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Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology [Internet]. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022. doi: 10.1101/glycobiology.4e.29
Glycans interact with many types of proteins including enzymes, antibodies, and lectins. Protein recognition and interactions with glycans represents a major way in which the information contained in glycan structures is deciphered and promotes biological activities. This chapter describes approaches to study the kinetics and thermodynamics of interactions between glycans and glycan-binding proteins (GBPs).
A tremendous variety of GBPs are known and many are discussed in Chapter 28 and in many other chapters in this book. GBPs differ in the types of glycans they recognize and in their binding affinity and kinetics. The underlying structural basis by which a GBP binds with specificity and high affinity to a very limited number of glycans (or even a single glycan) among the many thousands that are produced by a cell is discussed in Chapter 30. A wide variety of physical techniques are used to identify and quantify protein–glycan interactions. Differential affinities of glycans for different GBPs revealed by these approaches provide insight into the biological roles of glycans and their cognate GBPs. Characterization of protein–glycan recognition using such techniques, in combination with structural studies by nuclear magnetic resonance (NMR) and crystallography, is useful to identify novel antagonists or inhibitors of GBPs. Such approaches are being used, for example, to develop inhibitors of neuraminidases to treat influenza virus infections (Chapter 42) and to screen for high-affinity inhibitors of selectins for the treatment of inflammatory disorders (Chapter 34).
Much of the initial work on understanding protein–glycan interactions arose from studies on the combining sites of plant lectins and antibodies against specific blood group antigens. These studies led to the development of quantitative assays using glycans to inhibit binding interactions detected by cell agglutination or precipitation of targets, which provided early evidence for the importance of specific sugar structures in biological recognition events. Studies of protein–glycan interactions were instrumental in the development of techniques such as equilibrium dialysis and isothermal titration calorimetry, which are now widely used to analyze protein binding to a variety of types of ligands. On the other hand, methods used to study other types of protein–ligand interactions often need to be adapted to accommodate the specific properties of glycans and the proteins that interact with them.
Because many GBPs are oligomeric, with each subunit typically having a single carbohydrate-binding domain (carbohydrate-recognition domain [CRD]), many GBPs exhibit multivalent interactions with glycan ligands. Thus, although the CRD within a GBP may have a particular affinity for a ligand, the multivalent feature enhances binding through increased avidity and allows ligand cross-linking. Although the term “affinity” in this case, measured at equilibrium as a dissociation constant or affinity Kd, refers to the direct interaction of a single CRD with a monovalent ligand, “avidity,” measured as an avidity Kd, refers to the overall strength of multivalent interactions (Figure 29.1). Some researchers use the term “apparent” Kd to denote the nonequilibrium nature of the measurements. Examples of oligomeric and multivalent GBPs include plant lectins, galectins, which are soluble GBPs that typically associate into dimers and higher oligomers, and soluble C-type lectins, such as serum collectins, which are generally oligomeric. In fact, some GBPs, such as the mannose receptor, the mannose 6-phosphate receptors, and some galectins (Chapters 33, 34, and 36), have multiple CRDs within a single polypeptide and can bind multiple ligands. In such cases, the single protein–glycan interaction may be weak (mm–μm Kd), as for the mannose receptor, which binds α-linked mannose with affinity in the low millimolar range, but which can bind with high avidity to the surface of fungi or microbes that may be rich in mannose-containing ligands.
Monovalent and multivalent interactions of a glycan-binding protein (GBP) with monovalent or multivalent glycan ligands. A variety of interactions is possible, and these affect the equilibrium constant and contribute to the affinity Kd and the avidity (more...)
Membrane receptors can also function as oligomeric complexes. For example, the C-type lectin P-selectin, which is dimeric, and membrane Siglecs (Sia-binding immunoglobulin-like lectins) both cluster on the cell surface in the presence of glycan receptors on opposing cells. Similarly, the influenza virus hemagglutinin is trimeric and is present in multiple copies on a virion, whereas cholera toxin is a soluble protein that is an AB5 complex, consisting of a pentamer of glycan-binding B subunits associated with the catalytic and toxic subunit A. The glycan of ganglioside GM1 binds strongly to each B subunit of cholera toxin with high affinity because of specific and multiple interactions within each CRD (affinity Kd of ∼40 nm). As a pentamer in the AB5 form, cholera toxin has extremely high avidity (avidity Kd of ∼40 pm) for cells expressing GM1, which can occur in clusters at the cell surface.
