- Email: [email protected]
Determination of Glycolipid–Protein Interaction Specificity
[15]
glycolipid–protein interactions
205
[15] Determination of Glycolipid–Protein Interaction Specificity By PABLO H. H. LOPEZ and RONALD L. SCHNAAR Abstract
Glycolipids are found on all eukaryotic cells. Their expression varies among tissues, with the highest density found in the brain, where glycolipids are the most abundant of all glycoconjugate classes. In addition to playing roles in membrane structure, glycolipids also act as cell surface recognition molecules, mediating cell–cell interactions, as well as binding certain pathogens and toxins. Because of their amphipathic nature, underivatized glycolipids are amenable to immobilization on hydrophobic surfaces, where they can be probed with lectins, antibodies, pathogens, toxins, and intact cells to reveal their binding specificities and affinities. Three particularly useful methods to probe specific glycolipid‐mediated recognition events are microwell adsorption (ELISA), thin layer chromatography overlay, and surface plasmon resonance (SPR) spectroscopy.
Overview
Glycolipids are amphipathic molecules consisting of a hydrophilic oligosaccharide chain linked to a hydrophobic lipid that anchors the glycolipid in cell membranes (Hakomori, 2002). Two major classes of glycolipids, glycoglycerolipids and glycosphingolipids, are distinguished by their lipid moieties, diacylglycerol (or alkylacylglycerol) and ceramide, respectively. Whereas glycerol‐based glycolipids are most abundant in microbes and plants, glycosphingolipids are by far the most abundant and diverse class of glycolipids in animals. Animal glycosphingolipids are classified into families according to their core glycan sequences, on which many different terminal variations are elaborated (Stults et al., 1989). Glycosphingolipid families are expressed in tissue‐specific patterns. For example, ganglio‐series glycosphingolipids, based on the core structure Gal1‐3GalNAc1‐4Gal1‐4Glc1‐10 ceramide, predominate in the brain (Schnaar, 2000), whereas neolacto‐series glycolipids, based on the core structure Gal1‐4GlcNAc1‐3Gal1‐4Glc1‐10 ceramide are common on leukocytes (Stroud et al., 1996). Glycosphingolipids are further subclassified as neutral, sialylated (having one or more sialic acid residues), or sulfated (Stults et al., 1989). Sialylated glycosphingolipids are also known as gangliosides, which are widely distributed in vertebrates and METHODS IN ENZYMOLOGY, VOL. 417 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)17015-9
206
glycolipid interaction
[15]
are the most abundant glycoconjugates on nerve cells. Hundreds of distinct glycolipid structures have been reported (Stults et al., 1989), and their structural diversity underlies glycosphingolipid functions, being responsible for specific cell–cell interactions, pathogen/toxin tropism, and autoimmune pathologies (Hakomori, 2002; Schnaar, 2004; Willison, 2005). Whereas this chapter describes methods to determine glycolipid binding specificities using purified or partially purified natural glycolipids, the methods can also be applied to synthetic neoglycolipids (Feizi and Chai, 2004). Although the amphipathic nature of glycolipids poses challenges for their isolation and purification, their lipid moieties provide the means to readily immobilize and orient them on surfaces for probing with complementary binding molecules. In this chapter, methods to probe binding specificities of purified glycolipids by means of microplate adsorption and surface plasmon resonance spectroscopy (SPR) are described, as well as methods to identify binding components of glycolipid mixtures using thin layer chromatography (TLC) overlay.
