Sulfated glycosaminoglycans (Chapter 16) interact with a variety of proteins. This chapter focuses on examples of binding proteins, methods for measuring glycosaminoglycan-protein interaction, and information about three-dimensional structures of the complexes. Hyaluronan, a nonsulfated glycosaminoglycan, also engages in biologically important protein interactions, and this subject is covered in Chapter 15.

GLYCOSAMINOGLYCAN-BINDING PROTEINS ARE COMMON

More than 100 glycosaminoglycan (GAG)-binding proteins have been described in the literature, falling into the broad classes presented in Table 35.1. To a large extent, these studies have focused on protein interactions with heparin, which is a more highly sulfated, iduronic acid (IdoA)-rich form of heparan sulfate (HS; Chapter 16). This bias may reflect the commercial availability of heparin, which is frequently used for fractionation studies and heparin-Sepharose affinity chromatography. The binding of protein ligands to heparin is thought to mimic the physiological interaction of proteins with the HSs present on cell surfaces and in the extracellular matrix. In comparison, relatively few proteins are known to interact with chondroitin sulfate (CS) or keratan sulfate (KS) with comparable avidity and affinity. In some cases, CS and the related GAG dermatan sulfate (DS) may be physiologically relevant binding partners because these GAGs predominate in many tissues. Determining the physiological relevance of these interactions is a major area of research.

TABLE 35.1

Examples of glycosaminoglycan-binding proteins and their biological activity

The interaction between GAGs and proteins can have profound physiological effects on processes such as hemostasis, lipid transport and absorption, cell growth and migration, and development. Binding to GAGs can result in immobilization of proteins at their sites of production and in the matrix for future mobilization, regulation of enzyme activity, binding of ligands to their receptors, and protection of proteins against degradation. In some cases, the interaction may reflect complementarity of charge (e.g., histone-heparin interactions) rather than any specific biologically relevant interaction. In other cases, the interaction has been shown to depend on rare but very specific sequences of modified sugars in the GAG chain (e.g., antithrombin binding).

METHODS FOR MEASURING GLYCOSAMINOGLYCAN-PROTEIN BINDING

Numerous methods are available for analyzing GAG-protein interactions, and some provide direct measurement of K_d values. A common method involves affinity fractionation of proteins on Sepharose columns containing covalently linked GAG chains, usually heparin. The bound proteins are eluted with different concentrations of NaCl, and the concentration required for elution is generally proportional to the K_d. High-affinity interactions require at least 1 M NaCl to displace bound ligand, which translates into K_d values of 10⁻⁷–10⁻⁹ M (determined under physiological salt concentrations by equilibrium binding). Proteins with low affinity (10⁻⁴–10⁻⁶ M) either do not bind under normal conditions (0.15 M NaCl) or require only 0.3–0.5 M NaCl to elute. This method is based on the assumption that GAG-protein interaction is entirely ionic, which may not be entirely correct. Nevertheless, it can provide an assessment of relative affinity compared to other proteins.

A number of more sophisticated methods are now in use that provide detailed thermodynamic data (ΔH [change in enthalpy], ΔS [change in entropy], ΔCp [change in molar specific heat], etc.), kinetic data (on rate, off rate), and high-resolution data on atomic contacts in GAG-protein interactions (Table 35.2). Regardless of the technique one uses, it must be kept in mind that in vitro binding measurements are not likely to be the same as those of binding to proteoglycans on the cell surface or in the extracellular matrix, where the density of ligands, receptors, and other interacting factors varies greatly. To determine the physiological relevance of the interaction, one should consider measuring binding under conditions that can lead to a biological response. For example, one can measure binding to cells with altered GAG composition (Chapter 46) or after treatment with specific lyases to remove GAG chains from the cell surface (Chapter 16) and then determine whether the same response occurs as that in the presence of GAG chains. The interaction can then be studied more intensively using the in vitro assays described above.

