Primary contributions to this chapter were made by O. Hindsgaul (University of Alberta, Canada) and R.D. Cummings (The Oklahoma Health Sciences Center, Oklahoma).

NEARLY 100 CRYSTAL STRUCTURES of glycan-protein complexes have been solved, and several complexes have been deduced by NMR spectroscopy. The common features of these complexes are that the glycan-binding sites are small (spanning ~2.5 sugar residues), and interactions with proteins involve both hydrogen bonds to hydroxyl groups and hydrophobic interactions. Most glycans are bound in or near the minimum energy conformations observed in solution, but there are exceptions. Understanding the role of water in complex formation remains a major challenge. This chapter focuses on what is known about the detailed molecular mechanisms of these interactions and on the methods of analyzing glycan-protein interactions. Many different glycan-binding proteins have been described so far (see Chapters 22–30), and their interactions with glycans have been studied by a wide variety of approaches.

Historical Background ^(1–10)

Much of the early history related to glycan-protein interactions revolved around recognition of glycans by enzymes, such as the endoglycosidase lysozyme, which can degrade bacterial cell walls, and enzymes involved in intermediary metabolism, such as glycogen and starch synthases and phosphorylases. For the discovery of the antibacterial action of lysozyme and penicillin, Fleming, Chain, and Florey received the Nobel prize in 1945. Lysozyme was subsequently shown to be a highly specific endoglycosidase, capable of specifically cleaving β1–4 linkages in bacterial peptidoglycan (see Chapter 21). Glycogen synthetase was found to generate α1–4 glucosyl residues in glycogen, whereas other branching and debranching enzymes recognized α1–6-branched glucose residues. Studies on the metabolism of glycogen by these enzymes led to award of the Nobel prize in 1970 to Luis F. Leloir for his discovery of sugar nucleotides and their role in the biosynthesis of glycoconjugates and polysaccharides.

The concept of glycans being specifically recognized by proteins dates back to Emil Fischer, who used the phrase “lock and key” to refer to enzymes that recognize specific glycan substrates. The dream of specific interactions between sugars and proteins in three-dimensional space was realized by the determination of the crystal structure of lysozyme, which was the first “carbohydrate-binding protein” to be crystallized. Its structure was solved in complex with a tetrasaccharide in an elegant series of studies by Phillips and coworkers in the late 1960s. Lysozyme is an ellipsoidal protein which has a long cleft that runs for most of the length of one surface of the protein. This cleft is astonishingly large, considering that lysozyme has only 129 amino acids, and is capable of accommodating a hexasaccharide and cleaving it into a disaccharide product and a tetrasaccharide product. Other glycan-binding proteins whose three-dimensional structures are of historical significance are concanavalin A (crystal structure reported in 1972) and influenza virus hemagglutinin (crystal structure reported in 1981). In addition, critical information to the development of this field was gathered by Lemieux and Kabat and coworkers in studies on the combining sites of lectins and antibodies toward specific blood group antigens. From these and many recent studies, it is clear that the specific recognition of sugars by proteins occurs by several mechanisms. In discussing these interactions, the term lectin is now generally used to denote proteins with glycan-binding activity (see Chapters 22–30).

Recognition of Glycans by Proteins ^(11–13)

In today's terminology, the lock and key concept can be rephrased as follows: “What is the origin of the specificity seen in glycan-protein recognition?” In other words, why does a given bacterial adhesin, toxin, plant lectin, viral hemagglutinin, antibody, or selectin bind to only a very limited number of glycans among the thousands that are present on a cell surface? The fundamental question at the molecular level is why does a specific glycan leave the aqueous phase to enter a protein combining site? The forces involved in the binding of a glycan to a protein are the same as those for the binding of any ligand to its receptor: hydrogen bonding, van der Waals interactions, and charge and dipole attraction. So why are the dissociation constants (K_d) normally only in the 100 μm range and so difficult to calculate or predict?