Most plant lectins are dimers or tetramers and are thus multivalent. Of course, the density of glycans on glycoproteins can also affect the affinity of binding, as some glycoproteins carry multiple multiantennary N-linked chains, each of which may interact with a CRD of the GBP (Figure 29.1). In addition, some multivalent lectins GBPs, as seen for galectins, may “bind and jump” within a molecule from glycan to glycan, a type of internal diffusion that can alter the entropy of interactions and promote higher avidity.
The interaction of glycans with GBPs can be described thermodynamically and kinetically. Consider the simplest case in which a lectin (L) binds with a single site to a glycan (G) with a single binding determinant in a monovalent interaction. The interaction is governed by Equation 1 (Figure 29.2). At equilibrium the affinity constant, K, is defined as an association constant (or Ka) (Equation 2) and is equal to kon/koff, and the inverse of the Ka is the Kd or dissociation constant (Equation 3). Like any equilibrium constant, K is related to the standard free-energy change of the binding reaction at pH 7 (ΔGo) in kcal per mole (Equation 4), in which R is the gas constant (0.00198 kcal/mol-K) and T is the absolute temperature (in °K). The affinity constant K is related to the thermodynamic parameters ΔGo, ΔHo, and ΔSo (Equation 4), which represent the changes in free energy, enthalpy, and entropy of binding, respectively.
Equations governing the interactions of a glycan-binding protein or lectin (L) with a glycan ligand (G). The terms are defined in the text.
The on-rate kon is expressed in units of M−1sec−1 or M−1min−1, whereas koff is expressed in units of sec−1 or min−1. Although it is important to define Ka, kon, koff, ΔGo, ΔHo, and ΔSo for each binding phenomenon under consideration, investigators often discuss data in terms of the Kd (Equation 3), because the units are in concentration (millimolar, micromolar, nanomolar, etc.). It is also important to note that an equilibrium constant implies that the experimental setup for measuring it includes proof of actual equilibrium. In addition, the equilibrium constants are affected by temperature (increasing temperature decreases the equilibrium constant), buffer conditions, and potential cofactors, such as metals.
Whereas the binding of a monovalent GBP to a monovalent ligand is easily defined by the equilibrium kinetics described above, binding between multivalent ligands or GBPs involves multiple affinities, and the binding equilibria are more complex and more accurately described by a set of equilibrium constants. Typically, for multivalent ligands and GBPs, the values reported for affinity are apparent affinity constants and usually measure avidity.
There are many different ways to study the binding of glycans to proteins, and each approach has its advantages and disadvantages/limitations in terms of thermodynamic rigor, amounts of protein and glycan needed, and speed of analysis. Below is a discussion of some of the major ways in which the binding between a glycan and protein can be studied. Much of the available information about protein–glycan interactions derives from studies of relatively small glycan ligands interacting with a protein. In examining these interactions, two broad categories of techniques have been applied: (1) kinetic and near-equilibrium methods, such as equilibrium dialysis and titration calorimetry; and (2) nonequilibrium methods, such as glycan microarray screening, hapten inhibition, enzyme-linked immunoabsorbent assay (ELISA)-based approaches, and agglutination. In all of these approaches, the concepts of affinity versus avidity must be considered, and because of the multivalency of many GBPs and their ligands, it is difficult to precisely define the kinetic parameters, although the apparent affinity and avidity remain very useful measurements.
