Interactions of heparin/heparan sulfate with proteins: Appraisal of structural factors and experimental approaches

Andrew K. Powell1, Edwin A. Yates, David G. Fernig and Jeremy E. Turnbull

School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool, L69 7ZB, United Kingdom

Received on July 3, 2003; accepted on December 19, 2003


    Abstract
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Over the past decade, the glycosaminoglycans heparin and heparan sulfate have been shown to bind and regulate the activities of many proteins. Established techniques have provided both qualitative and quantitative information regarding these interactions, leading to a general view that proteins bind with a variety of affinities to particular sequences within heparin or heparan sulfate chains. The mechanism by which heparan sulfate regulates the activity of proteins through such interactions has, however, proved more elusive. We survey some relevant details of the structural characteristics of heparin/heparan sulfate and the approaches used to investigate their interactions with proteins. For the latter, the interactions of heparin/heparan sulfate with fibroblast growth factors and their receptors will be emphasized, because these proteins have been the subject of many studies. We reflect on the information that various techniques have provided, points regarding their use, and some relevant theoretical considerations regarding the study of protein–heparin/heparan sulfate interactions. A perspective of new and developing approaches, which may aid advances in this field, is also provided.

Key words: FGF / heparan sulfate / heparin / interaction / protein


    Introduction
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Heparin and heparan sulfate (HS) are negatively charged, polydisperse linear polysaccharides. They are composed of {alpha}1-4 linked disaccharide repeating units containing a uronic acid and an amino sugar. They share a common biosynthetic route, in which nonuniform modifications are made to the chains resulting in sequence diversity (reviewed in Esko and Lindahl, 2001Go). The uronic acid can be either ß D-glucuronic acid (GlcA) or its C-5 epimer {alpha} L-iduronic acid (IdoA), and these can be sulfated at position 2 (see Figure 1). The amino sugar is a D-glucosamine, as either N-deoxyacetamido (GlcNAc) or N-deoxysulfonamido (GlcNS), or, as some evidence suggests (Born et al., 1996Go), it may remain unsubstituted. Furthermore, glucosamine derivatives can be O-sulfated at position 6 and, more rarely, at position 3 (see Figure 1).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1. Monosaccharide units of heparin/HS chains.

 
Heparin and HS have been shown to bind and regulate the activities of many proteins, including enzymes, growth factors, extracellular matrix (ECM) proteins, and the cell surface proteins of pathogens (reviewed in Bernfield et al., 1999Go; Rostand and Esko, 1997Go). Genetic studies have recently shown that these properties reflect its ability to regulate biological processes, such as cell signaling and morphogenesis in vivo (reviewed in Lander and Selleck, 2000Go; Perrimon and Bernfield, 2000Go; Selleck, 2000Go). Several previous reviews have focused on the relationship between HS structure and activity (Capila and Linhardt, 2002Go; Casu and Lindahl, 2001Go; Conrad, 1998Go; Esko, 2001Go; Esko and Lindahl, 2001Go; Esko and Selleck, 2002Go; Park et al., 2000Go). This review focuses primarily on the approaches used to investigate the interactions of heparin/HS with proteins, including recent publications on developments in this arena. It reflects in depth on the information that various techniques have provided, points regarding their use, relevant details of the structural characteristics of heparin/HS, and theoretical considerations regarding the study of protein-heparin/HS interactions.


    Structural characteristics of heparin/HS
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Heparin is sometimes considered to be synonymous with HS, but this is an oversimplification. Heparin is found in vivo in the granules of connective tissue mast cells, whereas HS is found on the cell surface or in the ECM, covalently attached to several different core proteins forming heparan sulfate proteoglycans (HSPGs) (reviewed in Bernfield et al., 1999Go; Conrad, 1998Go). They have been shown to differ in their degree of sulfation, with heparin displaying higher N- and O-sulfation than HS (Gallagher and Walker, 1985Go). The high sulfation of liver HS (Lyon et al., 1994Go) and the isolation of HS resembling heparin from oligodendrocyte-type-2 astrocyte progenitor cells (Stringer et al., 1999Go), however, suggest that the two families of molecules may not be distinct in this respect and even that there may be a continuum of sulfation with heparin lying toward one end. Characterization of HS from cell lines has indicated that HS chains (unlike those of heparin) exhibit a domain structure: Regions of little or no sulfation (termed NA domains), consisting largely of GlcA-GlcNAc repeats, are interspersed with relatively small, more highly sulfated regions (termed S- or NS-domains), consisting largely of IdoA-GlcNS, and intervening mixed regions (termed NA/NS domains) (Figure 2) (Turnbull and Gallagher, 1991Go; reviewed in Lindahl et al., 1998Go). Importantly, both heparin and HS contain a wide range of sequences and, in addition, vary considerably in composition and sequence between sources. For HS, further diversity is seen in the overall chain organization with varying arrangements of S-domains (Brickman et al., 1998Go; Kreuger et al., 2002Go; Lyon et al., 1994Go; Stringer et al., 1999Go). The level of diversity found within HS so far is, however, only a small fraction of that theoretically possible if all combinations of known monosaccharides are considered, reflecting the substrate specificity and order of action of the biosynthetic enzymes.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Multidomain structure of HS. Schematic illustrating the three types of domains found in HS chains. Diversity is found within S-domains and potentially greater diversity within mixed (NA/NS) domains. Open squares, ClcNAc; open circles, GlcA; black squares, GlcNS; shaded large circles, IdoA; shaded small circles, 6-O-sulfate; black circles, 2-O-sulfate; black bars, IdoA-rich sequences.

 
Most work concerning the physicochemical characteristics of these molecules has been conducted on heparin, largely as a result of its abundance, commercial availability, use as an anticoagulant, and perceived simplicity. Importantly, it is now well established that the IdoA residue, when bearing a sulfate group at position 2, exists in equilibrium between a number of different conformations, the most important being the chair (1C4) and skew-boat (2S0) forms (Figure 3). Unsubstituted IdoA lacks this property and resides predominantly in the 1C4 chair form. In contrast, glucosamine and GlcA derivatives are stable in the 4C1 chair form (Figure 3) irrespective of substitution (Desai et al., 1993Go; Ferro et al., 1986Go; Yates et al., 1996Go). In addition, the sequence can influence the population of conformers (Ferro et al., 1986Go), and it has been suggested that substitution pattern may influence the conformation around the glycosidic linkages (Yates et al., 2000Go). These findings suggest that there are subtle, but important, conformational effects between residues in these chains.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 3. Conformations of iduronate, glucosamine, and glucuronic acid derivatives.

 
The dynamics of heparin have been investigated using nuclear magnetic resonance (NMR) (also reviewed in Mulloy and Forster, 2000Go; Capila and Linhardt, 2002Go). Early work (Mulloy et al., 1993Go) was undertaken employing a relatively simple mathematical model, in which the possibility of internal motion was not included. Furthermore, a lack of available knowledge concerning parameters, such as the distribution of charges on sulfate groups, meant that these factors could not be easily incorporated. The resultant model of heparin describes a relatively rigid molecule with clusters of sulfate groups distributed along the chain. In contrast, later work on a related compound, a chemically modified (epoxylated) heparin, concluded that these sequences were relatively flexible compared to a model compound (GlcA-GlcNAc repeating polysaccharide from Escherichia coli polysaccharide K5) representing the biosynthetic precursor of HS, heparosan (Hricovini et al., 1995Go, 1997Go). The degree of rigidity or flexibility present within a chain may have a significant impact on its behavior with any binding protein, although this is a difficult problem to tackle experimentally. In any case, the extreme descriptions flexible or rigid may not be appropriate because these molecules probably reside in the middle ground. It is also possible that the degree of flexibility within a single chain could vary considerably along the chain depending on the local sequence. The surrounding solvent water has been shown to have a profound effect on the conformation of hyaluronan and other polysaccharides (Almond and Sheehan, 2003Go) and it may also be an important factor in this case.

These long, semiflexible polyanions could have self- ordering properties at high concentrations (for example at the cell surface), but this aspect of HS has scarcely been investigated. The influence of bound cations (especially divalent) on the conformation of heparin/HS (Ayotte and Perlin, 1986Go; Rabenstein et al., 1995Go) and hence activity (Koenig et al., 1998Go), would also be an interesting field for further study.


    Approaches for qualitative analysis of the interactions of heparin/HS polysaccharides with proteins
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
An initial approach to investigate the structure–activity relationship of heparin/HS uses chemically modified derivatives of heparin. In these, one or more of the prevalent substitutions; (N-acetamido, N-sulfonamido, O-sulfoxy) is selectively undertaken by chemical treatment (more rarely, this may include carboxyl: when reduced to hydroxyl) to produce polysaccharides that are simpler than the parent. A correlation between a change in activity or binding and the structural modification has often been found (Belford et al., 1992Go; Guimond et al., 1993Go; Maccarana et al., 1993Go; Powell et al., 2002Go). Alternatively, a series of graded modifications can be employed and a trend in activity sought (Irie et al., 2002Go). If the compounds have been characterized carefully, typically by NMR (Yates et al., 1996Go) or exhaustive composition analysis (Linhardt et al., 1990Go), this approach can be used as an indicator of the involvement of partiular groups. However, caution should be exercised and the results not overinterpreted. Modifying the constituent monosaccharides can result in other changes, for example in chemical structure (Santini et al., 1997Go) or conformation, making it difficult to conclude definitively that a particular group is directly involved. It should also be noted that chemical re-O-sulfation of a desulfated HS analog or biosynthetic precursor is not possible with absolute selectivity.