The “Water Problem” ⁽¹²⁾

Few questions have generated such intense debate in the area of glycobiology. Unfortunately, most attempts to understand the energetics of glycan-protein binding have focused on calculating single contributing factors, such as determining the preferred conformation of a ligand or estimating the short-range interactions between the glycan and functional groups in the protein-combining site. Each such calculation is illuminating. However, as shown in Figure 4.1, the overall process of binding involves the meeting of a solvated polyhydroxylated glycan and a solvated protein-combining site. If a surface on the glycan is complementary to the protein-combining site, water can be displaced and binding occurs. When the complex finally forms, it presents a new surface to the surrounding medium which will also be hydrated. Solvation/desolvation energies are very large due to entropy and cannot be reliably calculated for hydrophilic compounds such as sugars. Thus, even though the energetic contributions of van der Waals and hydrogen-bonding interactions in the combining site can be estimated, errors in the estimation of the attendant solvation energy changes can be much larger, making the overall calculations of binding energy difficult.

Figure 4.1

Schematic of the binding of a glycan to a protein in water, resulting in displacement of water.

Atomic Details of Cholera Toxin Binding to the Pentasaccharide from G_M1 ⁽¹³⁾

Many complexes of glycans bound to proteins have been solved by X-ray crystallography. A consensus binding pattern has emerged where glycan surfaces of approximately 300–400 Ų make contact with the protein, corresponding to about 2.5 sugar residues. The complex of cholera toxin, a pentameric adhesion protein, bound to the G_M1 pentasaccharide illustrates the consensus binding pattern (Figure 4.2) (also see Chapter 28). Although G_M1 is a pentasaccharide, only three monosaccharides make any contact with the protein. The terminal β-linked galactose residue is almost completely buried in the protein-combining site, making numerous hydrogen-bonding interactions with protein functional groups, some via bound water molecules. Also very typical of glycan-protein complexes is the “stacking” interaction of an aromatic amino acid (Trp-88) with the hydrophobic “underside” of one of the sugar residues, in this case the terminal β-linked galactose residue. The penultimate β-linked GalNAc residue makes only minor contacts with the protein, whereas the Galβ1–4Glc reducing end disaccharide makes no contact at all. However, the purpose of this disaccharide is to hold the branching sialic acid residue in the proper orientation to make further contacts with the protein, as well as to the β-linked GalNAc residue. The net result is a very tight complex with a K_d value near 0.1 μm, one of the tightest known for monovalent glycan-protein binding.

Figure 4.2

Simplified structure of pentasaccharide G_M1 binding to cholera toxin. The amino acid residues in cholera toxin that contact the glycan directly or through H bonds with water are indicated.

Kinetics of Glycan-Protein Interactions

In addition to defining the three-dimensional structure of a glycan-binding protein, it is important to also define its specific interactions with a variety of glycans. The binding of a lectin (L) or an antibody to a glycan (G) is governed by the simple Equation 4.1. The affinity constant, K, is defined as an association constant (or K_a) by Equation 4.2; thus, K is also equal to k₁/k₂. Like any equilibrium constant, K is related to the standard free-energy change of the binding reaction at pH 7 (ΔG_o) in kcal per mole, as shown by Equation 4.3, where R is the gas constant (0.00198 kcal/mol-degree) and T is the absolute temperature (298°K).

The affinity constant K is related to thermodynamic parameters by Equation 4.4, where ΔG, ΔH, and ΔS represent the changes in free energy, enthalpy, and entropy of binding, respectively.

Although it is obviously important to define K, k₁, k₂, ΔG, ΔH, and ΔS for each binding phenomenon under consideration, many times investigators simply try to define K. Data are usually discussed in terms of the K_d (1/K_a) and terms of mm, μm, or nm are used. There are many different ways to study binding of glycans to proteins, and each approach has its advantages and disadvantages in terms of thermodynamic rigor, amounts of protein and glycan needed, and speed of analyses. A discussion follows of some of the major ways in which the binding between a glycan and protein is studied.

