaling. Secondary structures in solution complexes of polysaccharides with FGF-1 (which either supported signaling through FGFR1c or were inhibitory) were determined by SRCD. This allowed direct comparison of the two FGF-1-polysaccharide complexes in solution, containing identical molecular components and differing only in their cation content. Subtle structural differences were revealed, including a reduction in the level of disordered structure in the inhibitory complex.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulphate (HS) is a glycosaminoglycan present in all metazoan organisms. It is an unbranched chain made up of repeating disaccharide units of uronic acid and glucosamine sugars, and is present in both cells and the extracellular matrix. It is one of the most structurally diverse biological molecules and its biosynthesis involves a variety of enzymic modification steps. Unlike the genome and the transcriptome, HS synthesis is not template driven. Nevertheless, the HS structure and function are highly regulated with modification steps occurring in discrete regions of the polysaccharide chain to give rise to diverse structures interacting with, and regulating, many different proteins. The resulting variation leads to diverse biological roles of HS. To study this structural diversity, rapid isolation and characterization of HS from small amounts of tissues, followed by digestion with bacterially derived enzymes (heparitinases) and chromatography techniques can be used to separate HS oligosaccharides of different size and charge. However, this leads to complex datasets where comparison of just a few samples leads to difficulties in data analysis. Using automatically integrated peak data obtained from chromatographic software, one can apply the effective disc technique to the data points to obtain the centre of mass in each dataset, for example from different murine tissues. This allows facile comparative analysis of different datasets. When the cloud of points displays some preferential direction (anisotropy), it is preferable to compute its effective ellipse. Analysis of the dynamics of the cloud of points for repeated experiments allows the quantification of their reproducibility through evaluation of an average Lyapunov exponent characterizing the area-preserving nature of a sequence of effective ellipses. These basic mathematical approaches allow a more systematic comparison of datasets derived from structural analysis using basic spreadsheet software calculations and contribute to the development of system biology strategies for tackling biocomplexity of HS polysaccharides.
Journal of The Royal Society Interface 02/2009; 6(40):997-1004. DOI:10.1098/rsif.2008.0483 · 3.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The analysis of heparin/HS saccharides derived from small amounts of tissue or cells is a considerable technical challenge and the development of methods to characterise these carbohydrates has progressed comparatively slowly. A number of procedures have been devised to tag glycans selectively at the reducing end with a group that will enhance the sensitivity of detection and facilitate chromatographic separations. Outlined in this chapter are two useful strategies designed specifically for the analysis of heparin/HS saccharides. The first involves a fluorophore label, Bodipy-FL-hydrazide, which permits highly sensitive (fmol level) detection of saccharides utilising high performance strong anion exchange chromatography. The second facilitates oligosaccharide separation by gel-permeation chromatography and reverse phase high performance ion-pairing chromatography (RP-HPIPC) through the use of a phenylsemicarbazide tag. The latter also serves as an effective mass tag for electrospray mass spectrometry, permitting enhanced analysis of HS saccharides. These methods provide new opportunities for the development of glycomics approaches to study the structure and function of the heparan sulfate family of glycans.
[Show abstract][Hide abstract] ABSTRACT: The interactions between Cu(II) ions and heparin were investigated using several complementary spectroscopic techniques. NMR indicated an initial binding phase involving specific coordination to four points in the structure that recur in slightly different environments throughout the heparin chain; the carboxylic acid group and the ring oxygen of iduronate-2-O-sulfate, the glycosidic oxygen between this residue and the adjacent (towards the reducing end) glucosamine and the 6-O-sulfate group. In contrast, the later binding phase showed little structural specificity. One- and two-dimensional correlated FTIR revealed that complex out of phase (asynchronous) conformational changes also occurred during the titration of Cu(II) ions into heparin, involving the CO and N-H stretches. EPR demonstrated that the environments of the Cu(II) ions in the initial binding phase were tetragonal (with slightly varied geometry), while the later non-specific phases exhibited conventional coordination. Visible spectroscopy confirmed a shift of the absorbance maximum. Titration of Cu(II) ions into a solution of heparin indicated (both by analysis of FTIR and EPR spectra) that the initial binding phase was complete by 15-20 Cu(II) ions per chain; thereafter the ions bound in the non-specific mode. Hetero-correlation spectroscopy (FTIR-CD) improved resolution and assisted assignment of the broad CD features from the FTIR spectra and indicated both in-phase and more complex out of phase (synchronous and asynchronous, respectively) changes in interactions within the heparin molecule during the titration of Cu(II) ions.