Recently, a new strategy for investigating the role of functional groups of heparin/HS in protein interactions has been developed (Wu et al., 2002Go). Sulfates are added in vitro at specific positions to chemically modified heparins, using biosynthetic enzymes and a radiolabeled sulfate donor, and the effect of the modification on the ability of the carbohydrate to interact with proteins is determined using a polyacrylamide gel mobility shift assay (GMSA). This allows a correlation between protein binding and the specific sulfate groups added to be made (Wu et al., 2002Go, 2003Go). The ability of the strategy to distinguish between binary and ternary complexes also enables information regarding ternary complex formation to be obtained (Wu et al., 2003Go). Such an approach will extend the applications of chemically modified heparin substantially. However, an indirect effect of modification on conformation should always be considered. It will also be informative to discover the structural requirements of the substrate for these enzymes because this will determine the structures that are made. Both approaches have given important leads into the mechanism by which HS regulates protein activity (Guimond et al., 1993Go; Wu et al., 2003Go).


    Methods for qualitative analysis of the interactions of proteins with heparin/HS oligosaccharides
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
The multisite nature of full-length heparin/HS chains (polysaccharides) renders characterization of the interactions difficult, and interpretation of results must be cautious. Another widely used approach employs oligosaccharides obtained from chains by enzymatic or chemical cleavage. This enables the minimum size of heparin and HS sequences required for binding and activity to be investigated. Oligosaccharides can also be used to investigate the specificity of protein–HS interactions by determining the disaccharide composition or, more recently, the sequences of oligosaccharides that bind or activate particular proteins.

Oligosaccharide production
Production of oligosaccharides from heparin/HS chains utilizes partial chemical and/or enzymatic cleavage. The first occurs via modification of unsubstituted glucosamine or GlcNS residues by nitrous acid to form pair-wise oligosaccharides with 2,5 anhydromannose derivatives at the reducing end (Figure 4) (Guo and Conrad, 1989Go; Shively and Conrad, 1976Go). The useful property of increased reactivity toward nucleophiles at the reducing end anhydromannose residues is offset by the difficulty of detection because, in common with many carbohydrates, these saccharides lack a strong chromophore. Hence the investigator faces the choice of labeling the reducing end (for example, with fluorophores) to enhance the sensitivity of detection or to work with limited detection sensitivity and exploit the reactivity of the reducing end in other ways (for example, with saccharide immobilization). The second common degradative technique, employing bacterially derived lyase enzymes (Linhardt et al., 1990Go), produces pair-wise oligosaccharides with reducing ends that are intact but less reactive toward nucleophiles. The nonreducing end is modified to a {Delta} 4, 5-unsaturated uronic acid derivative (Figure 4). This has the advantage of being a chromophore (absorbance maximum of approximately 232 nm with a molar extinction coefficient of 5500 M–1cm–1; Linhardt et al., 1988Go), but the disadvantage of having lost its identity: both GlcA and IdoA degrade to a common structure. Both major degradative methods therefore result in unnatural terminal residues being produced.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Schematic showing the results of degradation of heparin/HS by two methods; (left) nitrous acid, results in oligosaccharides terminating in reactive anhydromannose derivatives and (right) heparitinase enzyme digestion results in oligosaccharides bearing an unsaturated bond at their nonreducing termini. In this case, D-glucuronic acid and L-iduronic acid both degrade to a common structure.

 
Neither the nitrous acid nor lyase methods produces unbiased oligosaccharide sets, and this limits the sequence diversity (sometimes called sequence space) attainable. One alternative method, which is rarely used, is that of free radical degradation. This can be performed with hydrogen peroxide mediated by Cu+ ions (Liu and Perlin, 1994Go) and produces fragments of any length rather than just even-numbered oligosaccharide repeats. If difficulties in separating such complex mixtures can be overcome, this could yield most structural information because the oligosasccharide sets produced contain low levels of structural bias.

Most of the studies characterizing the sequences in HS mediating binding to proteins have used tissue-derived HS. However, more recently, methodologies have been developed to enable HS sequences to be engineered in vitro. These have the advantage of expanding the amount of HS sequence space available for several reasons. First, potentially interesting sequences may be of low abundance in tissue-derived HS and are therefore not detected, in particular when using binding assays. In vitro library generation provides the opportunity of producing such sequences in greater abundance. Second, the overall sequence space available from tissue-derived HS is limited by cellular control of the biosynthesis process in vivo. The methods developed for engineering sequence diversity in vitro remove this control.

One strategy uses biosynthetic enzymes to modify templates, such as desulfated heparin or K5 capsular polysaccharide (N-acetylheparosan). For example, a modification procedure created diversity in the O-sulfation pattern of chemically O-desulfated heparin oligosaccharides using O-sulfotransferases (Jemth et al., 2002Go). Modification of heparin, HS, or K5 polysaccharide has also been achieved using other recombinant sulfotransferases (Wu et al., 2002Go), endosulfatases (Ai et al., 2003Go), and epimerase (Naggi et al., 2001Go), suggesting that these enzymes could be used in combination to create diverse oligosaccharide libraries. However, the degree of sequence space available may be restricted by the specificity of the enzymatic reactions used in each case and the ability to control them in vitro. Another methodology being developed enables diversity to be expanded in relation to all the major modifications found naturally (2-O, 6-O sulfation and N-sulfation/acetylation), through a combined partial desulfation strategy using heparin or HS as the starting template (Yates et al., 2004Go). An advantage of the methods of engineering sequences in vitro is that they overcome the obstacle of purifying sufficient quantities of bioactive sequences from tissue-derived HS for structural studies by using heparin or K5 as the starting material. However, for this purpose, the use of enzymatic methodology may be limited by availability and cost. Although these approaches may lead to the identification of HS sequences with higher affinity or bioactivity or identify active saccharides that approach minimum complexity, it will be important to confirm their occurrence in vivo by, for example, using sequence-specific phage display anti-HS antibodies whose epitopes are currently being characterized (Dennissen et al., 2002Go).

Chemical synthesis of HS oligosaccharides has also been achieved (Grootenhuis et al., 1995Go) but is a lengthy and specialized process best applied to a known structure. As the size of the oligosaccharides increases, the yield diminishes with each coupling step. Some improvements in both efficiency and the number of structures that can be made will stem from modern approaches to solid-phase synthesis and combinatorial chemistry (Seeberger and Haase, 2000Go) and a modular approach for synthesis of diverse HS saccharides has recently emerged (Prabhu et al., 2003Go). A further problem with this class of molecule, caused by the presence of both acid- and base-sensitive functional groups, is the relatively narrow range of conditions to which they can be exposed during the synthetic process. It is therefore likely to be a number of years before significant numbers of oligosaccharides can be generated by chemical synthetic approaches.

Similar to the strategy for engineering HS oligosaccharide libraries, in vitro synthesis of custom HS oligosaccharides could potentially be achieved using batteries of recombinant biosynthetic enzymes. Enzymatic elongation of K5 has been achieved using recombinant EXT proteins (glucuronyl/N-acetylglucosaminyltransferases), either alone (EXT1) or as a hetero-complex (EXT1/2) (Busse and Kusche-Gullberg, 2003Go), suggesting the possibility of synthesizing a template oligosaccharide for modification by epimerases, sulfotransferases, and endosulfatases. Continued characterization of the specificities of these enzymes, their tendency and requirements for forming macromolecular complexes (Pinhal et al., 2001Go), and the identification of methods for their regulation may provide sufficient knowledge to enable specific oligosaccharide sequences to be produced in vitro. Furthermore it is likely that creation of a repeat GlcA-GlcNAc oligosaccharide scaffold for further modification will require elongation of a primer sequence, hence controlled targeted cleavage of the primer from the custom sequence will also need to be developed.

An alternative approach may be to exploit cell lines overexpressing particular biosynthetic enzymes to enrich the expression of HS with particular structural features (Cheung et al., 1996Go; Liu et al., 1999Go; Pikas et al., 2000Go; Rong et al., 2001Go). Such a strategy would probably be limited by the need to purify oligosaccharides from the HS produced by the cell lines.

Screening
To investigate the relationship between structure and function, the relative abilities of oligosaccharides (produced by the methods described) to bind proteins of interest is determined. One method of characterizing protein binding sites within HS is to fractionate mixtures of oligosaccharides of similar hydrodynamic volume by the different ionic strength required for their elution from proteins (Kreuger et al., 1999Go, 2001Go; Loo et al., 2001Go; Turnbull et al., 1992Go). An alternative is to create oligosaccharide libraries by further prefractionating oligosaccharide mixtures and then assaying these fractions for activity (Guimond and Turnbull, 1999Go; Pye et al., 2000Go), or for protein binding.

Fractionation of oligosaccharide mixtures over proteins evaluates the ability of the oligosaccharides to bind the protein among many other competitor saccharides. This mixture fractionation approach may better reflect the in vivo situation in which the protein is, presumably, exposed to a mixture of sequences within the chains of HSPGs expressed in the ECM or on the surface of cells. A caveat is that these sequences, when analyzed as oligosaccharides, may not accurately reflect their behavior when placed in the context of an HS chain or proteoglycan at the cell surface (see later discussion). In contrast, assaying library fractions compares the intrinsic properties of each fraction of the library free of other potentially interfering interactions. Spacing considerations, which are frequently overlooked, may also be critical in providing high-affinity interactions between HS on the cell surface and a binding partner.

Recent developments in mixture fractionation have aimed at miniaturization. With surface noncovalent affinity mass spectrometry (SNA-MS), target proteins are adsorbed onto MS targets, oligosaccharides bound in situ and eluted using increased ionic strength (i.e., NaCl) wash discrimination steps, and the structures of eluting oligosaccharides immediately determined using matrix-assisted laser desorption ionization (MALDI) MS (Keiser et al., 2001Go). In this procedure, the amount of protein and oligosaccharide required is reduced to low picomole levels. Screening of oligosaccharide libraries for protein binding would also benefit from miniaturization. An approach has recently been developed for direct immobilization of HS oligosaccharides to glass slides exploiting microwave-enhanced reactions (Yates et al., 2003Go) and it should be possible to accurately interrogate these arrays using protein probes. Such oligosaccharide microarrays would enable the rapid parallel screening of libraries against multiple proteins. However, both these miniturization approaches may possess limitations. SNA-MS may be limited by the need for the protein to be actively immobilized on the target, either directly or via a label that is bound by an immobilized protein; immobilization and labeling of proteins often affects activity, and this must be considered. Similarly, surface display of oligosaccharides as, for example, microarrays may be limited by the tendency of proteins to bind to surfaces leading to high background binding for particular proteins. In certain cases, traditional filter trap methodology (Kett et al., 2003Go; Maccarana et al., 1993Go) may be preferred at the expense of miniaturization.