Since the affinity of single glycan-protein interactions is generally low, many naturally occurring animal lectins bind in a multivalent fashion. Multivalency raises questions of affinity versus avidity. Binding of a monovalent lectin to a monovalent ligand is easily defined by the equilibrium kinetics described in Equations 4.1 and 4.2. However, with multivalent ligands or lectins, multiple affinities occur and a more complex binding equilibrium, more accurately described by a set of equilibrium constants, must be used. The term avidity refers to the strength of multivalent ligand binding and obviously has complex kinetics. Typically, for multivalent ligands and lectins, the values reported for affinity are apparent affinity constants and usually measure the avidity. For divalent antibodies, where the situation has been more carefully analyzed, the avidity for antigen is typically 10–100 times that of the monovalent antibody. A similar situation may be true for most multivalent lectins. Another complication in studying some lectins is their membrane association. In the pure form, they probably occur as oligomers in detergent solutions. Another complication is that ligands for some lectins may be glycoproteins (e.g., mucins) or polysaccharides (e.g., heparin) with potentially multiple binding sites for the lectin. The density of binding sites on the ligand and the valency of the lectin may dramatically alter the apparent affinity of binding. All of these issues should be taken into account when studying the interactions of a lectin with glycans.

To understand the interactions between a glycan and a protein, it is important to define many parameters, including the precise molecular interactions and interatomic distances within a complex of glycan and protein, the equilibrium binding constant, the on and off rates of the interaction, and the entropy and enthalpy of the interaction. Such issues are usually related to a free glycan or small glycoconjugate interacting with a protein. However, in real biological situations, a glycan-binding protein may be interacting with a glycolipid or a glycoprotein that may be present at high density in the plane of the membrane, a polysaccharide with multiple repeating determinants, or a glycoprotein with many clustered determinants. Because of this complexity, most of our information about glycan-protein interactions derive from studies on relatively small glycan ligands interacting with a protein. In examining these interactions, there are three broad categories of approaches: biophysical methods, such as X-ray diffraction and NMR; kinetic methods, such as equilibrium dialysis and titration calorimetry; and indirect methods, such as hapten inhibition and ELISA (enzyme-linked immunosorbent assay)-based approaches.

Biophysical Methods: X-ray and NMR ^(14–18)

Two biophysical approaches for examining protein-glycan interactions at the molecular level are X-ray crystallography and nuclear magnetic resonance. Crystal complexes with glycans have been defined for a number of antibodies, plant and animal lectins, bacterial toxins, and enzymes that utilize carbohydrate. Approximately 100 three-dimensional structures of carbohydrate-binding proteins are listed in the Brookhaven Protein Data Bank. In addition, more than 1000 three-dimensional structures of mono- and oligosaccharides are stored in the Cambridge Structural Database. A resolution of at least 2–2.5 Å is required to accurately identify the positions and mode of binding of glycans. Such resolution is often difficult to obtain. In some cases, a crystal structure can be obtained for a glycan-binding protein independent of ligand, and its structure and potential binding site can be predicted using information from the three-dimensional structure of a homologous protein.

For the determination of the structure of a biomolecule in solution, NMR is the preferred technique. In general, monosaccharides are small and relatively rigid molecules, but an assembled glycan has a higher degree of flexibility, due to the general freedom of rotation about glycosidic bonds. In NMR, the proton-proton distances can be obtained following assignment of the proton resonances, through multidimensional techniques, such as the nuclear Overhauser effect (NOE). This information coupled with computational methods employing computer modeling allows the prediction of the free-state glycan conformation in solution. It is possible to extend this information to examine the interaction of a glycan with a protein in solution, using transfer NOE (TRNOE). This approach is based on the premise that the tumbling and rotation of a glycan in complex with a protein are much slower than that of free unbound glycans. In TRNOE, a small protein (<30 kD) is dissolved with a large excess of glycan that has been equilibrated with ²D₂O, and the resonances of the bound glycan can then be resolved independently from the free glycan. Available computer-assisted modeling strategies and information from glycan solution conformations and protein three-dimensional structures can be combined with NMR to provide even more information about the nature of the molecular details of the interactions between glycans and proteins. Although these approaches are highly informative, they are limited by the degree to which small glycans structurally mimic the larger macromolecule to which they are usually attached and by the problem that many of these interactions are often performed under nonequilibrium conditions.