Equilibrium dialysis is one of the earliest and simplest methods to study the binding of a GBP to a glycan. Although the technique is not used that often currently, understanding the principles of equilibrium dialysis helps to clarify the concept of measuring equilibrium constants. A solution of a GBP, such as a lectin or an antibody, is placed in a dialysis chamber of defined volume; the chamber must be permeable to glycans or other small molecules but not to the GBP. The chamber with the GBP inside is then placed in a larger known volume of buffer that contains the glycan in the concentration range of the expected Kd. After equilibrium is achieved, defined as no further changes in concentrations of the glycan either inside or outside the chamber, the total concentration of glycan in the chamber [In] is measured. The value [In] is a combination of bound glycan (associated with the GBP) plus free glycan versus the concentration of glycan outside the chamber [Out], which is the free glycan only. This difference in glycan [In] and [Out] will depend on the amount and affinity of the GBP. From this information, both the Ka and the valence n can be determined from the relationship
A plot of r/c versus r for different hapten concentrations approximates a straight line with a slope of −Ka. The valence of binding (number of binding sites per mole) is defined by the r intercept at an infinite hapten concentration. If such an analysis were performed with cholera toxin, for example, one would obtain five binding sites per mole of AB5 complex or 1 mol per mole of B subunit. Nonlinear curve fitting is used to determine equilibrium constants, because older methods using linear conversions have inherent deficiencies and may distort experimental error.
As in any technique for determining binding constants, a number of important assumptions are made and their validity must be considered. These include demonstrating that the protein and glycan(s) are stable and active during the course of the experiment, the glycan is freely diffusible, the complex is at equilibrium, and structurally unrelated small molecules—not expected to bind—show no apparent binding in the experimental setup. If such binding is observed, this may be considered nonspecific and may be subtracted from the specific binding.
There are several advantages to equilibrium dialysis: (1) the approach is relatively easy, and highly sophisticated equipment is not needed; (2) if the affinity is high, then relatively small amounts of protein are needed (typically a few milligrams); (3) if the affinity is high, only small amounts of glycan may be required; (4) if the protein and glycan are very stable, they may be recovered and reused; (5) radioactive or fluorescent-tagged glycans may be used; and (6) reliable equilibrium measurements can be made. Some drawbacks of the approach are that (1) it provides the Ka but not the rate constants (kon or koff); (2) if the affinity of the GBP for the glycan is low, then relatively large amounts of GBP and glycan may be required; (3) the technique is not very adaptable to high-throughput analyses with multiple samples, and many individual measurements with different ligands may be tedious; and (4) many different measurements must be made if the range of affinity is unknown, and this may require many days or weeks to complete.
A variation of this technique is illustrated by the equilibrium gel-filtration method developed by Hummel and Dreyer. In the Hummel–Dreyer method, a GBP is applied to a gel-filtration column that has been preequilibrated with a glycan of interest that is easily detectable (e.g., by radioactive or fluorescent tagging). As the protein binds to the 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) that 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 Hummel–Dreyer 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 for equilibrium dialysis, except that Hummel–Dreyer analyses are often quicker to perform and can be used with ligands of many different sizes. Such an approach has been invaluable in defining the equilibrium binding of selectins to their ligands.
Affinity chromatography is a technique typically used to identify interacting partners, but under some variations can be used to measure both affinity and specificity. In this affinity chromatography, a GBP is immobilized to an affinity support, such as Affi-Gel, CNBr-activated Sepharose, Ultralink, or some other activated support. If a glycan or a glycosylated macromolecule binds tightly to an immobilized GBP, a buffer containing a known glycan ligand may be added to force dissociation of the complex. For example, oligomannose-type and hybrid-type N-glycans will bind avidly to an agarose column containing the plant lectin concanavalin A (ConA-agarose) and 10–100 mm α-methyl mannoside is required to elute the bound material efficiently. In contrast, many highly branched complex-type N-glycans will not bind. Biantennary complex-type N-glycans bind to ConA-agarose, but they do not bind as tightly as high-mannose-type N-glycans and their elution can be achieved using 10 mm α-methyl glucoside. In this manner, one can assess the binding specificity of a GBP. In practice, this approach is rather crude, and although it gives valuable practical information about the capacity of an immobilized lectin to bind specific glycans, it does not provide quantitative affinity measurements. A variant of this method is to immobilize the glycan ligand through covalent linkage or by capturing a biotinylated glycan on a streptavidin-linked surface and then measuring GBP binding.