Materials
Glycolipids Organic solvents are used to solubilize endogenous glycolipids from tissues and cells, where they are typically expressed on plasma membranes. Extraction procedures have been optimized, often using defined chloroform‐methanol‐water mixtures added in specific solvent sequence and ratio, to maximize precipitation and removal of proteins and nucleic acids while maximizing solubilization of glycosphingolipids along with other lipids (Schnaar, 1994). Because glycosphingolipids aggregate with each other and other lipids in aqueous solution, organic solvents are used throughout subsequent purification steps, which typically involve solvent partition, ion exchange chromatography, and/or silicic acid chromatography. Details for glycolipid purifications, which vary depending on the source and specific nature of the target molecules, have been published elsewhere (Schnaar, 1994), and are beyond the scope of this chapter. Purified glycolipids dissolved in organic solvents (e.g., chloroform‐methanol‐water [4:8:3]) are typically stable when stored at 20 for years or even decades. Microplate (ELISA)‐Based Recognition 1. 96‐Well EIA/RIA clear flat‐bottom untreated polystyrene microplate (Corning Life Sciences, Acton, MA, product #9017) (see Note 1) 2. Reagent grade butanol (for prewashing microplates)
[15]
glycolipid–protein interactions
207
3. Reagent grade 100% ethanol 4. A 100‐fold stock of a lipid mixture consisting of 0.1 mM phosphatidylcholine (PC) and 0.4 mM cholesterol is prepared in ethanol. To 4.8 ml of 100% ethanol add 0.15 ml of a 3.3 mM stock of phosphatidylcholine in chloroform (Avanti Polar Lipids, Alabaster, AL, product 840053) and 50 l of 40 mM stock of cholesterol in chloroform (prepared from dry powder, Avanti product 700000). Store the lipid stock tightly capped in a glass tube with Teflon‐lined seal at 20 . 5. Dulbecco’s phosphate‐buffered saline (PBS) contains (g/l): NaCl (8.0), KCl (0.20), CaCl2 (0.10), MgCl26H2O (0.10), Na2HPO4 (2.3), and KH2PO4 (0.20) (Bashor, 1979). 6. Microplate blocking buffer is prepared by dissolving bovine serum albumin (BSA, e.g., Sigma‐Aldrich, St. Louis, MO, product A‐7030) in PBS at 1 mg/ml TLC Overlay 1. Aluminum‐backed silica gel high‐performance HPTLC plates (20 20 cm, E. Merck, Darmstadt, Germany, product 5547) (see Note 2) 2. TLC running solvent. A wide‐spectrum running solvent suitable for many applications is chloroform‐methanol‐aqueous 0.25% w/v KCl (60:35:8) (see Note 3) 3. Glass TLC chromatography developing tank (e.g., 12 5 12 cm, rectangular, Camag Scientific, Muttenz, Switzerland, product 022.5515). 4. Microliter TLC spotting syringe (Hamilton Co., Reno, NV, product 701N) 5. Glass petri dish 6. Poly(isobutyl methacrylate) (PIBM) solution. A stock solution of 5% w/v of PIBM (e.g., Sigma‐Aldrich, product 181544) dissolved in chloroform is prepared. The stock solution is diluted to working concentration (e.g., 0.1% w/v) in n‐hexane immediately before use (see Note 4) 7. Binding buffer is prepared by adding 1 mg/ml BSA and 0.05% (w/v) Tween‐20 (e.g., Pierce Chemical Co., Rockford, IL, product 28320) to PBS Surface Plasmon Resonance Spectroscopy 1. SPR‐based biosensor (e.g., Biacore 3000, Biacore AB, Uppsala, Sweden). 2. Lipid vesicle extrusion apparatus (Avanti Polar Lipids, product 610000) and 50‐nm polycarbonate filters (product 610003)
208
glycolipid interaction
[15]
3. 1,2‐Dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC, Avanti Polar Lipids product 850345) 4. SPR blocking buffer is prepared by dissolving 0.1 mg/ml BSA in 10 mM sodium phosphate, 150 mM sodium chloride, and adjusting to pH 7.4 (degassed and filtered daily) 5. Running buffer consists of 10 mM phosphate buffer, pH 7.4, 150 mM sodium chloride, pH 7.4 (degassed and filtered daily) 6. Sensor chip L1 (product BR‐1005‐58), Biacore AB 7. Washing solution consists of 20 mM CHAPS detergent (e.g., Sigma C‐5070) in water 8. Regenerating solution consists of 20 mM NaOH in water (degassed and filtered daily) Methods
The three methods for determining glycolipid–protein interactions described here—microplate, TLC, and SPR—provide a wide range of capabilities applicable to different experimental goals. In each case, glycolipids are non‐covalently adsorbed to a solid support (microwell, TLC plate or SPR chip), where they remain stably attached in aqueous solutions. Exposure of the adsorbed glycolipids to potential binding proteins (lectins, antibodies, toxins) or biological entities (viruses, bacteria, intact cells) results in specific interactions that are detected using direct or indirect methods. Microplate adsorbed purified glycolipids can be used to determine relative binding specificities of soluble lectins, antibodies, toxins, pathogens, or cells, or for high‐throughput screening, for example, of sera or hybridoma supernatants. TLC overlay is most useful for detecting and identifying binding species within mixtures of partially purified glycolipids from natural sources. SPR using purified glycolipids and unlabeled soluble glycan binding proteins (GBPs) provides real‐time binding kinetics for determination of affinities and specificities of glycolipid–protein interactions. Microplate (ELISA)‐Based Recognition Because of their amphipathic nature, glycolipids spontaneously and stably adsorb to hydrophobic surfaces under the appropriate conditions. Commercially available polystyrene microplates are excellent substrates for glycolipid immobilization and are amenable to subsequent incubation with GBPs and their detection by direct or indirect methods (Collins et al., 2000; Foxall et al., 1992; Schnaar et al., 2002). 1. To remove mold release agents used in their manufacture, microplates are prewashed (batchwise) with butanol and ethanol and then stored for