Equilibrium Dialysis ^(19–23)

In equilibrium dialysis, a concentrated solution of a glycan-binding protein (e.g., lectin and antibody) is placed in a dialysis chamber that is permeable to a ligand (glycan or other small hapten). The chamber is then placed in equilibrium with a known volume of buffer that contains the glycan in the concentration range of 1/K. At equilibrium, the concentration of bound plus free glycan inside the bag [In] and free glycan outside the bag [Out] will depend on the concentration and affinity of the protein inside the bag. From this information, both the association constant K and the lectin valence n can be determined from the relationship shown in Equation 4.5, where r is the ratio of mole of glycan bound per mole of lectin and c is the concentration of unbound glycan [Out]. The mole of glycan bound is determined by simply subtracting [Out] from [In].

A plot of r/c versus r for different hapten concentrations (Scatchard analysis) will approximate a straight line with a slope of -K. The association constant K can be determined as the negative of the slope, and the valence of binding is defined by the r intercept at an infinite hapten concentration.

A number of important assumptions are made in these experiments and their validity must be demonstrated. These include demonstrating that the protein and its hapten are stable and active over the course of the experiment, the hapten is freely diffusible, the complex is at equilibrium, and structurally unrelated haptens, not expected to bind, show no apparent binding in the experimental setup. The following are several advantages to equilibrium dialysis: (1) The approach is not complicated and sophisticated equipment is not necessary; (2) if the affinity is high, then relatively small amounts of protein are needed (≤1 μmole); (3) if the affinity is high enough, small amounts of hapten may be required; (4) if the protein and haptens are very stable, they may be recovered and reused; (5) radioactive haptens may be used; and (6) reliable equilibrium measurements can be made. The following are some drawbacks of the approach: (1) It can only provide K; (2) if the affinity of the lectin or antibody for the hapten is low, then relatively large amounts of both may be required; and (3) many different measurements must be made and this may require many days or weeks to complete for a single experiment.

A variation of this equilibrium technique is illustrated by equilibrium gel filtration developed by Hummel and Dreyer. In the Hummel-Dreyer method, a protein (e.g., lectin) is applied to a gel-filtration column that is preequilibrated with a ligand (e.g., glycan) of interest that is easily detectable, e.g., by radioactive or fluorescent tagging. As the protein binds to ligand, a complex is formed that emerges from the column as a “peak” above the baseline of ligand alone, followed by a “trough,” where the concentration of ligand is decreased below the baseline, which extends to the included or salt volume of the column. The amount of complex formed is easily determined by the known specific activity of the ligand. Because the amount of complex formed is directly proportional to the amount of protein (or ligand) applied, it is easy to calculate a binding curve from several different equilibrium gel filtration column profiles at different concentrations of either protein or ligand. This binding curve allows the calculation of the equilibrium constant of the interaction. The advantages and drawbacks of this technique are generally the same as those for equilibrium dialysis, except that Hummel-Dreyer analyses are often quicker to perform and can be used with ligands of many different sizes. Although this approach has not been used historically for lectin-glycan interactions, recent studies suggest that it is extremely useful in defining the interactions of soluble selectins with small ligands.