TABLE 35.2

Methods to measure glycosaminoglycan-protein interaction

CONFORMATIONAL AND SEQUENCE CONSIDERATIONS

As mentioned above, most GAG-binding proteins interact with HS or heparin. The likely basis for this preference is greater sequence heterogeneity and highly sulfated domains in HS and heparin. The unusual conformational flexibility of iduronic acid (IdoA) found in heparin, HS, and DS also has a role in their ability to bind proteins. GAGs are linear helical structures, consisting of alternating residues of N-acetylglucosamine or N-acetylgalactosamine with glucuronic acid or iduronic acid (except for keratan sulfates, which consist of alternating N-acetylglucosamine and galactose residues; see Chapter 16). Inspection of heparin oligosaccharides containing highly modified domains ([GlcNS6S-IdoA2S]n) shows that the N-sulfo and 6-O-sulfo groups of each disaccharide repeat lie on opposite sides of the helix from the 2-O-sulfo and carboxyl groups (Figure 35.1). Because of rotational restrictions about the anomeric linkages from the N-acetyl and carboxyl groups, the chains are relatively rigid with limited end-to-end bending possible compared to polypeptides. Analysis of the conformation of individual sugars shows that N-acetylglucosamine and glucuronic acid residues assume a preferred conformation in solution, designated ⁴C₁ (indicating that carbon 4 is above the plane defined by carbons 2, 3, and 5 and the ring oxygen, and that carbon 1 is below the plane). In contrast, IdoA2S assumes the ¹C₄ or the ²S₀ conformation (Figure 35.1), which reorients the position of the sulfo substituents and therefore creates a different orientation of charged groups. In many cases when a protein binds to an HS chain, it induces a change in conformation of the IdoA2S residue resulting in a better fit and enhanced binding. IdoA2S residues are always found in domains rich in N-sulfo and O-sulfo groups (for biosynthetic reasons; see Chapter 16), which is also where proteins usually bind. Thus, the greater degree of conformational flexibility in these modified regions may explain why so many more proteins bind with high affinity to heparin, HS, and DS than to other GAGs. The presence of an N-acetyl group in an N-acetyl-glucosamine residue changes the preferred conformation of the neighboring IdoA2S residue, showing that even minor modifications can influence conformation and chain flexibility. The evolution of GAG-binding proteins may be directed toward closely approximating the structures of their GAG ligands. Binding to GAGs that have a low degree of sulfation may require larger domains in the protein to interact with longer stretches of oligosaccharide. Molecular dynamic simulations on large heparin oligosaccharides have recently become possible with the availability of supercomputers (see Simulation 35.1 on the accompanying Web site). Such simulations can be used to predict the conformational flexibility of different domains within the chain, providing additional insights into GAG-protein interactions.

FIGURE 35.1

Conformation of heparin oligosaccharides. (A) Glucosamine (GlcN) and glucuronic acid (GlcA) exist in the ⁴C₁ conformation, whereas iduronic acid (IdoA) exists in equally energetic conformations designated ¹C₄ and ²S₀. (B) Space-filling model of a heparin (more...)

HOW SPECIFIC ARE GLYCOSAMINOGLYCAN-PROTEIN INTERACTIONS?

The discovery of multiple GAG-binding proteins led a number of investigators to examine whether a consensus amino acid sequence for GAG binding exists. In retrospect, this strategy was overly simplistic because it assumed that all GAG-binding proteins would recognize the same oligosaccharide sequence within heparin, or at least, sequences that would share many common features. We now know that some GAG-binding proteins interact with different oligosaccharide sequences (Table 35.3). The binding sites in the protein always contain basic amino acids (Lys and Arg) whose positive charges presumably interact with the negatively charged sulfates and carboxylates of the GAG chains. However, the arrangement of these basic amino acids can be quite variable, consistent with the variable positioning of sulfo groups in the GAG partner.