[15]
glycolipid–protein interactions
209
subsequent use. Microplates are immersed upright in a shallow glass dish (e.g., a glass baking dish) of butanol and allowed to incubate 15 min. Plates are drained and immersed in three sequential dishes of ethanol, draining between transfers. Finally, plates are drained, allowed to air dry, and are stored in sealed plastic bags. 2. Desired concentrations of glycolipid (typically 2–200 pmol/well) are adsorbed, along with a constant amount of PC (25 pmol/well) and cholesterol (100 pmol/well), as follows. PC/cholesterol working solution is prepared by diluting the stock 100‐fold in 100% ethanol (final concentration 1 M PC, 4 M cholesterol). The total amount of glycolipid required is calculated, and an aliquot containing a 10–20% excess (to account for handling losses) is transferred to a glass test tube and evaporated to dryness under a stream of nitrogen or in an evaporator (e.g., SpeedVac Concentrator, Thermo Electron Corp., Waltham, MA). The dried glycolipid is redissolved at the desired concentration(s) in the PC/cholesterol working solution. A bath sonicator (brief sonication) and/or vigorous vortex mixing are used to ensure that the glycolipids are efficiently redissolved. Glycolipid solutions are stored in screw‐capped glass tubes until ready for addition to microplate wells (see Note 5). 3. Immediately before addition to microplate wells, glycolipid solutions (from Step 2) are diluted with an equal volume of water and mixed vigorously. A 50‐l aliquot is then pipetted into each well of a 96‐well microplate using standard single or multichannel micropipettors with standard plastic disposable tips. The plate is then placed on a well‐ventilated bench or chemical fume hood and left uncovered at ambient temperature for 90 min to allow partial evaporation. After incubation, any remaining solvent is removed by inversion and vigorous shaking of the microplate, and the wells are washed three times by repeated immersion in water, inversion, and shaking. The plate is righted and water (200 l/well) added. Glycolipid‐adsorbed plates may be used immediately or stored in water for several hours (e.g., overnight) at ambient temperature without measurable loss of adsorbed lipids (see Note 6). 4. Water is removed from the glycolipid‐adsorbed wells by inverting and shaking the microplate, then is replaced with 200 l/well of microplate blocking buffer. The plate is incubated covered for 30 min at 37 , then the blocking buffer is removed by inversion and shaking, and the wells washed three times with PBS. 5. Binding and detection. Microplate‐adsorbed glycolipids are amenable to probing with lectins, proteins, and other biological entities that are either directly labeled or that can be detected with secondary reagents. Any standard ELISA‐type protocol can be applied to the microplates. One protocol for determining anti‐glycolipid antibody binding specificity follows (Schnaar et al., 2002). Primary antibodies (antisera, hybridoma
210
glycolipid interaction
[15]
supernatants, monoclonal antibodies, etc.) are diluted (typically 1 g/ml antibody) in microplate blocking buffer and 50 l is added to each glycolipid‐adsorbed well. After 90 min at ambient temperature, the microplate is washed three times by immersion in PBS, inversion, and shaking. Secondary antibody (e.g., alkaline phosphatase (AP)–conjugated anti‐human or anti‐mouse IgG, as appropriate) diluted (typically to 1 g/ml) in microplate blocking buffer, is then added at 50 l/well and the microplate incubated 45 min at ambient temperature. Finally, the microplate is washed by immersion in PBS, then water (twice) and AP substrate, consisting of 2 mg/ml p‐nitrophenylphosphate in developing buffer (100 mM Tris, 100 mM NaCl, 5 mM MgCl2) is added at 100 l/well. Color development is determined with a microplate reader. An example of the use of microplate‐adsorbed gangliosides to determine binding specificities of antiganglioside monoclonal antibodies is shown in Fig. 1 (Schnaar et al., 2002) (see Note 7). TLC Overlay TLC overlay is performed in two steps (Karlsson and Stromberg, 1987; Magnani et al., 1980). First, a glycolipid sample is applied to a silica gel TLC plate and developed to resolve the different species. Second, the developed plate is coated with a thin plastic film and overlain with aqueous solution containing soluble GBPs or other biological binding entities to be analyzed. After washing to remove unbound material, bound material is detected either directly or using secondary reagents. When combined with enzymatic and/or chemical modification in situ, TLC overlay is a powerful tool for the analysis of glycolipid–protein interactions (Schnaar and Needham, 1994). Thin Layer Chromatography 1. The chromatography tank is pre‐equilibrated by adding developing solvent (typically chloroform‐methanol‐aqueous mixtures, see ‘‘Materials’’) to a depth of 0.5 cm in the bottom of the chromatography developing tank, which is then covered with a grease‐free lid (glass or metal) and incubated for at least 30 min before introducing the spotted TLC plate. The tank is placed in an area of uniform temperature protected from drafts or heat sources. 2. TLC plates are prepared for spotting. Aluminum‐backed HPTLC plates are cut to desired sizes with a scissors, using care to avoid folding or flaking of the silica gel sorbent. The width of the plate should be 3 cm plus 1 cm per sample to be spotted. The silica gel sorbent along cut edges is smoothed using the dry tip of a gloved finger. Using a ruler and pencil,