Affinity Chromatography ^(24–27)

In this approach, a lectin is immobilized to an affinity support, such as Affi-Gel™, cyanogen-bromide-activated Sepharose, UltraLink™, or some other activated support. The immobilized lectin may be placed in a variety of column formats, ranging from old-fashioned gravity flow columns to HPLC. In the simplest approach, a lectin or other glycan-binding protein is immobilized to a support in a concentration range of 0.1 to 10 mg/ml. A buffer containing a glycan is added to the immobilized lectin, and binding to the lectin is measured, by virtue of its elution profile. If the glycan binds to the immobilized lectin relatively tightly, buffer containing a known hapten may be added to force dissociation of the complex. For example, high-mannose-type and hybrid-type N-glycans will bind avidly to a column of ConA-agarose and 500 mm α-methyl-Man will be required to elute the bound material efficiently (see Chapter 30). In contrast, many highly branched complex-type N-glycans will not bind. Biantennary complex-type N-glycans bind to ConA-agarose, but their elution can be effected by using 10 mm α-methyl-Man. In this manner, the binding specificity and approximate binding affinity of the lectin can be assessed, since the concentration of immobilized lectin is known and its capacity can also be defined. This approach is rather crude, and although it gives valuable practical information about the use of an immobilized lectin to bind specific glycans, it does not provide detailed affinity measurements. A variant of this method is to immobilize the glycan ligand and measure lectin binding. However, this is especially difficult to do with multiple ligands at multiple concentrations.

In a more sophisticated version of this approach, commonly termed frontal affinity chromatography, a solution containing a glycan of known concentration is applied to a column of immobilized glycan-binding protein, and the elution of the glycan from the column (the elution front) is monitored. Thus, the ligand is continuously applied to the column. The volume representing the elution front, i.e., when the ligand begins to present in the elution buffer, is a measure of the binding affinity. This can be directly calculated by Equation 4.6, where the binding capacity of the column is V_f[T]/V_t; V_f is the frontal volume; V_t is the total column volume; [O] is the concentration of the glycan in the added solution; and [L] is the lectin concentration in the matrix.

Various glycan concentrations are used, a series of graphs are obtained, and the 50% saturation points are derived (Figure 4.3). The data are plotted as 1/[O]_o (V_f-V_t) versus 1/[O]_o. From this plot, the value of the K_d can be derived from the intercept on the abscissa, which corresponds to -1/K_d and the intercept on the ordinate provides the concentration of active lectin [L]. This approach offers many advantages, which are similar to those discussed for equilibrium dialysis: (1) The approach is not complicated; (2) if the affinity is high, then relatively small amounts of protein are needed (≤1 μmole), and in fact a single column is required; (3) correspondingly small amounts of hapten may be used if the K_d is rather low (in the range of 10 nm to 10 μm); (4) if the haptens are stable, they may be recovered and reused; (5) radioactive haptens may be used; and (6) reliable equilibrium measurements can be made. However, the drawbacks to this approach are that (1) only K can be derived, (2) the conjugation of the glycan-binding protein to the matrix must be stable and the protein must retain reasonable activity for many different column runs, (3) many different column runs must be made with a single glycan, (4) if the K_d is high (>10 μm), then relatively large amounts of glycan may be required, and (5) the entire analysis can take weeks to months to complete.

Figure 4.3

Example of frontal affinity chromatography, where different concentrations of a glycan are applied to a column of immobilized lectin. At the lowest concentration (D), the elution of the glycan is retarded and the chromatographic front is indicated by (more...)

Titration Calorimetry ^(28–30)

The binding of a glycan to a lectin can be measured as a change in free energy through isothermal titration microcalorimetry using a commercial microcalorimeter. In this approach, a solution containing a glycan of interest is added via a syringe into a fixed concentration of lectin solution in a microcalorimetry cell (1–2 ml). Control experiments are done in which glycans are added to cells lacking protein. The 1–3 μl of glycan is added at many intervals and the heat evolved from binding is measured as μcal/sec. During the course of the experiment, the concentration of glycan is increased in the mixing cell over the range of a molar ratio of glycan to lectin from 0 to 10. The change of heat capacity of binding is determined, and the data are plotted as kcal/mole of injectant versus the molar ratio. These data are then analyzed by a Scatchard plot to obtain the K_d. In addition, the heat change is directly related to the enthalpy of reaction ΔH and is the heat of reaction (heat absorbed or given off) at constant temperature and pressure. From the knowledge the K_d and ΔH, and using Equation 4.4, it is possible to define the binding entropy ΔS.