Carbohydrate Research 09/2008; 343(12):2184-93. DOI:10.1016/j.carres.2007.12.019 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cleavage of beta-amyloid precursor protein (APP) by the protease beta-secretase (BACE1) is a key step in beta-amyloid peptide processing. We have described a novel role for heparan sulphate polysaccharides in Alzheimer's disease pathology as naturally occurring inhibitors of beta-secretase, suggesting new avenues for discovery of novel drugs for Alzheimer's disease based on heparins.
To evaluate engineered heparin analogues as novel beta-secretase inhibitors in vitro, including modifications to increase bioavailability.
We tested a number of selectively desulphated and chemically modified heparins for their ability to inhibit BACE1 and other proteases in vitro using APP fluorescent resonance energy transfer peptide substrates
Several lead compounds have been identified that are effective beta-secretase inhibitors, but have negligible activity as anticoagulants or as inhibitors of other aspartyl proteases structurally related to beta-secretase. In addition, the compounds studied also give some insight into the structural interaction between beta-secretase and heparin, indicating that the structure of the polysaccharide is much more important than charge.
We have demonstrated that modifications to increase bioavailability of chemically modified heparins have little effect on their efficacy as beta-secretase inhibitors. Therefore, these heparins show promise for development as a novel class of pharmaceuticals that target the underlying pathology of Alzheimer's disease. We have also found further evidence that it is the structure of the polysaccharide that is important for the interaction with beta-secretase, not simply the level of sulphation or charge.
[Show abstract][Hide abstract] ABSTRACT: Heparan sulfate proteoglycans (HSPGs) are essential to respiratory morphogenesis in species as diverse as Drosophila and mice; they play a role in the regulation of numerous HS-binding growth factors, e.g. fibroblast growth factors. Moreover, an HS analogue, heparin, modulates lung growth in vitro. However, it has been difficult to assess the roles of specific HS structures in lung development due to technical barriers to their spatial localisation. Lungs from Sprague-Dawley rats were harvested between E15.5 and E19.5 and immediately fixed in 4 % (w/v) paraformaldehyde (in 0.1 M phosphate-buffered saline (PBS), pH 7.4). Lungs were washed in PBS, cryoprotected with 20% (w/v) sucrose (in PBS), gelatin embedded [7.5% (w/v) gelatin, 15% (w/v) sucrose in PBS], before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap frozen at -40 degrees C. Cryosections were cut at 8 microm and stained with the HSPG core protein specific antibody 3G10 and a HS 'phage display antibody, EW4G2V. 3G10 and EW4G2V immunohistochemistry highlighted the presence of specific HS structures in lungs at all gestational ages examined. 3G10 strongly labelled airway basement membranes and the surrounding mesenchyme and showed weak staining of airway epithelial cells. EW4G2V, however, was far more selective, labelling the airway basement membranes only. Mesenchymal and epithelial cells did not appear to possess the HS epitope recognised by EW4G2V at these gestational ages. Novel 'phage display antibodies allow the spatial distribution of tissue HS to be analysed, and demonstrate in situ that distinct cellular compartments of a tissue possess different HS structures, possibly on the same proteoglycan core protein. These probes offer a new opportunity to determine the role of HS in the pathogenesis of congenital defects such as congenital diaphragmatic hernia (CDH), where lung development is aberrant, and the resulting pulmonary hypoplasia and hypertension are a primary cause of mortality.
Pediatric Surgery International 06/2007; 23(5):411-7. DOI:10.1007/s00383-006-1864-8 · 1.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eight Delta-disaccharide standards from heparan sulfate/heparin were derivatized with the fluorophore 4,4-difluoro-5,7- dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, hydrazide (BODIPY) via formation of a Schiff's base and separated using HPAEC on a Propac PA1 column with a linear salt gradient and isocratic 150 mM NaOH. Detection was with an in-line fluorescence detector. The standard deviation (sigma(n-1)) in retention times were 0.7-2% over nine runs. The limit of detection, was 100 fmol (100 x 10(-15)mol) of BODIPY labeled Delta-disaccharides, representing considerably improved detection compared to other fluorophore labeled derivatives and, unlike these, required no further purification steps. Separation and improved detection of BODIPY-Delta-disaccharide conjugates will assist the structural analysis of HS and the development of improved sequencing methodologies.