A common consideration for these methods is whether they select oligosaccharides directly based on their affinity for a particular protein. It is likely that selection of oligosaccharides through fractionation of mixtures over protein columns, or by filter trapping, will be dependent on the abundance of each oligosaccharide in the mixture. Moreover the fractionation of mixtures on the basis of the ionic strength required for elution may bias selection toward the ionic component of the interaction. The relative ionic strength required for disruption of protein–glycosaminoglycan (GAG) interactions has been interpreted to represent their relative affinities due to a correlation observed between the increasing equilibrium dissociation constant (Kd) values measured by isothermal calorimetry (ITC) for fibroblast growth factor (FGF) 2 mutants and the decreasing ionic strength required to elute the same mutant protein from a heparin affinity column (Thompson et al., 1994Go). However, deviations between the relative ionic strength elution of proteins from heparin-sepharose and direct affinity measurements have been observed (Lookene et al., 2000Go; Thompson et al., 1994Go), suggesting that differential ionic strength elution of oligosaccharides may not reflect selection based on their true relative affinities. Furthermore, the direct immobilization of proteins onto surfaces such as columns or filters may cause protein unfolding, and the concentrations of protein immobilized are often high; hence these techniques may bias the selection in other ways (Delehedde et al., 2001Go). Recent data dispute that ionic strength elution biases selection toward the ionic component of the interaction: HS oligosaccharides eluting at lower ionic strength from FGF1 and FGF2 affinity columns possessed a higher charge than some of those eluting with a higher concentration of NaCl (Kreuger et al., 2001Go) and a similar result was seen using SNA-MS (Keiser et al., 2001Go). However, it should also be noted that the presence of a number of charged groups does not necessarily implicate them all in the interaction. Interestingly, biosensor studies, which enable the immobilization level of streptavidin capture protein, hence captured biotinylated oligosaccharide, to be carefully controlled and monitored, identified a different minimum size of HS binding site than earlier studies using filter trap methodology (Delehedde et al., 2002aGo).

Screening the ability of library fractions to bind proteins may be best achieved by their immobilization on surfaces. One consideration is that the structure of the different oligosaccharides may effect the efficiency of their immobilization. Therefore, for the level of protein binding to the surface to be related to the affinity of the interaction, the level of immobilization for each library fraction must also be quantifiable. Alternatively, the fractions could be ranked on the basis of relative affinity if the level of immobilization is normalized.


    Structure–function studies of the interactions of proteins with HS/heparin oligosaccharides
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Information regarding the structural specificity of protein–HS interactions has been afforded by technical developments in the structural analysis of HS oligosaccharides, mutational analysis of protein HS-binding sites, molecular modeling, and, recently, crystal or NMR structures of protein–HS complexes.

Over the past few years a number of new strategies for the direct sequencing of HS oligosaccharides have emerged, augmenting the traditional structural characterization of HS, which relied on NMR spectroscopy (requiring milligrams of sample) or indirect analysis involving enzymatic and chemical degradation methods. These methods include integral glycan sequencing, using fluorescent or radiolabel end-tagging, exoenzyme digestions, and electrophoresis (Merry et al., 1999Go; Turnbull et al., 1999Go) or high-performance liquid chromatography (HPLC) separations (Vives et al., 1999Go) to provide direct sequence information; the sequencing of biosynthetically radiolabeled HS oligosaccharides using a step-sequencing HPLC method employing exoenzyme digestions (Merry et al., 1999Go); and MALDI-MS techniques for the mass measurement of HS oligosaccharides (Rhomberg et al., 1998Go), which in combination with specific chemical and enzymatic cleavage steps have been used for sequencing oligosaccharides (Shriver et al., 2000Go; Venkataraman et al., 1999Go). There have also been significant advances in the development of electrospray MS-based methods for separation of sulfated carbohydrates (Gunay et al., 2003Go; Hileman et al., 1998Go; Kuberan et al., 2002Go; Pope et al., 2001Go). The masses of oligosaccharides can be determined using these MS methods, which require small amounts of material and have the significant advantage of yielding accurate mass data, which provide an internal check on the validity of the sequence data.

Information regarding the topology of heparin–protein interactions has been obtained using molecular modeling, exploiting the crystal structure of the proteins involved (Raman et al., 2003Go; Sadir et al., 2001Go; Vives et al., 2002Go). A common modeling strategy used is to identify surface exposed residues on the protein and, from these, candidate binding-site residues (often those associated with sulfate ions in the crystal structure), model heparin oligosaccharides (built using NMR data for heparin, the coordinates of monosaccharide derived from crystal structures or more recently, known cocrystal structures) and to dock these oligosaccharides onto the protein to find the model with the minimum interaction energy. The effect of site-directed mutagenesis of residues implicated by such modeling studies provides information regarding the binding site for heparin/HS on the protein surface (Thompson et al., 1994Go).

Recently, crystal structures of binary or ternary oligosaccharide–protein complexes have begun to provide information concerning the precise geometry of the interactions (DiGabriele et al., 1998Go; Faham et al., 1996Go; Pellegrini, 2001Go; Pellegrini et al., 2000Go; Schlessinger et al., 2000Go). In general these studies agree with others regarding the identity of the amino acids and sulfates involved in the interactions. However, in such studies the structural details of the carbohydrate moieties can be difficult to ascertain, hence it is often necessary to employ a model of heparin in subsequent structural refinement. For the FGF system, only one structure has been determined with sufficient resolution to allow direct identification of the structural details of the glycan (Faham et al., 1996Go). Moreover, this structure (Faham et al., 1996Go) agrees with biochemical data regarding minimum size requirements (Delehedde et al., 2002aGo). An advance in this area would be the ability to select single HS species with known ability to activate through particular FGF/FGF receptor (FGFR) combinations. Interestingly, NMR studies of the dynamics of the conformation of heparin oligosaccharides binding to antithrombin III and FGFs suggest that the proposed structure of oligosaccharides complexed with proteins may be different to that of the oligosaccharide in solution (Hricovini et al., 1999Go, 2002Go). If this also applies to polysaccharides, this could have important consequences for the thermodynamics and hence affinity of these interactions (see later discussion).


    Approaches for quantitative analysis of the interactions of heparin/HS polysaccharides with proteins
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Interactions between heparin/HS and proteins have been characterized quantitatively using a number of techniques, including trapping and quantifying HS–protein complexes on surfaces, affinity coelectrophoresis (ACE), optical biosensors, and ITC, which are discussed here and summarized in Table I.


View this table:
[in this window]
[in a new window]
 
Table I. Techniques used to investigate the interactions of heparin/HS with proteins

 
Filter trapping (Loo et al., 2001Go; Sasaki et al., 1999Go) and immunoprecipitation (Kan et al., 1996Go; Wang et al., 1995Go; LaRochelle et al., 1999Go) have been used to separate heparin/HS–protein complexes from free molecules. The quantity of complex formed at equilibrium is determined through the use of labeled heparin/HS. Labeled heparin/HS species are also used in ACE, in which they are subjected to electrophoresis through agarose gel lanes containing a protein at various concentrations and the shifts in migration of the labeled materials determined. Optical biosensors detect a change in refractive index on complex formation at the surface where one of the partners is immobilized (Schuck, 1997Go). Using this technique, the binding of soluble heparin/HS to immobilized proteins has been observed (Rathore et al., 2001Go). However, this is often difficult because GAGs such as heparin and HS, in common with most naturally occurring carbohydrates, have low refractive indices, resulting in a small signal on a mass basis (Fernig, 2001Go). Furthermore the refractive index of GAGs may vary with the degree of sulfation (Fernig, 2001Go), an effect that may have its origin in complex conformational changes. As a result, it is not known whether the different chains present in heparin/HS give comparable signals on binding an immobilized protein, which may affect the quantitative analysis of the interaction. The use of GAGs as the soluble partner in biosensor experiments, unless quantitatively conjugated to a compound with a dominant refractive index, may therefore be problematic. Consequently, the common approach of immobilizing heparin/HS on the sensor surface is probably more appropriate. This requires labeling of the GAG to facilitate affinity capture via an immobilized partner because methods for efficient direct immobilization of unlabeled GAGs have yet to be developed.

Heparin and HS can be labeled either at the reducing end or along the chains. The former utilizes the attack of the carbonyl group by nucleophilic labels. The efficiency of labeling therefore is dependent on the presence of the reducing end carbonyl functional group, and this depends on the mode of preparation. The reducing end of untreated heparin/HS chains exists in equilibrium between the open and closed forms firmly in favor of the closed form (Figure 5), often resulting in low efficiency of labeling. Selective labeling of the free amino groups of unmodified glucosamine residues using 3H-acetic anhydride (Hook et al., 1982Go) or NHS ester derivatives (Lee and Conrad, 1984Go; Norgard-Sumnicht and Varki, 1995Go) has been extensively used for heparin chains. However, such residues are of low abundance, and tritium is an isotope of relatively low radioactivity. Furthermore, introducing bulky labels (such as LC-LC-biotin) may disrupt or alter binding sites, either by steric hindrance or by altering the conformation of sequences in proximity to the label. This may explain the observation that proteins bind with different properties to heparins biotinylated using different methods (Osmond et al., 2002Go). Careful control and quantification of the labeling may therefore be crucial.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 5. Conformations of the reducing end monosaccharide of heparin/HS chains.