[15]
glycolipid–protein interactions
211
FIG. 1. Specific binding of four different antiganglioside monoclonal antibodies1 to major brain gangliosides (Schnaar et al., 2002). The four major brain gangliosides GM1 (——), GD1a (—□—), GD1b (——), and GT1b (—▾—) were adsorbed to microplate wells, along with phosphatidylcholine and cholesterol, at the indicated ganglioside concentrations (pmol/well added). Highly specific binding of four different IgG1‐class antibodies (one for each of the gangliosides, as indicated) was demonstrated using the methods described. Data are presented as the mean and range for duplicate determinations. Sugar structures are represented using the symbol nomenclature of the Consortium for Functional Glycomics (http://functionalglycomics. org). The four major brain gangliosides shown have the same neutral glycan backbone (Gal1‐ 3GalNAc1‐4Gal1‐4Glc1‐10 ceramide) with different numbers and positions of sialic acids (diamonds) as shown. Modified from Schnaar et al. (2002), with permission.
light marks for sample application are drawn along a line 1 cm above and parallel to the bottom of the TLC plate. Each sample application line should be 0.5 cm long, leaving 1.5 cm unused on the lateral edges of the plate (where distortion may occur during development) and 0.5 cm between samples (see Note 8). 3. Glycolipid samples are dissolved in organic solvents for spotting. For pure samples, 100 pmol/lane (or less for high‐affinity binding proteins) is 1
Under a licensing agreement between Seikagaku America and the Johns Hopkins University, Dr. Schnaar is entitled to a share of royalty received by the University on sales of the monoclonal antibodies described in Figs. 1 and 2. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
212
glycolipid interaction
[15]
sufficient. For mixtures, 1 nmol (or more) may be spotted per lane (see Note 9). Optimally, each sample should be spotted in a total volume of 1 l, although larger volumes (up to 10 l) may be spotted with sufficient care and patience. Aliquots of glycolipid sample in organic storage solutions are evaporated to dryness under a nitrogen stream or in an evaporator then redissolved in a small volume of methanol or chloroform‐methanol‐water (4:8:3) for spotting. 4. Using a Hamilton syringe with the beveled edge of the needle closely apposed but not touching the TLC plate surface, samples are applied along the premarked pencil lines. For each lane, 0.5 l is applied, allowed to dry, then application and drying are repeated until the total desired volume has been spotted. Sufficient time is allowed after the last sample is applied for the spotting solvent to completely evaporate (see Note 10). 5. The TLC plate, with samples applied, is placed in the pre‐equilibrated developing tank, with the top of the plate resting against the tank wall. The tank is kept covered at uniform temperature away from drafts or heat sources until the solvent moves up the plate by capillary action to within a few millimeters from the top of the plate. To ensure reproducibility of TLC runs, used developing solvent is discarded after each run (see Note 11). TLC Overlay 1. After the solvent has reached the top of the TLC plate, it is removed from the developing tank and allowed to dry completely. Drying may be aided by unheated forced air (see Note 10). One corner of the aluminum‐ backed TLC plate is bent toward the sorbent‐adsorbed surface using a forceps to facilitate easier handling during subsequent steps. A glass petri dish is filled to 0.5‐cm depth with 0.1% PIBM in hexane. The TLC plate is slowly inserted, at an angle into the solution until it is completely immersed. The petri dish is covered and incubated for 90 sec. The TLC plate is removed and held vertically over adsorbent paper to allow excess PIBM solution to drain away from the surface. The treated TLC plate is dried completely, using unheated forced air to speed drying if desired (see Note 12). 2. A dilution of GBP (typically 1 g/ml) in binding buffer is placed in a small plastic tray whose size is close to that of the TLC plate. The TLC plate is immersed in the solution and pressed to the bottom of the container with a forceps to remove bubbles. The plate is incubated with GBP solution overnight at 4 with gentle shaking in a humidified atmosphere (see Note 13). 3. Bound proteins may be detected directly or with secondary reagents. For antibody binding (using sera or purified antibodies) after incubation of the TLC plate in GBP solution, the plate is washed three times for 1 min by immersion in PBS, then the plate is overlain with 0.5 g/ml of horseradish peroxidase–conjugated secondary antibody in binding buffer for 90 min at
[15]
glycolipid–protein interactions
213
FIG. 2. TLC immuno‐overlay of major brain gangliosides with antiganglioside monoclonal antibodies1 (Schnaar et al., 2002). A mixture of the four major brain gangliosides (GM1, GD1a, GD1b, GT1b) was spotted at the origin of replicate TLC lanes. After development, the plate was cut into sections, which were either stained with a chemical reagent to reveal the gangliosides (‘‘Std’’ lanes, top to bottom: GM1, GD1a, GD1b, GT1b) or immunostained with the indicated anti‐ganglioside monoclonal antibody, designated by its ganglioside binding specificity and IgG subclass (IgG1). Microplate binding data for the same four monoclonal antibodies is shown in Fig. 1. Modified from Schnaar et al. (2002), with permission.