A more sophisticated version of this approach, termed frontal affinity chromatography, can provide quantitative measurements of the equilibrium binding constants. In this technique, a solution containing a glycan of known concentration is continuously applied to a column of immobilized GBP, and the elution front of the glycan from the column is monitored. Eventually, enough ligand is added through continuous addition that its concentration in the eluant equals that in the starting material. If the glycan has no affinity for the GBP, it will elute in the void volume V0; if, however, the glycan interacts with the GBP, it will elute after the V0 and at a volume Vf (Figure 29.3).
Example of frontal affinity chromatography, in which different concentrations of a glycan are applied to a column of immobilized GBP. The profile depicts the elution of one glycan that binds the GBP and the elution of another glycan that does not bind (more...)
The advantages of frontal affinity chromatography are similar to those discussed for equilibrium dialysis: (1) the approach is easy and inexpensive; (2) if the affinity is high, then relatively small amounts of protein are needed (typically a few milligrams), and only a single column is required; (3) correspondingly, small amounts of glycan may be used if the Kd is in the range of 10 nm to 10 mm; (4) if the glycans are stable, they may be recovered and reused; (5) radioactive glycans may be used; and (6) reliable equilibrium measurements can be made. There are some limitations to this approach, including (1) only the Kd can be derived, not kon or koff; (2) the conjugation of the GBP to the matrix must be stable and the protein must retain reasonable activity for many different column runs; (3) the amount of GBP conjugated and active must be defined; (4) many different column runs must be made with a single glycan; and (5) if the Kd is high (>1 mm), this approach is typically not feasible. Overall, frontal affinity chromatography is quite useful and is automated.
Another variation is generally termed a “pull-down assay,” akin to a type of immunoprecipitation. In this approach a solution containing potential ligands is incubated with a GBP that may be immobilized on a surface (e.g., a bead). Afterward the bead-GBP is subjected to several steps (e.g., magnetic separation or centrifugation) to remove the unbound material. The material bound to the bead-GBP may then be eluted for measurement and further analyses. Using this pull-down setup, one can perform a concentration-dependent binding assay to obtain an apparent Ka of the ligand for the immobilized GBP.
Isothermal titration calorimetry (ITC) is one of the most rigorous means of defining the equilibrium binding constant between a glycan and a GBP or indeed any protein and its ligand. The binding of a glycan to the GBP is measured as a change in enthalpy using a commercial microcalorimeter. In this technique, a solution containing a glycan of interest is added in increments into a solution containing a fixed concentration of GBP. The glycan is added at many intervals and the heat evolved from binding is measured relative to a reference cell. Over the course of the experiment, the concentration of glycan is increased in the mixing cell over a glycan-to-GBP molar ratio of 0–10. The heat absorbed or evolved during binding is determined and the data are replotted as kcal/mole of injectant versus the molar ratio (Figure 29.4). These data are then analyzed by replotting data to obtain the Kd. The heat change is directly related to the enthalpy of reaction ΔHo. From knowledge of the Kd and ΔHo, and using Equation 4, it is possible to define the binding entropy ΔSo.
Example of isothermal titration calorimetry (ITC). (Top) Increasing amounts of a glycan are injected to a fixed amount of glycan-binding protein (GBP) in a cell, and the heat produced upon binding is measured as μcal/sec. (Bottom) The total kcal/mol (more...)
The major advantage of this approach is that it can provide thermodynamic information about the binding of a glycan to a GBP and is thus superior to equilibrium dialysis and affinity chromatography. The limitations of this approach are that (1) relatively large amounts of protein may be required to conduct multiple experiments (>10 mg); (2) relatively large amounts of glycans may be required; and (3) because of the above-mentioned problem, it is not typical for such analyses to use a wide range of different glycans. Nevertheless, this approach is rigorous and if the titration cell dimensions could be decreased in the future, then lower amounts of materials would be required.
