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
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Abstract |
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Key words: FGF / heparan sulfate / heparin / interaction / protein
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Introduction |
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Structural characteristics of heparin/HS |
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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, 1986; Rabenstein et al., 1995
) and hence activity (Koenig et al., 1998
), would also be an interesting field for further study.
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Approaches for qualitative analysis of the interactions of heparin/HS polysaccharides with proteins |
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Recently, a new strategy for investigating the role of functional groups of heparin/HS in protein interactions has been developed (Wu et al., 2002). 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., 2002
, 2003
). 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., 2003
). 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., 1993
; Wu et al., 2003
).
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Methods for qualitative analysis of the interactions of proteins with heparin/HS oligosaccharides |
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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, 1989; Shively and Conrad, 1976
). 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., 1990
), produces pair-wise oligosaccharides with reducing ends that are intact but less reactive toward nucleophiles. The nonreducing end is modified to a
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 M1cm1; Linhardt et al., 1988
), 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.
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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., 2002). Modification of heparin, HS, or K5 polysaccharide has also been achieved using other recombinant sulfotransferases (Wu et al., 2002
), endosulfatases (Ai et al., 2003
), and epimerase (Naggi et al., 2001
), 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., 2004
). 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., 2002
).
Chemical synthesis of HS oligosaccharides has also been achieved (Grootenhuis et al., 1995) 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, 2000
) and a modular approach for synthesis of diverse HS saccharides has recently emerged (Prabhu et al., 2003
). 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, 2003), 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., 2001
), 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., 1996; Liu et al., 1999
; Pikas et al., 2000
; Rong et al., 2001
). 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., 1999, 2001
; Loo et al., 2001
; Turnbull et al., 1992
). An alternative is to create oligosaccharide libraries by further prefractionating oligosaccharide mixtures and then assaying these fractions for activity (Guimond and Turnbull, 1999
; Pye et al., 2000
), 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., 2001). 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., 2003
) 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., 2003
; Maccarana et al., 1993
) 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 proteinglycosaminoglycan (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., 1994). However, deviations between the relative ionic strength elution of proteins from heparin-sepharose and direct affinity measurements have been observed (Lookene et al., 2000
; Thompson et al., 1994
), 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., 2001
). 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., 2001
) and a similar result was seen using SNA-MS (Keiser et al., 2001
). 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., 2002a
).
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.
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Structurefunction studies of the interactions of proteins with HS/heparin oligosaccharides |
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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., 1999; Turnbull et al., 1999
) or high-performance liquid chromatography (HPLC) separations (Vives et al., 1999
) to provide direct sequence information; the sequencing of biosynthetically radiolabeled HS oligosaccharides using a step-sequencing HPLC method employing exoenzyme digestions (Merry et al., 1999
); and MALDI-MS techniques for the mass measurement of HS oligosaccharides (Rhomberg et al., 1998
), which in combination with specific chemical and enzymatic cleavage steps have been used for sequencing oligosaccharides (Shriver et al., 2000
; Venkataraman et al., 1999
). There have also been significant advances in the development of electrospray MS-based methods for separation of sulfated carbohydrates (Gunay et al., 2003
; Hileman et al., 1998
; Kuberan et al., 2002
; Pope et al., 2001
). 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 heparinprotein interactions has been obtained using molecular modeling, exploiting the crystal structure of the proteins involved (Raman et al., 2003; Sadir et al., 2001
; Vives et al., 2002
). 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., 1994
).
Recently, crystal structures of binary or ternary oligosaccharideprotein complexes have begun to provide information concerning the precise geometry of the interactions (DiGabriele et al., 1998; Faham et al., 1996
; Pellegrini, 2001
; Pellegrini et al., 2000
; Schlessinger et al., 2000
). 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., 1996
). Moreover, this structure (Faham et al., 1996
) agrees with biochemical data regarding minimum size requirements (Delehedde et al., 2002a
). 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., 1999
, 2002
). If this also applies to polysaccharides, this could have important consequences for the thermodynamics and hence affinity of these interactions (see later discussion).
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Approaches for quantitative analysis of the interactions of heparin/HS polysaccharides with proteins |
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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., 1982) or NHS ester derivatives (Lee and Conrad, 1984
; Norgard-Sumnicht and Varki, 1995
) 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., 2002
). Careful control and quantification of the labeling may therefore be crucial.
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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, 1990; Munson and Rodbard, 1980
). 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, 1982
, 1985
) 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, 1996; Schuck, 1997
) 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., 1999
; Powell et al., 2002
; Rahmoune et al., 1998
). The consistency of affinity values determined by alternative methods (Schuck and Minton, 1996
) 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., 2002
; Vives et al., 2002
). This has also been observed when using higher concentrations of protein binding to the immobilized heparin/HS (Lookene et al., 2000
; Powell et al., 2002
). 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., 1998
), 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., 1994; Thompson et al., 1994
). 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 108109 M (Bundle and Sigurskjold, 1994; Pierce et al., 1999
; Wiseman et al., 1989
). 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, 2000
).
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/HSprotein 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.
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Methods for quantitative analysis of the interaction of proteins with HS oligosaccharides |
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The relative apparent affinities of the interaction between proteins and oligosaccharides can also be determined via competition assays with immobilized heparin (Delehedde et al., 2002a,b
). The observation that different methods of immobilizing heparin can affect the affinity of interactions (Osmond et al., 2002
) 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.
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Appraisal of heparin/HSprotein interaction studies and implications for elucidation of mechanisms |
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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., 2003). 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 46mer length, whereas activation required larger fragments (810mers). 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., 2002a), and promote formation of a ternary complex (Wu et al., 2003
) 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 proteinoligosaccharide 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., 1999). 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., 1999, 2002
), 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, 1997
). 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., 1997). 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., 1998
; Rahmoune et al., 1998
; Zhang et al., 2001
). 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., 1989
). 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, 2001
; Minton, 1993
; Zimmerman and Minton, 1993
), 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, 1997). 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., 2001
) may be questioned (Gallagher, 2001
). 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., 2002
). These sites need not necessarily have the same sequences.
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Conclusions |
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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