The major advantage of this approach is that it can provide all major thermodynamic information about the binding of a glycan to a glycan-binding protein, and thus, it is highly superior to equilibrium dialysis and affinity chromatography. The disadvantages of this approach are that (1) relatively large amounts of protein are typically required (>10 mg), (2) relatively large amounts of glycans are required, and (3) because of the foregoing problem, these analyses do not typically use a wide range of different glycans.

Surface Plasmon Resonance ^(31–36)

SPR is a technique in which the association of a free molecule (termed the analyte) with an immobilized ligand on a sensor chip induces a change in the refractive index of the chip surface. In SPR technology, light is reflected from the side of the surface not in direct contact with the sample, and the change in SPR causes a change in the reflected light intensity at a specific combination of angle and wavelength. This is measured as a change in the refractive index at the surface layer and is recorded as the SPR signal or resonance units (RU) (Figure 4.4A). In general, changes in refractive index for a given change of mass concentration at the surface layer are largely independent of the molecule of interest and are the same for proteins, glycans, lipids (liposomes), and nucleic acids. Typically, 1 RU approximates 1 pg/mm². Measurements may be conducted in a BIACORE™ instrument from Biacore AB. Binding is measured in real time in terms of 1–10 counts/sec (Figure 4.4B), and thus, information about the association and dissociation kinetics of the binding, and the overall K_d is obtainable.

Figure 4.4

Example of surface plasmon resonance. (A) In SPR, the reflective light is measured and is altered in response to binding of the analyte to the immobilized ligand. (B) An example of a sensorgram showing the binding of the analyte to the ligand and the (more...)

In a typical example, a solution of a protein (analyte) is passed continuously over a sensor chip microsurface containing the immobilized ligand (glycan), using a microfluidic system that precisely controls analyte delivery, all washing steps, etc. The BIACORE™ 2000 allows up to four channels or flow cells to be used simultaneously across a single sensor surface. One of these channels is used as the in-line reference or control. If high throughput is required, then different ligands can be immobilized in each channel. There are a variety of chemistries available for coupling of the ligand, including coupling by amine, thiols, aldehydes, and biotin capture. In some approaches, a glycoprotein ligand for a lectin is immobilized and the binding of the lectin is directly measured. It is also possible to sequentially degrade the immobilized glycoprotein ligand on the chip by passing through solutions containing exoglycosidases and reexamining the binding at each step to different lectins, thereby obtaining information about the glycosylation of the immobilized protein. The immobilized ligand is usually quite stable and can be used repeatedly for hundreds of runs over a period of months.

The advantages of this approach are that (1) affinities in the range from millimolar to picomolar can be measured; (2) complete measurements of k₁ and k₂ (Equation 4.1) are routine; (3) for immobilization of a molecule using amine coupling, 1–5 μg is normally sufficient; (4) typically, the concentration range of analyte is 0.1–100 × K_d and the typical volumes needed are in the range of 50–150 μl; and (5) measurements are extremely rapid, and complete experimental results can be obtained within a few days. The drawbacks of this approach are that analytes must have sufficient mass (>2000 daltons) to cause a significant change in SPR upon binding (thus, the glycan is usually immobilized instead of the protein) and coupling of free glycans to the chip surface is inefficient; thus, neoglycoproteins or some other type of large conjugate must be immobilized. Another drawback of SPR is that there may be some nonhomogeneity in conditions on the BIACORE™ due to mass transport effects, allowing rebinding of analyte to the solid-phase surface, although this may in fact mimic some biological conditions, such as those occurring in cell-cell interactions. At high analyte concentrations, this problem could considerably lower the dissociation rate k₂ (Equation 4.2), and thus provide a K_d that may be low. This problem is usually addressed by performing many measurements at different analyte concentrations and relying on those in the low analyte concentration range.