TABLE 35.3

Examples of oligosaccharides preferentially recognized by glycosaminoglycan-binding proteins

Most proteins are formed from α-helices, β-strands, and loops. Therefore, to engage a linear GAG chain, the positively charged amino acid residues would have to line up along the same side of the protein segment. α-Helices have periodicities of 3.4 residues per turn, which would require the basic residues to occur every third or fourth position along the helix in order to align with an oligosaccharide. In β-strands, the side chains alternate sides every other residue. Thus, positively charged residues should be located very differently if the peptide chain folds into a β-strand.

On the basis of the structure of several heparin-binding proteins that were available in 1991, Alan Cardin and Herschel Weintraub proposed that typical heparin-binding sites had the sequence XBBXBX or XBBBXXBX, where B is lysine or arginine and X is any other amino acid. From the structural arguments provided above, it should be obvious that only some of the basic residues in these sequences could participate in GAG binding, the actual number being determined by whether the peptide sequence exists as an α-helix or a β-sheet. We now know that the presence of these sequences in a protein merely suggests a possible interaction with heparin (or another GAG chain), but it does not prove that the interaction occurs under physiological conditions. In fact, the predicted binding sites for heparin in fibroblast growth factor 2 (FGF2) turned out to be incorrect once the crystal structure was determined. It is likely that binding involves multiple protein segments that juxtapose positively charged residues into a three-dimensional turn-rich recognition site. The specific arrangement of residues should vary according to the type and fine structure of those oligosaccharides involved in binding.

In plant and animal lectins and antibodies that recognize glycans, the glycan recognition domains are typically shallow pockets that engage the terminal sugars of the oligosaccharide chain (Chapters 27 and 34). In GAG-binding proteins, the protein usually binds to sugar residues that lie within the chain or near the terminus. Therefore, the binding sites in GAG-binding proteins consist of clefts or sets of juxtaposed surface residues rather than pockets. Given that GAG chains generally exist in a helical conformation, only those residues on the face toward the protein interact with amino acid residues; the ones on the other side of the helix might be free to interact with a second ligand. Alternatively, residues in a binding cleft could interact with both sides of the helix. Finally, one should keep in mind that the oligosaccharides that bind represent only a small segment of the GAG chain. Thus, a single glycan chain can bind multiple protein ligands.

Using phage display technology, a library of antiheparin and antiheparan sulfate antibodies was prepared. These antibodies are unreactive with other GAGs, showing specificity toward heparin and HS. Furthermore, they recognize specific structural features, such as heparin’s antithrombin-binding site, and show tissue and organ specificity. These antibodies will undoubtedly be useful in future studies aimed at understanding the specificity of GAG-protein interactions.

ANTITHROMBIN-HEPARIN: A PARADIGM FOR STUDYING GLYCOSAMINOGLYCAN-BINDING PROTEINS

The best-studied example of protein-GAG interaction is the binding of antithrombin to heparin and HS. This interaction is of great pharmacological importance in hemostasis because heparin is used clinically as an anticoagulant. Antithrombin is a member of the serpin family of protease inhibitors, many of which bind to heparin. Binding has a twofold effect: First, it causes a conformational change in the protein and activation of the protease inhibiting action, resulting in a 1000-fold enhancement in the rate at which it inactivates thrombin and Factor Xa. Second, the heparin chain acts as a template, enhancing the physical approximation of thrombin and antithrombin. Thus, both the protease (thrombin) and the inhibitor have GAG-binding sites.

Heparin acts as a catalyst in these reactions by enhancing the rate of the reaction through approximation of substrates and conformational change. After the inactivation of thrombin by antithrombin occurs, the complex loses affinity for heparin and dissociates. The heparin is then available to participate in another activation/inactivation cycle.

Early studies using affinity fractionation schemes showed that only about one third of the chains in a heparin preparation actually bind with high affinity to antithrombin. Comparing the sequence of the bound chains with those that did not bind failed to reveal any substantial differences in structure, consistent with the later discovery that the binding site consists of only five sugar residues (Figure 35.2) (the average heparin chain is about 50 sugar residues). This observation can be extended to virtually all GAG-binding proteins, inferring that the binding sites represent a very small segment of the chains (Table 35.3).