Journal of Chromatography A 12/2006; 1135(1):52-6. DOI:10.1016/j.chroma.2006.09.064 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The potent gliogenic and neurotrophic fibroblast growth factor (FGF)-2 signals through a receptor complex comprising high-affinity FGF receptor (FGFR)1 with heparan sulphate proteoglycans (HSPGs) as co-receptors. We examined the intracellular dynamics of FGF-2, FGFR1 and the HSPGs syndecan-2 and -3, glypican-1 and -2, and perlecan in neurones and glia in and around adult rat cerebral wounds. In the intact cerebral cortex, FGF-2 and FGFR1 mRNA and protein were constitutively expressed in astrocytes and neurones respectively. FGF-2 protein was localized exclusively to astrocyte nuclei. After injury, expression of FGF-2 mRNA was up-regulated only in astrocytes, whereas FGFR1 mRNA expression was increased in both glia and neurones, a disparity indicating that FGF-2 may act as a paracrine and autocrine factor for neurones and glia respectively. FGF-2 protein localized to both cytoplasm and nuclei of injury-responsive neurones and glia. There was weak or no staining of HSPGs in the normal cerebral neuropil and glia nuclei, with a few immunopositive neurones. Specific HSPGs responded to injury by differentially co-localizing with trafficked intracellular FGF-2 and FGFR1. The spatiotemporal dynamics of FGF-2-FGFR1-HSPG complex formation implies a role for individual HSPGs in regulating FGF-2 storage, nuclear trafficking and cell-specific injury responses in CNS wounds.
Journal of Neurochemistry 03/2006; 96(4):1189-200. DOI:10.1111/j.1471-4159.2005.03632.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The role of HS (heparan sulphate) in the pathology of AD (Alzheimer's disease) is multifaceted. HS and other glycosaminoglycans have been widely reported to be associated with neuritic plaques. HS has also been shown to promote the aggregation of Abeta (amyloid beta-peptide), the proteinaceous component of neuritic plaques. Recently, we described a novel and contrasting role for HS in the pathology of AD: HS can inhibit the formation of Abeta, by directly interacting with the protease BACE1 (beta-site amyloid precursor protein cleaving enzyme 1; beta-secretase 1), that cleaves the amyloid precursor protein and is the rate limiting step in the generation of Abeta. Here, we review the current roles of HS and the potential for HS-derivatives in the treatment of AD.
[Show abstract][Hide abstract] ABSTRACT: Introduction Embryoid bodies (Ebs) are layered, ordered aggregates of cells which form when murine embryonic stem (ES) cells are grown in suspension. Ebs are made up of primitive endoderm cells overlaying an epithelium of epiblast cells, separated by a basement membrane and surrounding a central cavity. The generation of Ebs from undifferentiated ES cells has been used as a model for early development. The process of EB formation has been shown to require signalling through fibroblast growth factor (FGF) receptor 2, implicating heparan sulfate (HS) which is a necessary coreceptor for the formation of a signalling complex.Heparan sulfate proteoglycans are found both on the surface of cells and in the extracellular matrix. Within the cells, a newly synthesized HS chain is partially modified by a family of enzymes, many of which have multiple isoforms with differing substrate preferences. These modifications include the addition of sulfate groups at up to four positions on each disaccharide unit. The pattern of modifications that a HS saccharide undergoes alters its structure, affecting the ability of the HS to bind and regulate different FGF-FGF receptor combinations, and so regulate signalling outcomes.It is hypothesized that the complement of modification enzymes that a cell expresses determines the structure of the HS that the cell produces, thereby altering the signalling response of the cell to HS-binding factors such as FGFs. In this way, an ES cell could alter its HS to allow signalling by, for example, a particular FGF, setting in motion a chain of intracellular events leading to differentiation. Materials and methods Methods used in this study include tissue culture, RT-PCR and immunocytochemistry. Results We have found that undifferentiated ES cells express HS as observed by cell staining with anti-HS antibodies. They also display particular expression patterns of the different HS modification enzyme isoforms as measured by RT-PCR. Studies on alterations in HS synthesis and structure during the process of EB formation are underway. In addition, the effect of the addition of exogenous heparin (a highly sulfated analogue of HS) and HS saccharides with variant structures on EB formation is being investigated. Discussion Alterations in the profile of modification enzymes expressed by ES cells as they differentiate may correlate with changes in the structure of the HS they make. These changes may allow the differentiating cells to control the way in which they respond to their environment.These studies will allow the dissection of the structural requirements for a functional role of HS in embryoid body formation and stem cell differentiation.