 
To analyze the interactions quantitatively, a mathematical model is applied. The level of nonspecific binding is often determined to enable calculation of the level of specific binding for use in the models. However, common approaches for calculating nonspecific binding are probably inaccurate (Mendel and Mendel, 1985Go; van Zoelen, 1989Go). Instead, it may be more appropriate to consider nonspecific binding as consisting of interactions with different affinities to those of interest and to analyze data using nonlinear regression with equations incorporating terms for specific and nonspecific binding as separate interactions (Mendel and Mendel, 1985Go). The accuracy of quantitative analysis of molecular interactions is also influenced by the mathematical model employed to describe them. For example, the simplest way to describe an interaction between two binding entities is obviously a 1:1 complex. However, applying this simple one-site model to heparin/HS chains requires care because of the nature of heparin/HS chains. First, the chains probably possess multiple heterogeneous binding sites exhibiting different kinetics and affinities. This innate heterogeneity may be enhanced by any labeling procedure as a result of nonuniform labeling. Second, individual HS chains, and in particular the more uniformly highly sulfated heparin chains, are likely to possess multiple protein binding sites, which may overlap. As a result, proximal binding sites may behave differently resulting in cooperativity in protein binding (McGhee and von Hippel, 1974Go). For example, the binding partners at one site may reorganize by sliding, or dissociation/reassociation, to reveal additional binding sites. Binding to these will appear different to the initial interactions because of the rearrangement processes involved. Finally, like other carbohydrate chains (Edwards et al., 1995Go) HS may be flexible, resulting in variable accessibility to proteins. These properties of heparin/HS chains suggest a requirement for the use of multisite models to describe their interactions with proteins.

Despite the difficulties associated with quantitatively analyzing the interactions of heparin/HS chains with proteins using a one-site model, there are many examples in the literature of such a procedure being used to determine affinities (Kd values) from concentration-response data. However, the accuracy of such values may be limited if they are derived directly from linear transformation of the model or by applying the model to data producing nonlinear Scatchard graphs (Leatherbarrow, 1990Go; Munson and Rodbard, 1980Go). Also, it is frequently difficult to assess the validity of the use of a one-site model because the semilogarithmic graphs required to prove saturability (Klotz, 1982Go, 1985Go) are omitted.

Several studies have quantified the rate constants as well as the affinities of interactions between proteins and heparin or HS chains using optical biosensors. In many, careful experimental design (O'Shannessy and Winzor, 1996Go; Schuck, 1997Go) resulted in kinetically homogeneous binding of proteins to heparin or HS chains, overcoming the problems associated with the heterogeneous and multidentate nature of the chains (Amara et al., 1999Go; Powell et al., 2002Go; Rahmoune et al., 1998Go). The consistency of affinity values determined by alternative methods (Schuck and Minton, 1996Go) in some of these studies helped confirm the validity of the results. However, the experimental precautions employed to avoid artifacts may mean that the rate constants calculated by such studies describe the binding of proteins to sites within the heparin/HS chains that possess the fastest association and dissociation rate constants. These may not be characteristics of the same sequence. Other reports commented that the interaction between proteins and heparin or HS chains could not be characterized by a single-site-binding model and hence were not kinetically homogeneous (Rux et al., 2002Go; Vives et al., 2002Go). This has also been observed when using higher concentrations of protein binding to the immobilized heparin/HS (Lookene et al., 2000Go; Powell et al., 2002Go). The ability to characterize the interaction using single-site models is probably protein dependent and may be less possible with HS because of the greater heterogeneity of the chains. Deriving kinetic rate constants using multisite-binding models has been achieved (Rahmoune et al., 1998Go), but careful analysis is required to be able to conclude with confidence that artifacts are not causing the heterogeneous kinetics.

ITC has also been used for the quantitative analysis of the interaction of proteins with low-molecular-weight heparin (Pantoliano et al., 1994Go; Thompson et al., 1994Go). ITC monitors an interaction between two unlabeled molecules in solution from the heat change that occurs, and hence does not require labeling of either molecule. Titration of one of the molecules into a solution of the other enables Kd values and thermodynamic parameters of interactions all to be calculated directly by applying single- or multisite models to the binding isotherms produced from the data of a single experiment. The calculation of affinities directly from the data is, however, currently limited to interactions with Kd values greater than 10–8–10–9 M (Bundle and Sigurskjold, 1994; Pierce et al., 1999Go; Wiseman et al., 1989Go). Interactions of higher affinity may be open to characterization using displacement titration calorimetry in which a ligand of lower affinity competitively inhibits one of higher affinity (Sigurskjold, 2000Go).

ITC monitors interactions between molecules in solution, similar to filter trap and immunoprecipitation methodology, which only use surfaces to capture preexisting complexes. Optical biosensors measure interactions between molecules where one is in solution and the other immobilized on a surface. Such immobilization will reduce the degrees of freedom of one of the interacting molecules and as a result may affect the kinetics and affinity of the interaction. Values for the affinities of heparin/HS–protein interactions may therefore vary depending on the technique used. Immobilization may be more representative of the in vivo situation where HS chains are covalently attached to core proteins as HSPGs.


    Methods for quantitative analysis of the interaction of proteins with HS oligosaccharides
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Quantitative analysis of the interaction of proteins with heparin oligosaccharides using ITC has been reported (Capila et al., 1999Go; Dong et al., 2001Go; Fath et al., 1998Go). Recently an optical biosensor has been used to characterize the interaction of growth factors with reducing-end-biotinylated heparin oligosaccharides, enabling kinetics as well as affinities to be calculated (Delehedde et al., 2002aGo,bGo). Such information was obtained by the application of mathematical models, as described. In these studies a one-site-binding model could be applied easily, possibly reflecting the unidentate nature of oligosaccharides. The use of oligosaccharides characterized as being the minimal length for adequate binding to a particular protein may enable interactions that display kinetically heterogeneous binding to heparin/HS chains to be characterized quantitatively (although considerations regarding the use of oligosaccharides and chains should be noted; see later discussion). The current development of array optical biosensors will facilitate rapid screening of oligosaccharide–protein interactions based on affinity or kinetic characteristics.

The relative apparent affinities of the interaction between proteins and oligosaccharides can also be determined via competition assays with immobilized heparin (Delehedde et al., 2002aGo,bGo). The observation that different methods of immobilizing heparin can affect the affinity of interactions (Osmond et al., 2002Go) and that proteins often bind with heterogeneous kinetics to heparin chains (see earlier discussion) may make the use of a pure heparin oligosaccharide immobilized via reducing-end biotinylation/streptavidin more appropriate. Such an approach may provide a simple automated assay for screening HS oligosaccharide libraries and their ranking based on their effectiveness as competitors.


    Appraisal of heparin/HS–protein interaction studies and implications for elucidation of mechanisms
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
The methodologies have provided a wealth of information regarding the structure–function relationships of HS and heparin. However, as exemplified by the FGF system, the mechanism by which HS regulates protein function often remains elusive, and this may partly reflect the difficulty of gaining an understanding of the physicochemical properties of HS. Many studies of HS binding sites in FGFs and FGFRs have indicated that the sites contain stretches of GlcNS and a high proportion of IdoA. The predominance of highly sulfated sequences isolated by binding assays may reflect bias of the methods used to prepare and fractionate oligosaccharides, as already discussed. In HS, the regions bordering NA- and S-domains appear on the basis of simple combinatorial possibilities to be those most varied in structure but have rarely been the focus of attention. The investigation of these sequences, accessed by producing HS oligosaccharides using subtle partial heparitinase cleavage or alternative cleavage methods, in binding assays may well provide an alternative picture of the structural features involved. Interestingly, recent results investigating the activity of oligosaccharide fractions suggest that active sequences often exhibit very different structural motifs to those mentioned; examples include sequences rich in GlcNAc (some exhibiting higher activities than heparin itself) (Guimond and Turnbull, 1999Go) and bacterial K5 sequences, containing a high proportion of GlcA instead of IdoA (Leali et al., 2001Go). Importantly, to our knowledge, HS sequences isolated on their binding properties have not been shown to possess activity in signaling or mitogenesis assays.

The activity of oligosaccharides may reflect their interactions with both FGFs and FGFRs to form a ternary complex, whereas binding assays reflect an interaction with a single protein, hence comparison of HS sequences that bind to different FGFs and FGFRs may not be fully informative regarding how HS regulates the activity of FGFs. The combination of enzymatic sulfation of chemically desulfated heparins and GMSA showed that specifically modified heparins that were active in bioassays could facilitate ternary complex formation but could not bind to FGFs or FGFRs alone (Wu et al., 2003Go). The use of GMSA or similar assays, in conjunction with oligosaccharide libraries, would enable the correlation of the ability of oligosaccharides to activate FGF2 signaling with their ability to promote ternary complex formation.

Similarly, filter trap, affinity chromatography, and activity assays have been used to characterize the minimum size requirement for binding and activity. Until recently it was generally held that binding to FGFs required oligosaccharides of 4–6mer length, whereas activation required larger fragments (8–10mers). However, the demonstration that 4mer heparin-derived oligosaccharides can bind FGF2 with high affinity, activate sustained intracellular signaling and DNA synthesis (the latter albeit with a lower potency than 10mer and larger oligosaccharides) (Delehedde et al., 2002aGo), and promote formation of a ternary complex (Wu et al., 2003Go) questions this conclusion. The length dependence observed by the different studies may be influenced by the method of generating the oligosaccharides. The importance of rigorously purifying oligosaccharides by several methods selecting on the degree of polymerization, and demonstrating that larger contaminating oligosaccharides are not present in sufficient quantities to account for this activity must also be stressed.

There has been a tendency to characterize interactions from a simple perspective in terms of the sulfates required rather than the conformation adopted by each sequence. Attempts at structural elucidation of protein–oligosaccharide complexes may provide this information in the future but to date have depended on the application of models of oligosaccharide structure. The crystal structures published have also used highly sulfated heparin oligosaccharides, which may mask the fine specificity of complexes formed with less sulfated HS oligosaccharides. Furthermore sequencing of oligosaccharides from the S-domains of fibroblast HS chains suggests that heparin oligosaccharides are unlikely to be an accurate model for those in the S-domains of HS. The latter exhibit, in particular, a lower degree of 6-O sulfation and tightly controlled position of 6-O sulfoxy groups in the center of the domains (Merry et al., 1999Go). It will be important to focus on determining the structures of complexes containing homogenous, active, but less sulfated HS oligosaccharides. The development of in vitro methods for generating libraries of HS sequences is likely to facilitate this process. Crystallographic studies as well as traditional techniques provide information regarding the state of the complex at equilibrium (endpoint). The increasing use of more dynamic and quantitative techniques is important because they may reveal likely signaling mechanisms. Both of these approaches will be facilitated by methods enabling greater purification of oligosaccharide mixtures. Separation of structural isomers with similar physicochemical properties but different sequences, which are potentially able to interact with the same protein, is currently difficult. The use of multiple techniques based on different physicochemical properties, including oligosaccharide conformation, may overcome such difficulties.