Surface plasmon resonance (SPR) is used to measure association and dissociation kinetics of ligands (analytes) with a receptor. In SPR, the association of the analyte and receptor, with one or the other immobilized on a sensor chip, which incorporates a critical metal sensing surface, induces a change in total surface plasmon waves resulting in a change in the refractive index of the layer in contact with a gold film (Figure 29.5). This change is recorded as the SPR signal or resonance units (RUs). Binding is measured in real time, and information about the association and dissociation kinetics can thus be obtained, which in turn can be used to obtain Ka and Kd from Equations 2 and 3. There are several instruments available from various companies based on the principle of SPR.
Example of surface plasmon resonance (SPR). (A) In SPR, the reflected light is measured and is altered in response to binding of the analyte in the flow cell to the immobilized GBP. (B) An example of a sensorgram showing the binding of the analyte to (more...)
A variety of chemistries are available for coupling ligand or receptor to the surface of the chip, including reaction with amines, thiols, or aldehydes and noncovalent biotin capture. In some approaches, a glycoprotein ligand for a GBP is immobilized and binding of the GBP is measured directly. It is also possible to degrade the immobilized glycoprotein ligand on the chip sequentially by passing over solutions containing exoglycosidases and reexamining at each step the binding to different GBPs, thereby obtaining structural information about the ligand. The immobilized ligand is usually quite stable and can be used repeatedly for hundreds of runs during 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 kon and koff are routine (see Equations 2 and 3), making calculations of Kd straightforward; (3) for immobilization of a molecule using amine coupling, only 1–5 µg is normally sufficient; (4) typically, the concentration range of the analyte is 0.1–100×Kd 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 (1) analytes must have sufficient mass to cause a significant change in SPR on binding (thus, the glycan is usually immobilized instead of the protein); (2) coupling of free glycans to the chip surface may be inefficient, and thus neoglycoproteins or some other type of large conjugate may be needed; and (3) there may be inhomogeneity in conditions on the SPR instrument because of mass transport effects, which could affect the dissociation rate and thus provide an inaccurate Kd measurement.
A related technology is biolayer interferometry (BLI), in which an optical biosensor analyzes the interference pattern of white light reflected from two surface. One surface carries the GBP immobilized in a biosensor tip, and the other surface is used as a reference. Binding of molecules to the biosensor tip changes the interference pattern in real time, thus allowing measurement of the kinetics of binding and dissociation. A clear advantage of this technique is that it does not require labels, such as fluorescent tags for detection (i.e., it is label-free). Additionally, BLI is generally performed in a 96- or 384-well format enabling it to be high-throughput. It is not quite as sensitive, however, as SPR, and thus small ligands, such as small oligosaccharides, may not provide robust signals. Like SPR, one of the interacting partners (e.g., the GBP) must be immobilized; with multiple tips it is difficult to ensure equal levels of immobilization. Nevertheless, this technique is easy to use and less costly compared to SPR, and when employed with the Cheng–Prusoff model in assays to determine oligosaccharide inhibition, BLI has been shown to generate solution Kd values that agree with those from NMR assays.