International Journal of Experimental Pathology 08/2004; 85(4). DOI:10.1111/j.0959-9673.2004.0390t.x · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glial cell line-derived neurotrophic factor (GDNF) has many functions including regulation of kidney morphogenesis and of neuron growth and survival in the enteric, sensory and central nervous systems. Reports of GDNF being used against Parkinson's disease in human patients have sparked intense clinical interest in GDNF signalling. We recently showed that GDNF signalling requires cell surface heparan sulphate glycosaminoglycans (Barnett et al., 2002, J. Cell Sci. 115, 4495-4503). Here we use exogenous modified heparins to determine those structural features required to inhibit GDNF signalling in ex vivo assays. 2-O-sulphate groups were found to impart high activity but were not absolute requirements for the inhibition of GDNF signalling. These findings may explain the similarities between the phenotypes of transgenic mice lacking GDNF and those lacking heparan sulphate 2-sulphotransferase, the enzyme responsible for achieving 2-O-sulphation of uronic acids in vivo.
[Show abstract][Hide abstract] ABSTRACT: The functions of the heparan sulfates (HSs) are determined by specific saccharide motifs within HS chains. These sequences
confer selective protein binding properties and the ability to modulate protein activities (1,2). HS chains consist of an alternating disaccharide repeat of glucosamine (GlcN; N-acetylated or N-sulfated) and uronic acid (glucuronic [GlcA] or iduronic acid [IdoA]). The initial biosynthetic product containing N-acetylglucosamine
(GlcNAc) and GlcA is modified by N-sulfation of the GlcN, ester (O)-sulfation (at positions 3 and 6 on the GlcN and position 2 on the uronic acids) and by epimerization of GlcA to IdoA. The
extent of these modifications is incomplete and their degree and distribution varies in HS between different cell types. In
HS chains N- and O-sulfated sugars are predominantly clustered in sequences of up to eight disaccharide units separated by N-acetyl-rich regions with relatively low sulfate content (3).
[Show abstract][Hide abstract] ABSTRACT: The functions of heparan sulfate (HS) are determined by specific saccharide motifs within HS chains. These sequences confer
selective protein-binding properties and the ability to modulate protein bioactivities (1,2). HS chains consist of an alternating disaccharide repeat of glucosamine (GlcN; N-acetylated or N-sulfated) and uronic acid
(glucuronic [GlcA] or iduronic acid [IdoA]). The initial biosynthetic product containing N-acetylglucosamine (GlcNAc) and
GlcA is modified by N-sulfation of the GlcN, ester (O)-sulfation (at positions 3 and 6 on the GlcN and at position 2 on the
uronic acids) and by epimerization of GlcA to IdoA. The extent of these modifications is incomplete, and their degree and
distribution varies in HS between different cell types. In HS chains, N- and O-sulfated sugars are predominantly clustered
in sequences of up to 8 disaccharides separated by N-acetyl-rich regions with a low sulfate content (3).
[Show abstract][Hide abstract] ABSTRACT: The sequencing of heparan sulfate oligosaccharides has recently become possible using integral Glycan Sequencing, which utilizes a combination of chemical and enzymatic degradation steps followed by polyacrylamide gel electrophoresis. This technique has previously employed the fluorescent label, anthranilic acid, and has been used to sequence low nmol amounts of purified saccharides. Here, we present an improved method, which uses the alternative label, 7-aminonapthalene-1,3-disulfonic acid, the reducing agent sodium triacetoxyborohydride and optimizes the nitrous acid step in heparin/heparan sulfate degradation. These improvements increase the sensitivity at least ten-fold taking the amount of starting material required into the pmol range. We show that this label is compatible with the integral glycan sequencing methodology and demonstrate its application to the sequencing of chemically modified heparin derivatives. Advances in sequencing techniques for heparan sulfate saccharides will permit detailed structure-function studies and will in the future underpin novel proteomics-based approaches aimed at studying their diverse functional roles as protein regulators.
[Show abstract][Hide abstract] ABSTRACT: Studies on the structure-function relationships of the complex linear polysaccha-rides, known as glycosaminoglycans (GAGs), are becoming increasingly important as biological functions are established for them. However, structural analysis of GAGs presents a difficult technical problem, particularly in the case of the N-sulfated GAGs heparan sulfate (HS) and heparin, which display remarkable structural diversity (1). A widely used and effective approach is to degrade the chains into smaller saccharide units that can then be separated and analyzed. In this regard, strong anion-exchange (SAX) HPLC techniques have proved particularly useful for both the analysis of disac-charide composition (2,3) and the separation of complex mixtures of larger saccharides (3–6). However, in many methods the columns used have been silica-based and suffer from drawbacks related to poor stability of the support (e.g., inconsistency of run times, peak broadening, and short column life). There is clearly a need for improvements in column performance, especially for the purification of larger saccharides to homogeneity for sequencing and bioactivity testing. This chapter describes how a single type of polymer-based SAX column, ProPac PA1, can be used to provide high-resolution separations of both disaccharides and larger oligosaccharides derived from HS and heparin, with consistent elution times and excellent column performance characteristics (see
Notes 1 and 2). Disaccharides from chondroitin sulfate and dermatan sulfate can also be separated (see
Note 3). The improved resolution of saccharides compared to other SAX-HPLC methods, combined with the versatility and longevity of the columns, makes them a valuable tool for purification and structural analysis of HS/heparin and other GAG saccharides.