Thermodynamic and affinity considerations of using heparin/HS chains or oligosaccharides
The interactions of HS with proteins of the FGF system have often been examined from the viewpoint of affinity. However, it is difficult to infer mechanism from affinity and to obtain an accurate picture of the driving forces involved from a thermodynamic perspective. Particularly problematic is the use of oligosaccharides or mixtures of oligosaccharides over polysaccharides. Although usually an attempt to simplify the problem, it is likely to overemphasize the enthalpic contribution of bond formation on HS binding to a protein because several factors are usually ignored. These include possible changes in entropy of the polysaccharide on binding, if the bound conformation is significantly different from that in solution, as has been observed in the case of oligosaccharides (Hricovini et al., 1999Go, 2002Go), and related rearrangement of the solvent. These parameters may be significantly different for polysaccharides and oligosaccharides. This problem is compounded by the assumption that there are no cooperative effects arising from changes in the secondary structure of the interacting species. The affinity of an interaction does not just represent the complementarity of a ligand to its partner, but also the degree of reorganization that has occurred for all the participating species (Szwajkajzer and Carey, 1997Go). Consequently, the apparent affinity may be misinterpreted in mechanistic terms; various combinations of affinity and specificity are possible and can compensate each other.

In addition, protein-binding sites are found within HS chains that also possess domains of low sulfation, which, as mentioned, are relatively rigid (Hricovini et al., 1997Go). The influence of considering binding sites within the context of the chain is illustrated by the observation that full-length HS chains can exhibit different activities when compared to fragments derived from them (Kato et al., 1998Go; Rahmoune et al., 1998Go; Zhang et al., 2001Go). This observation could have steric, avidity (many active sites) and/or thermodynamic causes. In a similar vein, protein binding may be affected by HS chains being covalently attached at one end to core proteins in vivo. Evidence for such an effect is a reduction in the ability of HS chains to potentiate and inhibit FGF1 activity when released from core proteins (Gordon et al., 1989Go). This may be due to the core proteins displaying multiple chains and increasing their local concentration, a reduction in the degrees of freedom of the chains, or a reduction in the glycosidic linkage rotational flexibility near the core protein. Other considerations are that the physicochemical conditions (e.g., pH, redox potential, cation and ionic strength) in vivo are usually known only in vague terms. Moreover, the molecular environment in the ECM and on the cell surface is extremely crowded (Ellis, 2001Go; Minton, 1993Go; Zimmerman and Minton, 1993Go), a fact ignored in all analyses to date but that will have profound consequences on the interaction between a protein and HS.

On balance, it appears unlikely that the mechanism for the regulation of FGF signaling through interaction of HS involves large-scale reorganization of one or several components (e.g., FGF or FGFR to fit the HS conformation exquisitely). This would reduce the affinity on account of a large thermodynamic penalty. This could, however, be partially compensated by accepting a lower degree of specificity. A second type of mechanism involves little reorganization. In this model, some leeway in the stringency of fit would still produce reasonably high affinity (Szwajkajzer and Carey, 1997Go). Other mechanisms, especially those containing several distinct phases, are more difficult to assess and will be dependent on the order and rate of these events (i.e., kinetics) as well as the degree of reorientation needed. If models that propose only moderate to poor specificity for HS are found to be correct, then the degree of information that HS encodes (Turnbull et al., 2001Go) may be questioned (Gallagher, 2001Go). An alternative explanation is one of competition in terms of kinetics between sites on different chains and between those on the same chain (Powell et al., 2002Go). These sites need not necessarily have the same sequences.


    Conclusions
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
A range of techniques have been used to investigate protein–heparin/HS interactions and have provided a wealth of information using both heparin/HS chains and oligosaccharides. The use of the latter facilitates identification of the structural features required for a particular interaction and simplifies quantitative analysis because simple mathematical models can be used to describe the interaction. Traditional approaches in combination with the development of new methods for rapidly screening and sequencing oligosaccharide libraries and further development of methods for in vitro engineering of HS oligosaccharide sequences will no doubt continue to provide information on the sequence characteristics of heparin/HS–protein interactions. It is crucial that improved purification techniques are developed to facilitate such studies. The information obtained using oligosaccharides may, however, be less relevant to the in vivo situation, where such sequences are embedded and restricted in longer chains. Their principal use may be for the discovery of novel glycotherapeutics that target disease-related protein–HS interactions. Analysis of the more physiologically relevant chains is difficult because of their heterogeneity and multidentate nature. Information regarding the qualitative and quantitative parameters of interactions has been obtained for several proteins despite this innate complexity. Global chemical modification provides preliminary information on the link between structure and binding characteristics or activity, but more sophisticated approaches are required to fully understand relationships between sequence and function, taking into account the conformation of different sequences. This is an area in which advances should be particularly informative. Similarly, quantitative information regarding the interactions of proteins with chains can be acquired, but the need to eliminate artifacts may limit the information available. We believe, however, that to determine the mechanism by which a sequence achieves a biological function in vivo, new approaches are required that place active sequences in the context of a chain, ideally one closely similar to that in which it exists in vivo. Recent developments in chemical/enzymatic modifications of chains (Rong et al., 1999Go) coupled with synthetic chemistry may enable an active oligosaccharide to be built into a homogeneous chain, hence addressing some of these considerations. This would provide, in particular, a more realistic molecular context for an active sequence. Such approaches should lead to valuable information regarding heparin/HS–protein interactions.


    Acknowledgements
 
The authors wish to acknowledge Scott Guimond for many helpful discussions and the funding of the U.K. Biotechnology and Biological Sciences Research Council (research grants to A.K.P., E.A.Y., D.F.G., and J.E.T.) and the U.K. Medical Research Council (senior fellowship to J.E.T.).


    Footnotes
 
1 To whom correspondence should be addressed; e-mail: andrew.powell{at}liverpool.ac.uk


    Abbreviations
 
ACE, affinity coelectrophoresis; ECM, extracellular matrix; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GAG, glycosaminoglycan; GMSA, gel mobility shift assay; HPLC, high-performance liquid chromatography; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; ITC, isothermal titration calorimetry; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; NMR, nuclear magnetic resonance; SNA, surface noncovalent affinity


    References
 Top
 Abstract
 Introduction
 Structural characteristics of...
 Approaches for qualitative...
 Methods for qualitative analysis...
 Structure-function studies of...
 Approaches for quantitative...
 Methods for quantitative...
 Appraisal of heparin/HS-protein...
 Conclusions
 References
 
Ai, X., Do, A.T., Lozynska, O., Kusche-Gullberg, M., Lindahl, U., and Emerson, C.P. Jr. (2003) QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling. J. Cell Biol., 162, 341–351.[Abstract/Free Full Text]

Almond, A. and Sheehan, J.K. (2003) Predicting the molecular shape of polysaccharides from dynamic interactions with water. Glycobiology, 13, 255–264.[Abstract/Free Full Text]

Amara, A., Lorthioir, O., Valenzuela, A., Magerus, A., Thelen, M., Montes, M., Virelizier, J.L., Delepierre, M., Baleux, F., Lortat-Jacob, H., and Arenzana-Seisdedos, F. (1999) Stromal cell-derived factor-1alpha associates with heparan sulfates through the first beta-strand of the chemokine. J. Biol. Chem., 274, 23916–23925.[Abstract/Free Full Text]

Ayotte, L. and Perlin, A.S. (1986) N.m.r. spectroscopic observations related to the function of sulfate groups in heparin. Calcium binding vs. biological activity. Carbohydr. Res., 145, 267–277.[CrossRef][ISI][Medline]

Belford, D.A., Hendry, I.A., and Parish, C.R. (1992) Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding. Biochemistry, 31, 6498–6503.[ISI][Medline]

Bernfield, M., Gotte, M., Park, P.W., Reizes, O., Fitzgerald, M.L., Lincecum, J., and Zako, M. (1999) Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem., 68, 729–777.[CrossRef][ISI][Medline]

Born, J., Jann, K., Assmann, K.J., Lindahl, U., and Berden, J. H. (1996) N-Acetylated domains in heparan sulfates revealed by a monoclonal antibody against the Escherichia coli K5 capsular polysaccharide. Distribution of the cognate epitope in normal human kidney and transplant kidney with chronic vascular rejection. J. Biol. Chem., 271, 22802–22809.[Abstract/Free Full Text]

Brickman, Y.G., Ford, M.D., Gallagher, J.T., Nurcombe, V., Bartlett, P.F. and Turnbull, J.E. (1998) Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development. J. Biol. Chem., 273, 4350–4359.[Abstract/Free Full Text]

Bundle, D.R. and Sigurskjold, B. W. (1994) Determination of accurate thermodynamics of binding by titration microcalorimetry. Methods Enzymol., 247, 288–305.[ISI][Medline]

Busse, M. and Kusche-Gullberg, M. (2003) In vitro polymerization of heparan sulfate backbone by the EXT proteins. J. Biol. Chem., 278, 41333–41337.[Abstract/Free Full Text]

Capila, I. and Linhardt, R.J. (2002) Heparin-protein interactions. Angew Chem. Int. Ed. Engl., 41, 391–412.[ISI][Medline]

Capila, I., VanderNoot, V.A., Mealy, T.R., Seaton, B.A., and Linhardt, R.J. (1999) Interaction of heparin with annexin V. FEBS Lett., 446, 327–330.[CrossRef][ISI][Medline]

Casu, B. and Lindahl, U. (2001) Structure and biological interactions of heparin and heparan sulfate. Adv. Carbohydr. Chem. Biochem., 57, 159–206.[ISI][Medline]

Cheung, W.F., Eriksson, I., Kusche-Gullberg, M., Lindhal, U., and Kjellen, L. (1996) Expression of the mouse mastocytoma glucosaminyl N-deacetylase/N-sulfotransferase in human kidney 293 cells results in increased N-sulfation of heparan sulfate. Biochemistry, 35, 5250–5256.[CrossRef][ISI][Medline]

Conrad, H.E. (1998) Heparin-binding proteins. San Diego, Academic Press.