Fluorescence polarization is an established technique but only relatively recently has it been applied to measure the binding constants of glycans to GBPs. This approach is based on the reduced rotational motion of a relatively small glycan when it is bound to a relatively large protein compared with the rotation of the free glycan in solution. Light absorbed by the fluorophore is emitted as fluorescence, but the angle of the emission relative to the incident light is depolarized by rotation of the molecule in solution, as measured through a filter to select molecules oriented close to the plane of incident polarized light. In practice, a fluorescently labeled glycan is incubated with increasing concentrations of a GBP and the fluorescence depolarization is measured. In the absence of the GBP, the fluorescently labeled glycan tumbles randomly and the degree of polarization is low. However, if the fluorescently labeled glycan binds to the GBP, its rate of rotation is diminished and the polarization remains high. By this approach, one can measure directly the Kd of the interaction as a function of the GBP concentration. The advantages of this technique are that (1) it is a homogeneous assay and provides direct measurements of the Kd in solution without derivatization of the GBP; (2) it is relatively simple and can provide rapid measurements of many compounds using microtiter plate–based approaches; (3) it uses relatively small amounts of glycan; (4) the concentrations of all the molecules are known; (5) it avoids complications of multivalent interactions because the glycans are monovalent and free in solution; and (6) it is amenable to inhibition by competitive agents and can be used to determine relative potency of compounds as inhibitors of GBPs. In the latter approach, a single fluorescently labeled glycan is mixed with the GBP, increasing concentrations of inhibitor glycans (which are not fluorescently labeled) are added, and the inhibition of binding is measured. Because the interactions are simple single-site competition, it is possible to use the concentration that causes 50% inhibition (IC50) to derive the approximate Kd for the inhibitor. Some of the disadvantages are that (1) the technique is limited to small glycans (≤2000 Da); (2) it requires fluorescence derivatization of the glycan (the fluorophore may alter the properties of the glycan); and (3) preparation of the glycan and chemical derivatization may be tedious and require large amounts of glycans. However, once a fluorescently labeled glycan has been generated, there is usually enough for many assays.
More complex approaches to measure noncovalent complexes between proteins and glycan ligands include mass spectrometry (MS) and NMR. Soft ionization methods are used to detect protein–glycan complexes in electrospray ionization MS and in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Although such methods have several advantages, including sensitivity and the ability to measure binding to multiple ligands in a single experiment, they may also be complicated to interpret, because the noncovalent complex may dissociate during the measurement, the detection may not be quantitative, and the ionization efficiency of the complex versus the free protein or ligand may differ. Another MS approach is hydrogen deuterium exchange (HDX) mass spectrometry, in which the bound ligand alters the deuterium uptake kinetics on the protein at the binding site of the ligand. Thus, the increased association of ligand decreases deuterium uptake, which can be measured on appropriate peptides proteolytically prepared from the protein after the exchange, and this can be plotted to obtain a binding isotherm. This method has the advantages of sensitivity and ease of use, but it also has drawbacks, including the expense of the approach and instrument time, the problem that relatively small glycans may have limited impact on deuterium exchange, and the problem that conformational changes can affect exchange rates in regions outside the CRD.
Several types of NMR spectroscopy measurements are useful for measuring protein–glycan interactions. Interactions can be measured in real time in solution and there is no need to separate the protein–glycan complex from the unassociated GBP or glycan. When weakly bound ligands are in fast exchange with free ligand, both line broadening and changes in chemical shifts can be used; however, the protein–glycan complex has to have clear and detectable chemical shifts distinct from the unbound materials. Saturation transfer difference (STD) measurements can also be used. Here, 1H-NMR is used to detect glycans which are in large excess over GBP. Often these techniques work best for relatively low affinity interactions.
Another spectroscopic approach to measuring glycan binding to a protein relies on changes in intrinsic fluorescence on interacting with a glycan. Finally, atomic force microscopy (AFM) can also be used to define protein–glycan or glycan–glycan interactions. In this approach, the force required to separate a glycan-coated bead from an immobilized GBP can be measured in highly quantitative methods. Remarkably, single-molecule force measurements can be made by AFM. This method not only gives information about the strength of binding, but also provides insight into the molecular nature of noncovalent bond formation between the protein and its ligand.
The conventional ELISA has been adapted for studying glycans and GBPs in a variety of formats. Of course, many glycans are antigens and antibodies to them can be analyzed in the conventional ELISA format. Some of the earliest ELISA-type approaches used biotinylated bacterial polysaccharides captured on streptavidin-coated microtiter plates to measure interactions of antibodies to the polysaccharides. In most types of ELISAs used in glycobiology, either an antibody or a GBP of interest is immobilized and the binding of a soluble glycan to the protein is measured, or the reagents are reversed and the glycan or glycoconjugate is immobilized. In either approach, the glycans are modified in some way, such as addition of biotin or a fluorophore, to allow their detection, or they may be coupled to another protein with an attached reporter group (e.g., a fluorescent moiety or an enzyme such as peroxidase).