FIGURE 35.2

Crystal structure of the antithrombin-pentasaccharide complex (from Protein Data Bank). (A,D) α-Helices that make contact with heparin; (RCL) the reactive center loop that inactivates thrombin and Factor X; (F) another α-helix in the protein. (more...)

Crystals of antithrombin were prepared and analyzed by X-ray diffraction to 2.6-Å resolution. The docking site for the heparin pentasaccharide is formed by the apposition of helices A and D, which both contain critical arginine and lysine residues at the interface. The sequence in the D helix (₁₂₄AKLNCRLYRKANKSSKLVSANR₁₄₅) places many of the positively charged residues on one face of the helix, in proximity to the arginine residues in the A helix (₄₁PEATNRRVW₄₉) (Figure 35.2). The pentasaccharide is sufficient to activate antithrombin binding toward Factor Xa, but it will not facilitate the inactivation of thrombin. For this to occur, a larger oligosaccharide of at least 18 residues is needed. As mentioned above, thrombin also contains a heparin-binding site, and the larger heparin oligosaccharide is thought to act as a template for the formation of a ternary complex with thrombin and antithrombin. In contrast to antithrombin, thrombin exhibits little oligosaccharide specificity. As might be expected, adding high concentrations of heparin actually inhibits the reaction, because the formation of binary complexes of heparin and thrombin or heparin and antithrombin predominate. This important principle of “activation at low concentrations and inhibition at high concentrations” also occurs in other systems where ternary complexes form (Chapter 27).

Heparin is a pharmaceutical formulation produced by partial fractionation of natural GAGs derived primarily from porcine intestines (Chapter 16). Mast cells are known to produce a highly sulfated version of HS resembling heparin, although highly sulfated, iduronic acid–rich heparin oligosaccharides are present in HS isolated from other tissues, especially the liver. Although heparin has proven to be of great therapeutic use, its role in vivo remains unclear. Mast cells degranulate in response to specific antigen stimulation, resulting in release of stored heparin, histamine, and proteases. When this occurs, local anticoagulation might occur, but localized coagulation defects have not been described in animals bearing mutations that alter mast cells or heparin. Antithrombin-binding sequences are also found in ovarian granulosa cell HS, where they may have a role in regulating extravascular coagulation around ovulatory follicles. A small percentage of endothelial cell HS contains antithrombin-binding sequences as well. However, these binding sites appear to be located on the ablumenal side of blood vessels, and mice lacking the central 3-O-sulfated GlcNS unit, a hallmark of the antithrombin-binding sequence (Figure 35.2), do not exhibit any systemic coagulopathy after birth. Nevertheless, antithrombin deficiency causes massive disseminated coagulopathy. Perhaps these findings indicate that lower-affinity binding sequences are sufficient to activate antithrombin. This system illustrates an important caveat: One cannot necessarily ascribe functions to endogenous proteoglycans based on the effects of GAGs added to experimental systems.

Heparin cofactor II (HCII), another thrombin inhibitor, will bind to DS as well as heparin, albeit to different sites on the protein. The structure of HCII and its thrombin complex is very similar to antithrombin and its kinetically competent complex. However, unlike antithrombin, HCII is the only serpin known to associate with DS. As shown in Table 35.3, the DS that binds HCII consists of a repeating structure rich in 2-O-sul-foiduronic acid. Furthermore, the kinetic mechanism of inactivating thrombin differs in HCII and antithrombin. HCII function as an anticoagulant is believed to be restricted to damaged tissue in which DS proteoglycans in the matrix become exposed.