ELISA-type Assays ^(37–40)

The conventional ELISA has been adapted for studying glycans and glycan-binding proteins in a variety of formats. Of course, many glycans are antigens, and antibodies to them can be analyzed in the conventional ELISA format. In the first adaptation of this approach by Saul Roseman's group, sugars were attached to a polyacrylamide matrix and actual cell adhesion through the hepatocyte Gal/GalNAc (asialoglycoprotein) receptor was measured. In most adaptations, an antibody or a glycan-binding protein of interest is immobilized and the binding of a glycan to the protein is measured. For this to succeed, the glycans are conjugated in some way, such as to biotin or another protein with an attached reporter group (e.g., fluorescent moiety or enzyme, such as peroxidase). Competition ELISA-type assays have recently been developed to probe the binding site of a lectin or an antibody. In this approach, a glycan is coupled to a carrier protein (the target) and its binding to an immobilized protein in the solid phase is detected directly. Competitive glycans or antibodies to components in the assay are added to the wells and their competition for the target is observed and defined as an IC₅₀, i.e., the concentration at which they inhibit by 50% the binding of the target. The major advantage of this approach is its ease and the ability to define the relative binding activity of a panel of glycoconjugates. The major disadvantage is that it does not provide direct information about affinity constants or other thermodynamic parameters.

IC₅₀-Hapten Inhibition^(41–46)

In this approach, the ability of a soluable hapten to block the binding of a lectin or antibody to a target glycoconjugate is measured. (In glycobiology, the term hapten is often used to denote a small glycan that competitively binds to a lectin and competes for its binding to a more complex ligand.) The target may be cells, as in a visual agglutination assay, or immobilized, as in ELISA-type formats discussed above. The concentration of added hapten that provides 50% inhibition of binding of the lectin or antibody is taken as the inhibitory concentration (IC₅₀). Such approaches have been used for many years in studies on lectin agglutination of cells and were useful in elucidating the nature of the human blood group substances. If a sufficiently diverse panel of soluble haptens is used, then the relative efficacies of each glycan can be measured and defined as the IC₅₀. The IC₅₀ does not, however, relate directly to the binding affinity, since what is being measured is inhibition, and the amount of inhibitor needed is determined by the affinity of the lectin or antibody to the immobilized target. The actual binding affinity must be defined by other techniques described earlier in this chapter. However, the ease of deriving the IC₅₀ and the direct comparisons that arise from using a large panel of haptens make this an attractive method for many in the field.

Precipitation ^(47–51)

The interaction of a multivalent lectin or antibody with a multivalent ligand allows for the formation of cross-linked complexes. In many cases, these complexes are insoluble and can be identified as precipitates. The formation of precipitates using antibodies to multivalent ligands was of great historical significance in the development of the field of immunology. In this technique, a fixed amount of lectin or antibody (receptor) is titrated with a glycoprotein or a glycan. At a precise ratio of ligand to receptor, a precipitate is formed. Such precipitation is highly specific to the affinity constant of the ligand to the receptor. The amount of protein or ligand in the precipitate can be measured directly by chemical means, using assays for glycan or protein. The technique of precipitation is still useful for studying those potentially multivalent ligands and has been used to demonstrate recently that each branch of terminally galactosylated complex-type di-, tri-, and tetraantennary N-glycans is independently recognized by galactose-binding lectins. Another precipitation approach takes advantage of the fact that a complex between a lectin and a glycan can be “salted” out or precipitated by ammonium sulfate. A variation of this approach was used in early studies on the characterization of the hepatocyte Gal/GalNAc receptor (asialoglycoprotein receptor), in which the ligand (in this case ¹²⁵I-labeled asialoorosomucoid) was incubated with a preparation of receptor. The sample was treated with an amount of ammonium sulfate capable of precipitating the complex but not the free unbound ligand. The precipitated complex was captured as a precipitate on a filter and the amount of ligand in the complex was directly determined by γ-counting.