Competition ELISA-type assays are also used to probe the binding site of a GBP. In this approach, a glycan is coupled to a carrier protein (the target), and its binding to an immobilized GBP is detected directly. Competitive glycans are added to the wells and their competition for the GBP is measured as a function of concentration to obtain an IC50. Under appropriate conditions and concentrations of the ligand, the Ki values and Kd values are similar. The major advantages of this approach are that (1) it is relatively easy; (2) it has high-throughput capability and can be used in an automated fashion by robotic handling; (3) it can provide relative Kd values if the GBP concentration is varied appropriately over a large range and binding is saturable; and (4) it has the capacity to define the relative binding activity of a panel of glycoconjugates. The major limitations of this approach are that (1) it does not provide direct information about affinity constants or other thermodynamic parameters; (2) it can require relatively high amounts of GBP and glycans if used as a general screening array; and (3) it usually requires chemical derivatization of glycans or GBPs.
Glycan microarrays are an extension of both ELISA-type formats and modern DNA and protein microarray technology. In a glycan microarray, glycans are linked, usually covalently, to a solid surface through reaction with N-hydroxysuccinimide (NHS)-esters or epoxide-containing supports on a glass slide. Glycans are prepared to contain reactive primary amines at their reducing termini, but other chemical coupling methods are available. In addition, noncovalent immobilization can be used in which lipid-derivatized glycans or glycolipids are deposited onto nitrocellulose-coated slides. Also, neoglycoconjugates (e.g., glycans covalently attached to protein carriers as in neoglycoproteins) can be generated and then coupled to a surface to present glycans for interactions with GBPs. The glycans or glycan-containing materials are printed, much like DNA is printed for DNA microarrays, using contact printers or piezoelectric (noncontact) printing (Figure 29.6). Usually, a few nanoliters of solutions containing glycans in concentrations of 1–100 µm are deposited by a robotic printer on the glass surface in ∼100-µm-diameter spots. Slides are incubated for several hours to allow the chemical reactions to covalently fix the samples on the slides. The slides are then blocked to prevent nonspecific binding of reagents, and these microarrays overlaid with a buffer containing the GBP and incubated for several hours to allow equilibrium binding to occur. The slides are washed to remove unbound GBP and then analyzed. Analyses involve fluorescence detection, which means that either the GBP has to be directly fluorescent-labeled or a fluorescent-tagged antibody to the GBP must be used.
Preparation of covalent glycan microarrays printed on N-hydroxysuccinimide (NHS)- or epoxide-activated glass slides. In this example, the glycans have a free amine at the reducing end and are coupled to the glass slide. After washing to remove unattached (more...)
The chief features of the successful microarrays are the variety of glycans they contain. However, the clustered and relatively high amount of glycans densely packed that can bind detectable amounts of GBPs, may also promote binding of even relatively low affinity multivalent GBPs. Thus, the density of the ligand should be taken into account when interpreting the results. The type of linker used and the state of the monosaccharide at the reducing end (i.e., open ring, open or closed ring form attached to the linker) can also affect binding.
Binding is visualized or imaged as intensely fluorescent spots against a dark background. The data are visually imaged on a scanner and then graphically represented. In a typical successful analysis a GBP may bind to several glycans that share structural features, often termed glycan-binding determinants or motifs. If desired, the GBP can then be tested for its binding to the identified candidates by other methods such as titration microcalorimetry or fluorescence polarization, to define the Kd as discussed above. The use of microarrays in characterizing GBPs is a central component of functional glycomics (Chapter 51). Publicly available glycan microarray binding data repositories are increasingly used in the field.