FGF-HEPARIN INTERACTIONS ENHANCE STIMULATION OF FGF RECEPTOR SIGNAL TRANSDUCTION

A large number of growth factors can be purified based on their affinity for heparin. The heparin-binding family of fibroblast growth factors has grown to more than 22 members and includes the prototype FGF2, otherwise known as basic fibroblast growth factor. FGF2 has a very high affinity for heparin (Kd ~ 10⁻⁹ M) and requires 1.5–2 M NaCl to elute from heparin-Sepharose. FGF2 has potent mitogenic activity in cells that express one of the FGF signaling receptors (four FGFR genes are known and multiple splice variants exist). Cell-surface HS binds to both FGF2 and FGFR, facilitating the formation of a ternary complex. Both binding and the mitogenic response are greatly stimulated by heparin or HS, which promote dimerization of the ligand-receptor complex.

The costimulatory role of HS (and heparin) in this system is reminiscent of the heparin/antithrombin/thrombin story. Indeed, the minimal binding sequence for FGF2 also consists of a pentasaccharide (Table 35.3). However, this pentasaccharide is not sufficient to trigger a biological response (mitogenesis). For this to occur, a longer oligosaccharide (10 mer) containing the minimal sequence and additional 6-O-sulfo groups are needed to bind FGFR. The sequence that binds to both FGF2 and FGFR is prevalent in heparin but rare in HS. The requirement for this rare binding sequence reduces the probability of finding this particular arrangement in naturally occurring HSs. Thus, some preparations of HS are inactive in mitogenesis, and those containing only one half of the bipartite binding sequence are actually inhibitory.

The structure of FGF2 cocrystallized with a heparin hexasaccharide has been obtained (Figure 35.3). The heparin fragment ([GlcNS6Sα1-4IdoA2Sα1-4]₃) was helical and bound to a turn-rich heparin-binding site on the surface of FGF2. Only one N-sulfo group and the 2-O-sulfo group from the adjacent iduronic acid are bound to the growth factor in the turn-rich binding domain, and the next GlcNS residue is bound to a second site, consistent with the minimal binding sequence determined with oligosaccharide fragments (Table 35.3). No significant conformational change in FGF2 occurs upon heparin binding, consistent with the idea that heparin primarily serves to dimerize FGF2 and juxtapose components of the FGF signal-transduction pathway. The crystal structure of acidic FGF (FGF1) has also been solved and shows similar sequences on its surface. However, the oligosaccharide sequence that binds with high affinity to FGF1 contains 6-O-sulfo groups.

FIGURE 35.3

Stereo view of the crystal structure of FGF2 with a heparin hexasaccharide (shown at the top of the figure; yellow balls indicate sulfur atoms). The stereo rendering was made with RASMOL using data from the Molecular Modeling Database (MMDB Id: 4322, (more...)

The cocrystal structure of the complex of (FGF2-FGFR)₂, first solved in the absence of heparin/HS ligand, showed a canyon of positively charged amino acid residues, suggestive of an unoccupied heparin-binding site. Subsequently, the heparin-oligosaccharide-containing complex was solved. The stoichiometry is still controversial, but recent biochemical evidence supports a 2:2:2 complex of HS: FGF2:FGFR for signaling (Figure 35.4).

FIGURE 35.4

Formation of complexes among heparan sulfate, FGF2, and FGFR. Two alternate crystal structures have been described for the complex.

OTHER ATTRIBUTES OF GLYCOSAMINOGLYCAN-PROTEIN INTERACTIONS

Chapters 34 and 39 discuss how sulfated GAGs can serve as receptors for microbes, viruses, and parasites. Chapters 38–44 discuss how GAG deficiencies can affect physiology and how aberrations in GAG composition can cause disease.

In some cases, the interaction of GAG chains with proteins may depend on metal cofactors. For example, L- and P-selectins have been shown to bind to a subfraction of HS chains and heparin in a divalent-cation-dependent manner. This observation raises the possibility that other types of cation-dependent receptors for GAG chains may exist. Recent studies show that GAG binding to L-selectin helps in leukocyte rolling. Furthermore, the interaction can be pharmacologically manipulated by exogenous heparin, including chemically modified derivatives that lack anticoagulant activity.