Delehedde, M., Lyon, M., Sergeant, N., Rahmoune, H., and Fernig, D.G. (2001) Proteoglycans: pericellular and cell surface multireceptors that integrate external stimuli in the mammary gland. J. Mamm. Gland Biol. Neoplasia, 6, 253–273.[CrossRef][ISI][Medline]

Delehedde, M., Lyon, M., Gallagher, J.T., Rudland, P.S., and Fernig, D.G. (2002a) Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts. Biochem. J., 366, 235–244.[ISI][Medline]

Delehedde, M., Lyon, M., Vidyasagar, R., McDonnell, T.J., and Fernig, D.G. (2002b) Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes. J. Biol. Chem., 277, 12456–12462.[Abstract/Free Full Text]

Dennissen, M.A., Jenniskens, G.J., Pieffers, M., Versteeg, E.M., Petitou, M., Veerkamp, J.H., and van Kuppevelt, T.H. (2002) Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies. J. Biol. Chem., 277, 10982–10986.[Abstract/Free Full Text]

Desai, U.R., Wang, H.M., Kelly, T.R., and Linhardt, R.J. (1993) Structure elucidation of a novel acidic tetrasaccharide and hexasaccharide derived from a chemically modified heparin. Carbohydr. Res., 241, 249–259.[ISI][Medline]

DiGabriele, A.D., Lax, I., Chen, D.I., Svahn, C.M., Jaye, M., Schlessinger, J., and Hendrickson, W.A. (1998) Structure of a heparin-linked biologically active dimer of fibroblast growth factor. Nature, 393, 812–817.[CrossRef][ISI][Medline]

Dong, J., Peters-Libeu, C.A., Weisgraber, K.H., Segelke, B.W., Rupp, B., Capila, I., Hernaiz, M.J., LeBrun, L.A., and Linhardt, R.J. (2001) Interaction of the N-terminal domain of apolipoprotein E4 with heparin. Biochemistry, 40, 2826–2834.[CrossRef][ISI][Medline]

Edwards, P.R., Gill, A., Pollard-Knight, D.V., Hoare, M., Buckle, P.E., Lowe, P.A., and Leatherbarrow, R.J. (1995) Kinetics of protein–protein interactions at the surface of an optical biosensor. Anal. Biochem., 231, 210–217.[CrossRef][ISI][Medline]

Ellis, R.J. (2001) Macromolecular crowding: obvious but underappreciated. Trends Biochem. Sci. 26, 597–604.[CrossRef][ISI][Medline]

Esko, J.D. (2001) Glycosaminoglycan binding proteins. In Varki, A. (Ed.), Essentials of glycobiology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Esko, J.D. and Lindahl, U. (2001) Molecular diversity of heparan sulfate. J. Clin. Invest., 108, 169–173.[Free Full Text]

Esko, J.D. and Selleck, S.B. (2002) Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem., 71, 435–471.[CrossRef][ISI][Medline]

Faham, S., Hileman, R.E., Fromm, J.R., Linhardt, R.J., and Rees, D.C. (1996) Heparin structure and interactions with basic fibroblast growth factor. Science, 271, 1116–1120.[Abstract]

Fath, M.A., Wu, X., Hileman, R.E., Linhardt, R.J., Kashem, M.A., Nelson, R.M., Wright, C.D., and Abraham, W.M. (1998) Interaction of secretory leukocyte protease inhibitor with heparin inhibits proteases involved in asthma. J. Biol. Chem., 273, 13563–13569.[Abstract/Free Full Text]

Fernig, D.G. (2001) Optical biosensor techniques to analyse protein-polysaccharide interactions. In Iozzo, R.V. (Ed.), Proteoglycan protocols. Humana Press, NJ, pp. 505–518.

Ferro, D.R., Provasoli, A., Ragazzi, M., Torri, G., Casu, B., Gatti, G., Jacquinet, J., Sinay, P., Petitou, M., and Choay, J. (1986) Evidence for conformation equilibrium of the sulfated L-iduronate residue in heparin and in synthetic heparin mono- and oligosaccharides: NMR and force-field studies. J. Am. Chem. Soc., 108, 6773–6778.[ISI]

Gallagher, J.T. (2001) Heparan sulfate: growth control with a restricted sequence menu. J. Clin. Invest., 108, 357–361.[Free Full Text]

Gallagher, J.T. and Walker, A. (1985) Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides. Biochem. J., 230, 665–674.[ISI][Medline]

Gordon, P.B., Choi, H.U., Conn, G., Ahmed, A., Ehrmann, B., Rosenberg, L., and Hatcher, V.B. (1989) Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor. J. Cell. Physiol., 140, 584–592.[ISI][Medline]

Grootenhuis, P.D., Westerduin, P., Meuleman, D., Petitou, M., and van Boeckel, C.A. (1995) Rational design of synthetic heparin analogues with tailor-made coagulation factor inhibitory activity. Nat. Struct. Biol., 2, 736–739.[Medline]

Guimond, S.E. and Turnbull, J.E. (1999) Fibroblast growth factor receptor signalling is dictated by specific heparan sulphate saccharides. Curr. Biol., 9, 1343–1346.[CrossRef][ISI][Medline]

Guimond, S., Maccarana, M., Olwin, B.B., Lindahl, U., and Rapraeger, A.C. (1993) Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4. J. Biol. Chem., 268, 23906–23914.[Abstract/Free Full Text]

Gunay, N.S., Tadano-Aritomi, K., Toida, T., Ishizuka, I., and Linhardt, R.J. (2003) Evaluation of counterions for electrospray ionization mass spectral analysis of a highly sulfated carbohydrate, sucrose octasulfate. Anal. Chem., 75, 3226–3231.[CrossRef][ISI][Medline]

Guo, Y.C. and Conrad, H.E. (1989) The disaccharide composition of heparins and heparan sulfates. Anal. Biochem., 176, 96–104.[ISI][Medline]

Hileman, R.E., Siegel, M.M., Tabei, K., Balagurunathan, K., and Linhardt, R.J. (1998) Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization-mass spectrometry. Electrophoresis, 19, 2677–2681.[ISI][Medline]

Hook, M., Riesenfeld, J., and Lindahl, U. (1982) N-[3H]acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans. Anal. Biochem., 119, 236–245.[ISI][Medline]

Hricovini, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11–23.[CrossRef][ISI][Medline]

Hricovini, M., Guerrini, M., Torri, G., and Casu, B. (1997) Motional properties of E. coli polysaccharide K5 in aqueous solution analyzed by NMR relaxation measurements. Carbohydr. Res., 300, 69–76.[CrossRef][ISI][Medline]

Hricovini, M., Guerrini, M., and Bisio, A. (1999) Structure of heparin-derived tetrasaccharide complexed to the plasma protein antithrombin derived from NOEs, J-couplings and chemical shifts. Eur. J. Biochem., 261, 789–801.[Abstract/Free Full Text]

Hricovini, M., Guerrini, M., Bisio, A., Torri, G., Naggi, A., and Casu, B. (2002) Active conformations of glycosaminoglycans. NMR determination of the conformation of heparin sequences complexed with antithrombin and fibroblast growth factors in solution. Semin. Thromb. Hemost., 28, 325–334.[CrossRef][ISI][Medline]

Irie, A., Yates, E.A., Turnbull, J.E., and Holt, C.E. (2002) Specific heparan sulfate structures involved in retinal axon targeting. Development, 129, 61–70.[Abstract/Free Full Text]

Jemth, P., Kreuger, J., Kusche-Gullberg, M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2002) Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs. Exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding. J. Biol. Chem., 277, 30567–30573.[Abstract/Free Full Text]

Kan, M., Wang, F., To, B., Gabriel, J.L., and McKeehan, W.L. (1996) Divalent cations and heparin/heparan sulfate cooperate to control assembly and activity of the fibroblast growth factor receptor complex. J. Biol. Chem., 271, 26143–26148.[Abstract/Free Full Text]

Kato, M., Wang, H., Kainulainen, V., Fitzgerald, M.L., Ledbetter, S., Ornitz, D.M., and Bernfield, M. (1998) Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2. Nat. Med., 4, 691–697.[ISI][Medline]

Keiser, N., Venkataraman, G., Shriver, Z., and Sasisekharan, R. (2001) Direct isolation and sequencing of specific protein-binding glycosaminoglycans. Nat. Med., 7, 123–128.[CrossRef][ISI][Medline]

Kett, W.C., Osmond, R.I., Moe, L., Skett, S.E., Kinnear, B.F., and Coombe, D.R. (2003) Avidin is a heparin-binding protein. Affinity, specificity and structural analysis. Biochim. Biophys. Acta, 1620, 225–234.[ISI][Medline]

Klotz, I.M. (1982) Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science, 217, 1247–1249.[ISI][Medline]

Klotz, I.M. (1985) Ligand–receptor interactions: facts and fantasies. Q. Rev. Biophys., 18, 227–259.[ISI][Medline]

Koenig, A., Norgard-Sumnicht, K., Linhardt, R., and Varki, A. (1998) Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents. J. Clin. Invest., 101, 877–889.[Abstract/Free Full Text]

Kreuger, J., Prydz, K., Pettersson, R.F., Lindahl, U., and Salmivirta, M. (1999) Characterization of fibroblast growth factor 1 binding heparan sulfate domain. Glycobiology, 9, 723–729.[Abstract/Free Full Text]