A variation of glycan microarrays is a bead-based Luminex-type assay. In this high-throughput approach glycans are covalently immobilized on Luminex beads of different fluorophore properties. Thus, a multiplexed approach is taken where a GBP is mixed with a set of conjugated beads each identifiable by its fluorescent properties. The GBP may be biotinylated, allowing detection by fluorescent streptavidin, or the GBP may be detected by binding to specific antibody or another reagent if the GBP is epitope-tagged. The degree of binding of the GBP is a measure of its affinity and specificity for a particular glycan. The advantages of this approach is that it is high-throughput, allowing thousands of assays to be performed automatically, as well as utilizing small amounts of both glycan and GBP. The collection of data and its analysis are also automated. A limitation of the approach is that each glycan to be derivatized must be activated by having a primary amine or other chemistry for conjugation, and each conjugation of a glycan needs to be independently validated.
Multivalent GBPs interacting with multivalent ligands, as expressed on cells, can cause the cells to agglutinate. This can be exploited by allowing one to measure the ability of a soluble glycan to block the agglutination activity of the GBP. The concentration of the soluble glycan that provides 50% inhibition of agglutination is taken as the inhibitory concentration (IC50). Such approaches have been used for many years in studies on lectin-induced agglutination of cells and were useful in elucidating the nature of the human blood group substances. If a sufficiently large panel of soluble glycans is used, then the relative efficacies of each of these can be measured to help define the specificity of the GBP. A major advantage of this technique is that it does not require tagging of the glycans. Furthermore, polystyrene or dextran beads modified with discrete glycans can be used in lieu of cells. In this case, the glycans on the agglutinating particle are better defined. Usually, the IC50 does not relate directly to the binding affinity, because inhibition is being measured. The actual binding affinity must be defined by other techniques described earlier in this chapter.
The interaction of a multivalent GBP or antibody with a multivalent ligand allows formation of cross-linked complexes in solution. In many cases, these complexes become insoluble and precipitate. Precipitation may be highly specific and reflects the affinity constant of the ligand for the receptor. To quantify this interaction a fixed amount of GBP or antibody is titrated against a glycoprotein or a glycan to which it binds and a precipitate will form at a precise ratio of ligand to receptor. The amount of protein or ligand in the precipitate can be measured directly by chemical means, using assays for glycans or proteins. The technique of precipitation is useful for studying potentially multivalent ligands, and it has been used recently to show that each branch of terminally galactosylated complex-type di-, tri-, and tetra-antennary N-glycans is independently recognized by galactose-binding lectins. Another precipitation approach takes advantage of the fact that a complex between a GBP 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 asialoglycoprotein receptor (AMR), in which the ligand 125I-labeled asialoorosomucoid was incubated with a preparation of receptor. The sample was treated with sufficient ammonium sulfate to precipitate the complex but not the unbound ligand. The precipitated complex was captured on a filter and the amount of ligand in the complex was determined by γ-counting.
In this approach, a glycoprotein (or ligand) is mixed with a GBP or antibody and the mixture is electrophoretically separated in polyacrylamide. For glycosaminoglycans, this technique is termed affinity co-electrophoresis (ACE). This method is particularly useful in defining the apparent Kd of the interaction and allows identification of subpopulations of glycosaminoglycans that differentially interact with the GBP. In another method, termed crossed affinity immunoelectrophoresis, a second step of electrophoresis is conducted in the second perpendicular dimension across an agarose gel that contains precipitating monospecific antibody to the glycoprotein or ligand. The gel is then stained with Coomassie Brilliant Blue and an immunoelectrophoretogram is obtained. Glycoproteins not interacting with the GBP 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 second 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.
An indirect approach to studying protein–glycan interactions is to express a copy DNA (cDNA) encoding a glycosyltransferase in an animal or bacterial cell (Chapter 56). The adhesion of the modified cell (either transiently or stably transfected) to a GBP or antibody is then measured and taken to reflect the binding of the GBP or antibody to the new glycans (neoglycans) on the cell surface. Conversely, a cDNA encoding a GBP is expressed in cells and their ability to bind glycan ligands is tested. The expression of selectins, Siglecs, and other GBPs on the cell surface of transfected cells has been helpful in evaluating the roles of GBPs in cell adhesion under physiological flow conditions.
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