Electrophoresis ^(52–54)

In this approach, a glycoprotein (or ligand) is mixed with a glycan-binding protein or antibody, and the mixture is electrophoretically separated in polyacrylamide. For glycosaminoglycans, this technique is termed affinity coelectrophoresis (ACE) (see Chapter 29). This method is particularly useful in defining the apparent K_d of the interaction and allows for identification of subpopulations of glycosaminoglycans that differentially interact with the glycan-binding protein. In another method, termed crossed affinity immunoelectrophoresis, a second step of electrophoresis is conducted in the second perpendicular dimension in the presence of a precipitating monospecific antibody to the glycoprotein or ligand in 1% agarose gels. The gel is then stained with Coomassie brilliant blue and an immunoelectrophoretogram is obtained. Glycoproteins not interacting with the lectin or antibody have faster mobility than the complex. The amount of glycoprotein or ligand is determined by the area under the curves obtained in the two-dimensional analysis. This method is useful for studying glycoforms of proteins and has been particularly valuable in analyzing glycoforms of α1-acid glycoprotein, an acute phase glycoprotein, in serum and changes in its α1–3-fucosylation.

Expression of cDNAs for Ligands and Receptors ^(55–58)

Another modern approach to studying glycan-protein interactions is to express the cDNA encoding either a glycosyltransferase or a lectin in an animal or bacterial cell. The adhesion of the modified cell to a target is then measured. For example, such approaches have been highly valuable in studying selectins and their ligands. It helped lead to the identification of sialyl Lewis X and sialyl Lewis A as important recognition determinants for selectins and the expression cloning of the cDNA encoding the P-selectin glycoprotein ligand-1 (see Chapter 26). The expression of glycan-binding proteins, such as selectins or I-type lectins, on the cell surface of transfected cells has been helpful in evaulating the specific role of lectins in cell adhesion under physiological flow conditions (see Chapters 24 and 26).

Future Directions

The detailed interactions of glycans and proteins allow for “decoding” glycan structural information. However, the complex mechanistic details of these interactions are just beginning to be unraveled. Furthermore, increasing numbers of glycan-binding proteins identified to date suggest that each glycan may have a functional receptor, either in the animal or in a pathogenic organism attacking the animal. Understanding the molecular interactions between glycans and proteins is essential for the development of a real molecular-level appreciation of the biology of glycoconjugates. There are many approaches currently being used, and each has its advantages and disadvantages. Obviously, a combination of methods provides a better overall description than any single method alone. It is anticipated that new synthetic approaches to glycans will allow more detailed studies in the future, since the amounts of naturally occurring glycans of interest may be low and difficult to obtain in sufficient quantity. In addition, the increasing availability of recombinant forms of glycan-binding proteins will enhance the evolution of this area of glycobiology. These recent developments in understanding the molecular details of glycan-protein interactions may allow for the generation of specifically tailored compounds that mimic a glycan-binding site, which may allow formulation of new families of pharmaceutical agents. Finally, much of the present information derives from studies on small glycans and lectins, and little is known about the very complex interactions between a glycan-binding protein and its macromolecular glycoconjugate ligand. Although there are many methods of studying glycan-protein interactions, great difficulty still exists in translating the “one-dimensional” information based on free diffusion to the two- and three-dimensional nature of cell-cell adhesion or cell-matrix adhesion. In addition, exciting new developments in several fields suggest that higher-order structures of glycoconjugates may also be important in regulating glycan-protein interactions. As examples, glycosphingolipids may cluster in “rafts” and some glycoproteins have important carbohydrate determinants adjacent to noncarbohydrate determinants, such as tyrosine sulfate. The coordinated interactions between such organized glycan determinants may promote high-affinity interactions and present an even greater challenge to future research in this field.

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