Kreuger, J., Salmivirta, M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) Sequence analysis of heparan sulfate epitopes with graded affinities for fibroblast growth factors 1 and 2. J. Biol. Chem., 276, 30744–30752.[Abstract/Free Full Text]

Kreuger, J., Matsumoto, T., Vanwildemeersch, M., Sasaki, T., Timpl, R., Claesson-Welsh, L., Spillmann, D., and Lindahl, U. (2002) Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function. EMBO J., 21, 6303–6311.[Abstract/Free Full Text]

Kuberan, B., Lech, M., Zhang, L., Wu, Z.L., Beeler, D.L., and Rosenberg, R.D. (2002) Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry. J. Am. Chem. Soc., 124, 8707–8718.[CrossRef][ISI][Medline]

Lander, A.D. and Selleck, S.B. (2000) The elusive functions of proteoglycans: in vivo veritas. J. Cell. Biol., 148, 227–232.[Abstract/Free Full Text]

LaRochelle, W.J., Sakaguchi, K., Atabey, N., Cheon, H.G., Takagi, Y., Kinaia, T., Day, R.M., Miki, T., Burgess, W.H., and Bottaro, D.P. (1999) Heparan sulfate proteoglycan modulates keratinocyte growth factor signaling through interaction with both ligand and receptor. Biochemistry, 38, 1765–1771.[CrossRef][ISI][Medline]

Leali, D., Belleri, M., Urbinati, C., Coltrini, D., Oreste, P., Zoppetti, G., Ribatti, D., Rusnati, M., and Presta, M. (2001) Fibroblast growth factor-2 antagonist activity and angiostatic capacity of sulfated Escherichia coli K5 polysaccharide derivatives. J. Biol. Chem., 276, 37900–37908.[Abstract/Free Full Text]

Leatherbarrow, R.J. (1990) Using linear and non-linear regression to fit biochemical data. Trends Biochem. Sci., 15, 455–458.[CrossRef][ISI][Medline]

Lee, W.T. and Conrad, D.H. (1984) The murine lymphocyte receptor for IgE. II. Characterization of the multivalent nature of the B lymphocyte receptor for IgE. J. Exp. Med., 159, 1790–1795.[Abstract]

Lindahl, U., Kusche-Gullberg, M., and Kjellen, L. (1998) Regulated diversity of heparan sulfate. J. Biol. Chem., 273, 24979–24982.[Free Full Text]

Linhardt, R.J., Rice, K.G., Kim, Y.S., Lohse, D.L., Wang, H.M., and Loganathan, D. (1988) Mapping and quantification of the major oligosaccharide components of heparin. Biochem. J., 254, 781–787.[ISI][Medline]

Linhardt, R.J., Turnbull, J.E., Wang, H.M., Loganathan, D., and Gallagher, J.T. (1990) Examination of the substrate specificity of heparin and heparan sulfate lyases. Biochemistry, 29, 2611–2617.[ISI][Medline]

Liu, J., Shworak, N.W., Sinay, P., Schwartz, J.J., Zhang, L., Fritze, L.M., and Rosenberg, R.D. (1999) Expression of heparan sulfate D-glucosaminyl 3-O-sulfotransferase isoforms reveals novel substrate specificities. J. Biol. Chem., 274, 5185–5192.[Abstract/Free Full Text]

Liu, Z. and Perlin, A.S. (1994) Evidence of a selective free radical degradation of heparin, mediated by cupric ion. Carbohydr. Res., 255, 183–191.[CrossRef][ISI][Medline]

Loo, B.M., Kreuger, J., Jalkanen, M., Lindahl, U., and Salmivirta, M. (2001) Binding of heparin/heparan sulfate to fibroblast growth factor receptor 4. J. Biol. Chem., 276, 16868–16876.[Abstract/Free Full Text]

Lookene, A., Stenlund, P., and Tibell, L.A. (2000) Characterization of heparin binding of human extracellular superoxide dismutase. Biochemistry, 39, 230–236.[CrossRef][ISI][Medline]

Lyon, M., Deakin, J.A., and Gallagher, J.T. (1994) Liver heparan sulfate structure. A novel molecular design. J. Biol. Chem., 269, 11208–11215.[Abstract/Free Full Text]

Maccarana, M., Casu, B., and Lindahl, U. (1993) Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor. J. Biol. Chem., 268, 23898–23905.[Abstract/Free Full Text]

McGhee, J.D. and von Hippel, P H. (1974) Theoretical aspects of DNA–protein interactions: co-operative and non-co-operative binding of large ligands to a one-dimensional homogeneous lattice. J. Mol. Biol., 86, 469–489.[ISI][Medline]

Mendel, C.M. and Mendel, D.B. (1985) "Non-specific" binding. The problem, and a solution. Biochem. J., 228, 269–272.[ISI][Medline]

Merry, C.L., Lyon, M., Deakin, J.A., Hopwood, J.J., and Gallagher, J.T. (1999) Highly sensitive sequencing of the sulfated domains of heparan sulfate. J. Biol. Chem., 274, 18455–18462.[Abstract/Free Full Text]

Minton, A.P. (1993) Macromolecular crowding and molecular recognition. J. Mol. Recognit., 6, 211–214.[Medline]

Mulloy, B. and Forster, M.J. (2000) Conformation and dynamics of heparin and heparan sulfate. Glycobiology, 10, 1147–1156.[Abstract/Free Full Text]

Mulloy, B., Forster, M.J., Jones, C., and Davies, D.B. (1993) N.m.r. and molecular-modelling studies of the solution conformation of heparin. Biochem. J., 293(pt 3), 849–858.[ISI][Medline]

Munson, P.J. and Rodbard, D. (1980) Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem., 107, 220–239.[ISI][Medline]

Naggi, A., Torri, G., Casu, B., Oreste, P., Zoppetti, G., Li, J.P., and Lindahl, U. (2001) Toward a biotechnological heparin through combined chemical and enzymatic modification of the Escherichia coli K5 polysaccharide. Semin. Thromb. Hemost., 27, 437–443.[CrossRef][ISI][Medline]

Norgard-Sumnicht, K. and Varki, A. (1995) Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups. J. Biol. Chem., 270, 12012–12024.[Abstract/Free Full Text]

O'Shannessy, D.J. and Winzor, D.J. (1996) Interpretation of deviations from pseudo-first-order kinetic behavior in the characterization of ligand binding by biosensor technology. Anal. Biochem., 236, 275–283.[CrossRef][ISI][Medline]

Osmond, R.I., Kett, W.C., Skett, S.E., and Coombe, D.R. (2002) Protein-heparin interactions measured by BIAcore 2000 are affected by the method of heparin immobilization. Anal. Biochem., 310, 199–207.[ISI][Medline]

Pantoliano, M.W., Horlick, R.A., Springer, B.A., Van Dyk, D.E., Tobery, T., Wetmore, D.R., Lear, J.D., Nahapetian, A.T., Bradley, J.D., and Sisk, W.P. (1994) Multivalent ligand-receptor binding interactions in the fibroblast growth factor system produce a cooperative growth factor and heparin mechanism for receptor dimerization. Biochemistry, 33, 10229–10248.[ISI][Medline]

Park, P.W., Reizes, O., and Bernfield, M. (2000) Cell surface heparan sulfate proteoglycans: selective regulators of ligand-receptor encounters. J. Biol. Chem., 275, 29923–29926.[Free Full Text]

Pellegrini, L. (2001) Role of heparan sulfate in fibroblast growth factor signalling: a structural view. Curr. Opin. Struct. Biol., 11, 629–634.[CrossRef][ISI][Medline]

Pellegrini, L., Burke, D.F., von Delft, F., Mulloy, B., and Blundell, T.L. (2000) Crystal structure of fibroblast growth factor receptor ectodomain bound to ligand and heparin. Nature. 407, 1029–1034.[CrossRef][ISI][Medline]

Perrimon, N. and Bernfield, M. (2000) Specificities of heparan sulphate proteoglycans in developmental processes. Nature, 404, 725–728.[CrossRef][ISI][Medline]

Pierce, M.M., Raman, C.S., and Nall, B.T. (1999) Isothermal titration calorimetry of protein–protein interactions. Methods, 19, 213–221.[CrossRef][ISI][Medline]

Pikas, D.S., Eriksson, I., and Kjellen, L. (2000) Overexpression of different isoforms of glucosaminyl N-deacetylase/N-sulfotransferase results in distinct heparan sulfate N-sulfation patterns. Biochemistry, 39, 4552–4558.[CrossRef][ISI][Medline]

Pinhal, M.A., Smith, B., Olson, S., Aikawa, J., Kimata, K., and Esko, J. D. (2001) Enzyme interactions in heparan sulfate biosynthesis: uronosyl 5-epimerase and 2-O-sulfotransferase interact in vivo. Proc. Natl Acad. Sci. USA, 98, 12984–12989.[Abstract/Free Full Text]

Pope, R.M., Raska, C.S., Thorp, S.C., and Liu, J. (2001) Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry. Glycobiology, 11, 505–513.[Abstract/Free Full Text]

Powell, A.K., Fernig, D.G., and Turnbull, J.E. (2002) Fibroblast growth factor receptors 1 and 2 interact differently with heparin/heparan sulfate. Implications for dynamic assembly of a ternary signaling complex. J. Biol. Chem., 277, 28554–28563.[Abstract/Free Full Text]

Prabhu, A., Venot, A., and Boons G.-J. (2003) New set of orthogonal protecting groups for the modular synthesis of heparan sulfate fragments. Org. Lew., 5, 4975–4978.