[Show abstract][Hide abstract] ABSTRACT: Signalling by fibroblast growth factors (FGFs) through FGF receptors (FGFRs) depends on the cell-surface polysaccharide heparan sulphate (HS)  . HS has an ordered domain structure of highly diverse saccharide motifs that present unique displays of sulphate, carboxyl and hydroxyl groups . These motifs interact with many proteins, particularly growth factors. HS binds both to FGFs    and FGFRs , and probably activates signalling by facilitating ligand-induced receptor dimerisation  . Nevertheless, the extent to which specific HS saccharide sequences play a regulatory role has not been established. By screening a library of structurally diverse HS decasaccharides in bioassays of FGF signalling mediated by three different FGFR isoforms, we found that saccharides showed specificity for both ligands and receptors; some saccharides selectively activated FGF signalling through different FGFR isoforms, others acted as negative regulators. We conclude that HS saccharides play critical roles in dictating the specificity of ligand-receptor interactions in FGFR signalling. Controlled alterations in HS structures  would provide a mechanism for regulation of cellular responsiveness to growth factors that bind HS.
Current Biology 12/1999; 9(22):1343-6. DOI:10.1016/S0960-9822(00)80060-3 · 9.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and variable patterns of sulfation, especially in heparan sulfate (HS). HS and the structurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is accumulating that these interactions depend on specific saccharide sequences, but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress. We describe how HS and heparin saccharides can be sequenced rapidly by using an integrated strategy with chemical and enzymic steps. Attachment of a reducing-end fluorescent tag establishes a reading frame. Partial selective chemical cleavage at internal N-sulfoglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations of exosulfatases and exoglycosidases permits the selective removal of specific sulfates and monosaccharides from their nonreducing ends. PAGE of the products yields a pattern of fluorescent bands from which the saccharide sequence can be read directly. Data are presented on sequencing of heparin tetrasaccharides and hexasaccharides of known structure; these data show the accuracy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an HS decasaccharide of unknown structure. Application and further development of this sequencing strategy, called integral glycan sequencing, will accelerate progress in defining the structure-activity relationships of these complex GAGs and lead to important insights into their biological functions.
Proceedings of the National Academy of Sciences 04/1999; 96(6):2698-703. DOI:10.1073/pnas.96.6.2698 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction of heparan sulfate (HS) with basic fibroblast growth factor (bFGF) is influential in enabling the growth factor to bind to its cell surface tyrosine kinase receptor. In this study, we have investigated further the structural properties of HS required to mediate the activity of bFGF in a mitogenic assay. We have prepared a library of heparinase III-generated HS oligosaccharides fractionated by both their size (dp6-dp12) and sulfate content. The ability of these oligosaccharides to activate bFGF in a mitogenic assay was then correlated with their length and disaccharide composition. All octa- and hexasaccharide fractions tested were unable to activate bFGF. Dodeca- and decasaccharide fractions were found to contain both activating and non-activating oligosaccharides, and showed a clear correlation between total sulfate content and the level of activatory activity. Disaccharide analysis of a range of dodeca- and decasaccharide fractions showed that both activating and non-activating oligosaccharides were composed mainly of N-sulfated and IdoA(2S)-containing disaccharides. The only significant difference between activating and non-activating oligosaccharides was the content of 6-O-sulfated disaccharides, in particular the disaccharide IdoA(2S)alpha1,4GlcNSO3(6S). These results show that there is a requirement for 6-O-sulfation of N-sulfated glucosamine residues, in addition to the 2-O-sulfation of IdoA, for the promotion of bFGF mitogenic activity by naturally occurring HS oligosaccharides. Analysis of the structure-activity relationships in the dodecasaccharide fractions in particular, suggests that a minimum bFGF activation sequence exists which is dependent on the positioning of at least one 6-O-sulfate group.