HS proteoglycans are often expressed in a spatially and temporally limited fashion. The temporary placement of an HS proteoglycan at a specific tissue site might or might not coincide with the presence of its appropriate protein ligand. Furthermore, if the binding partner has no access to the HS proteoglycan, it cannot interact—adding an additional level of specificity. Recent studies demonstrate that the fine structure of HS chains also changes during development, thus enabling or disabling specific associations between ligands and receptors.

Gradients of morphogens, factors that determine cell fates based on concentration, also determine the patterns of cell and tissue organization during development (see Chapter 24). The mechanism of gradient formation is controversial, but interestingly, virtually all morphogens can interact with heparin and HS. These interactions can affect transport of ligands, receptor interactions, endocytosis, and degradation, which together may have a role in determining the robustness of the gradient. The GAG chains of proteoglycans also offer a linear domain over which ligand proteins can diffuse. By limiting the space available to these proteins from the three-dimensional space of extracellular fluids and the extracellular matrix to one-dimensional space along the chains, the chance of encounters among heparin-binding proteins, such as FGF and its receptor (FGFR), may be enhanced. Thus, HS proteoglycans may have their most important role in controlling the kinetics of protein–protein interactions rather than the thermodynamics of such encounters.

FURTHER READING

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2. Maimone MM, Tollefsen DM. Structure of a dermatan sulfate hexasaccharide that binds to heparin cofactor II with high affinity. J Biol Chem. 1990;265:18263–18271. [PubMed: 2211700]
3. Conrad HE. Heparin-binding proteins. Academic Press; San Diego: 1998.
4. Mulloy B, Forster MJ. Conformation and dynamics of heparin and heparan sulfate. Glycobiology. 2000;10:1147–1156. [PubMed: 11087707]
5. Casu B, Lindahl U. Structure and biological interactions of heparin and heparan sulfate. Adv Carbohydr Chem Biochem. 2001;57:159–206. [PubMed: 11836942]
6. Capila I, Linhardt RJ. Heparin–protein interactions. Angew Chem Int Ed Engl. 2002;41:390–412. [PubMed: 12491369]
7. Esko JD, Selleck SB. Order out of chaos: Assembly of ligand binding sites in heparan sulfate. Annu Rev Biochem. 2002;71:435–471. [PubMed: 12045103]
8. Lander AD, Nie Q, Wan FY. Do morphogen gradients arise by diffusion? Dev. Cell. 2002;2:785–796. [PubMed: 12062090]
9. Li W, Johnson DJ, Esmon CT, Huntington JA. Structure of the antithrombin-thrombin-heparin ternary complex reveals the antithrombotic mechanism of heparin. Nat Struct Mol Biol. 2004;11:857–862. [PubMed: 15311269]
10. Mohammadi M, Olsen SK, Goetz R. A protein canyon in the FGF–FGF receptor dimer selects from an à la carte menu of heparan sulfate motifs. Curr Opin Struct Biol. 2005;15:506–516. [PubMed: 16154740]
11. Fernandez C, Hattan CM, Kerns RJ. Semi-synthetic heparin derivatives: Chemical modifications of heparin beyond chain length, sulfate substitution pattern and N-sulfo/N-acetyl groups. Carbohydr Res. 2006;341:1253–1265. [PubMed: 16712822]
12. Kreuger J, Spillmann D, Li JP, Lindahl U. Interactions between heparan sulfate and proteins: The concept of specificity. J Cell Biol. 2006;174:323–327. [PMC free article: PMC2064228] [PubMed: 16880267]
13. Nandini CD, Sugahara K. Role of the sulfation pattern of chondroitin sulfate in its biological activities and in the binding of growth factors. Adv Pharmocol. 2006;53:253–279. [PubMed: 17239770]
14. Kurup S, Wijnhoven TJ, Jenniskens GJ, Kimata K, Habuchi H, Li JP, Lindahl U, van Kuppevelt TH, Spillmann D. Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4. J Biol Chem. 2007;282:21032–21042. [PubMed: 17517889]