Pye, D.A., Vives, R.R., Hyde, P., and Gallagher, J.T. (2000) Regulation of FGF-1 mitogenic activity by heparan sulfate oligosaccharides is dependent on specific structural features: differential requirements for the modulation of FGF-1 and FGF-2. Glycobiology, 10, 1183–1192.[Abstract/Free Full Text]

Rabenstein, D.L., Robert, J.M., and Peng, J. (1995) Multinuclear magnetic resonance studies of the interaction of inorganic cations with heparin. Carbohydr. Res., 278, 239–256.[ISI][Medline]

Rahmoune, H., Chen, H.L., Gallagher, J.T., Rudland, P.S., and Fernig, D.G. (1998) Interaction of heparan sulfate from mammary cells with acidic fibroblast growth factor (FGF) and basic FGF. Regulation of the activity of basic FGF by high and low affinity binding sites in heparan sulfate. J. Biol. Chem., 273, 7303–7310.[Abstract/Free Full Text]

Raman, R., Venkataraman, G., Ernst, S., Sasisekharan, V. and Sasisekharan, R. (2003) Structural specificity of heparin binding in the fibroblast growth factor family of proteins. Proc. Natl Acad. Sci. USA, 100, 2357–2362.[Abstract/Free Full Text]

Rathore, D., McCutchan, T.F., Garboczi, D.N., Toida, T., Hernaiz, M.J., LeBrun, L.A., Lang, S.C., and Linhardt, R.J. (2001) Direct measurement of the interactions of glycosaminoglycans and a heparin decasaccharide with the malaria circumsporozoite protein. Biochemistry, 40, 11518–11524.[CrossRef][ISI][Medline]

Rhomberg, A.J., Ernst, S., Sasisekharan, R., and Biemann, K. (1998) Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans. Proc. Natl Acad. Sci. USA, 95, 4176–4181.[Abstract/Free Full Text]

Rong, J., Nordling, K., Bjork, I., and Lindahl, U. (1999) A novel strategy to generate biologically active neo-glycosaminoglycan conjugates. Glycobiology, 9, 1331–1336.[Abstract/Free Full Text]

Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2001) Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase. Biochemistry, 40, 5548–5555.[CrossRef][ISI][Medline]

Rostand, K.S. and Esko, J.D. (1997) Microbial adherence to and invasion through proteoglycans. Infect. Immun., 65, 1–8.[Free Full Text]

Rux, A.H., Lou, H., Lambris, J.D., Friedman, H.M., Eisenberg, R.J., and Cohen, G.H. (2002) Kinetic analysis of glycoprotein C of herpes simplex virus types 1 and 2 binding to heparin, heparan sulfate, and complement component C3b. Virology, 294, 324–332.[CrossRef][ISI][Medline]

Sadir, R., Baleux, F., Grosdidier, A., Imberty, A., and Lortat-Jacob, H. (2001) Characterization of the stromal cell-derived factor-1alpha-heparin complex. J. Biol. Chem., 276, 8288–8296.[Abstract/Free Full Text]

Santini, F., Bisio, A., Guerrini, M., and Yates, E.A. (1997) Modifications under basic conditions of the minor sequences of heparin containing 2,3 or 2,3,6 sulfated D-glucosamine residues. Carbohydr. Res., 302, 103–108.[CrossRef]

Sasaki, T., Larsson, H., Kreuger, J., Salmivirta, M., Claesson-Welsh, L., Lindahl, U., Hohenester, E., and Timpl, R. (1999) Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin. EMBO J., 18, 6240–6248.[Abstract/Free Full Text]

Schlessinger, J., Plotnikov, A.N., Ibrahimi, O.A., Eliseenkova, A.V., Yeh, B.K., Yayon, A., Linhardt, R.J., and Mohammadi, M. (2000) Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell., 6, 743–750.[ISI][Medline]

Schuck, P. (1997) Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct., 26, 541–566.[CrossRef][ISI][Medline]

Schuck, P. and Minton, A.P. (1996) Kinetic analysis of biosensor data: elementary tests for self-consistency. Trends Biochem. Sci., 21, 458–460.[CrossRef][ISI][Medline]

Seeberger, P.H. and Haase, W.C. (2000) Solid-phase oligosaccharide synthesis and combinatorial carbohydrate libraries. Chem. Rev., 100, 4349–4394.[CrossRef][ISI][Medline]

Selleck, S.B. (2000) Proteoglycans and pattern formation: sugar biochemistry meets developmental genetics. Trends Genet., 16, 206–212.[CrossRef][ISI][Medline]

Shively, J.E. and Conrad, H.E. (1976) Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry, 15, 3932–3942.[ISI][Medline]

Shriver, Z., Sundaram, M., Venkataraman, G., Fareed, J., Linhardt, R., Biemann, K., and Sasisekharan, R. (2000) Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin. Proc. Natl Acad. Sci. USA, 97, 10365–10370.[Abstract/Free Full Text]

Sigurskjold, B.W. (2000) Exact analysis of competition ligand binding by displacement isothermal titration calorimetry. Anal. Biochem., 277, 260–266.[CrossRef][ISI][Medline]

Stringer, S. E., Mayer-Proschel, M., Kalyani, A., Rao, M., and Gallagher, J.T. (1999) Heparin is a unique marker of progenitors in the glial cell lineage. J. Biol. Chem., 274, 25455–25460.[Abstract/Free Full Text]

Szwajkajzer, D. and Carey, J. (1997) Molecular and biological constraints on ligand-binding affinity and specificity. Biopolymers, 44, 181–198.[CrossRef][ISI][Medline]

Thompson, L.D., Pantoliano, M.W., and Springer, B.A. (1994) Energetic characterization of the basic fibroblast growth factor-heparin interaction: identification of the heparin binding domain. Biochemistry, 33, 3831–3840.[ISI][Medline]

Turnbull, J.E. and Gallagher, J.T. (1991) Distribution of iduronate 2-sulphate residues in heparan sulphate. Evidence for an ordered polymeric structure. Biochem. J., 273, 553–559.[ISI][Medline]

Turnbull, J.E., Fernig, D.G., Ke, Y., Wilkinson, M.C., and Gallagher, J.T. (1992) Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J. Biol. Chem., 267, 10337–10341.[Abstract/Free Full Text]

Turnbull, J.E., Hopwood, J.J. and Gallagher, J.T. (1999) A strategy for rapid sequencing of heparan sulfate and heparin saccharides. Proc. Natl Acad. Sci. USA, 96, 2698–2703.[Abstract/Free Full Text]

Turnbull, J., Powell, A., and Guimond, S. (2001) Heparan sulfate: decoding a dynamic multifunctional cell regulator. Trends Cell Biol., 11, 75–82.[CrossRef][ISI][Medline]

van Zoelen, E.J. (1989) Receptor-ligand interaction: a new method for determining binding parameters without a priori assumptions on non-specific binding. Biochem. J., 262, 549–556.[ISI][Medline]

Venkataraman, G., Shriver, Z., Raman, R., and Sasisekharan, R. (1999) Sequencing complex polysaccharides. Science, 286, 537–542.[Abstract/Free Full Text]

Vives, R.R., Pye, D.A., Salmivirta, M., Hopwood, J.J., Lindahl, U., and Gallagher, J.T. (1999) Sequence analysis of heparan sulphate and heparin oligosaccharides. Biochem. J., 339, 767–773.[CrossRef][ISI][Medline]

Vives, R.R., Sadir, R., Imberty, A., Rencurosi, A., and Lortat-Jacob, H. (2002) A kinetics and modeling study of RANTES(9–68) binding to heparin reveals a mechanism of cooperative oligomerization. Biochemistry, 41, 14779–14789.[CrossRef][ISI][Medline]

Wang, F., Kan, M., Yan, G., Xu, J., and McKeehan, W. L. (1995) Alternately spliced NH2-terminal immunoglobulin-like Loop I in the ectodomain of the fibroblast growth factor (FGF) receptor 1 lowers affinity for both heparin and FGF-1. J. Biol. Chem., 270, 10231–10235.[Abstract/Free Full Text]

Wiseman, T., Williston, S., Brandts, J.F., and Lin, L.N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem., 179, 131–137.[ISI][Medline]

Wu, Z.L., Zhang, L., Beeler, D.L., Kuberan, B., and Rosenberg, R.D. (2002) A new strategy for defining critical functional groups on heparan sulfate. FASEB J., 16, 539–545.[Abstract/Free Full Text]

Wu, Z.L., Zhang, L., Yabe, T., Kuberan, B., Beeler, D.L., Love, A., and Rosenberg, R.D. (2003) The involvement of heparan sulfate (HS) in FGF1/HS/FGFR1 signaling complex. J. Biol. Chem., 278, 17121–17129.[Abstract/Free Full Text]

Yates, E.A., Santini, F., Guerrini, M., Naggi, A., Torri, G., and Casu, B. (1996) 1H and 13C NMR spectral assignments of the major sequences of twelve systematically modified heparin derivatives. Carbohydr. Res., 294, 15–27.[CrossRef][ISI][Medline]

Yates, E.A., Santini, F., De Cristofano, B., Payre, N., Cosentino, C., Guerrini, M., Naggi, A., Torri, G., and Hricovini, M. (2000) Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1 J(CH) coupling constants in heparin derivatives. Carbohydr. Res., 329, 239–247.[CrossRef][ISI][Medline]

Yates, E.A., Guimond, S.E., and Turnbull J.E. (2004) Highly diverse heparan sulfate libraries providing access to expanded areas of sequence space for bioactivity screening. J. Med. Chem., 47, 277–280.[CrossRef][ISI][Medline]

Yates, E.A., Jones, M.O., Clarke, C.E., Powell, A.K., Johnson, S.R., Porch, A., Edwards, P.P., and Turnbull, J.E. (2003) Microwave enhanced reaction of carbohydrates with amino-derivatised labels and glass surfaces. J. Mater. Chem., 13, 2061–2063.[CrossRef][ISI]

Zhang, Z., Coomans, C., and David, G. (2001) Membrane heparan sulfate proteoglycan-supported FGF2-FGFR1 signaling: evidence in support of the ìcooperative end structures model. J. Biol. Chem., 276, 41921–41929.[Abstract/Free Full Text]

Zimmerman, S.B. and Minton, A.P. (1993) Macromolecular crowding: biochemical, biophysical, and physiological consequences. Annu. Rev. Biophys. Biomol. Struct., 22, 27–65.[CrossRef][ISI][Medline]