ambient temperature. The plate is then washed three times for 1 min in PBS. Bound secondary antibody is detected by dipping the plate in substrate solution containing 2.8 mM 4‐chloro‐1‐naphthol, 0.01% H2O2 and 3.3% methanol in 20 mM Tris–HCl buffer, pH 7.4. A positive reaction results in a purple band. The reaction is stopped by washing the plate in PBS; then the plate is allowed to air dry. An example of TLC immunooverlay is shown in Fig. 2 (see Note 14). TLC Blotting. An alternative to direct TLC overlay is TLC blotting, in which TLC‐resolved glycolipids are transferred quantitatively to polyvinylidene fluoride (PVDF) membranes (Taki et al., 1994). Once transferred, glycolipids remain stably adsorbed to the hydrophobic membrane and are amenable to the same procedures used in Western blotting of PVDF‐transferred proteins. 1. Glycolipids are resolved using glass‐backed silica gel HPTLC plates using the protocols described above. 2. After developing and drying, the plate is dipped in blotting solvent (isopropanol‐0.2% aqueous CaCl2‐methanol [40:20:7]) for 20 sec, placed sorbent face up, and covered with a PVDF membrane sheet (Immobilon‐P, Millipore, Billerica, MA) cut to the same size. A glass fiber filter sheet (type GF/A, Whatman PLC, Middlesex, UK) is placed on top of the PVDF. 3. An iron preheated to 180 is pressed with even pressure on top of the glass fiber filter for 30 sec. The PVDF membrane is removed, washed with PBS, and probed using standard Western‐blotting techniques (see Note 15).
214
glycolipid interaction
[15]
Surface Plasmon Resonance (SPR) Spectroscopy SPR is performed by immobilizing glycolipids on a commercial SPR sensor chip, followed by exposure to unlabeled soluble GBPs (Kuziemko et al., 1996; MacKenzie et al., 1997; MacKenzie and Hirama, 2000; Nakajima et al., 2001). Binding is performed in a biosensor designed to detect mass bound to the sensor chip surface. Two biosensor chip surface chemistries are typically used to study lipid‐mediated binding events: a gold chip derivatized with a long‐chain thioalkanes to generate a flat hydrophobic monolayer on which lipids are immobilized (e.g., HPA chip, Biacore AB) and a chip derivatized with alkane‐derivatized dextran (e.g., L1 chip, Biacore AB), to which preformed glycolipid‐containing liposomes (bilayer lipid vesicles) are anchored. The later method is described here because of its high capacity and design flexibility (see Note 16). Glycolipid‐Containing Liposomes 1. The glycolipid of interest and carrier phospholipid (DMPC) are combined in organic solvent in a glass tube and evaporated to dryness under a stream of nitrogen or using a rotary evaporator. For example, 0.04 mg of glycolipid in storage solvent (chloroform‐methanol‐water [4:8:3]) is added to a glass tube, dried under nitrogen, then redissolved in 200 l of chloroform‐methanol (2:1) containing 2 mg of DMPC. The lipid mixture is again dried under nitrogen, then placed under vacuum for 2 h. As a control, identical liposomes are prepared without added glycolipid. 2. Thoroughly dried lipids are rehydrated by suspension in running buffer prewarmed to 37 at a final lipid concentration of 10 mM (300 l for the previous example). The suspension is mixed vigorously for 30 min, avoiding the generation of bubbles and keeping the solution at 25 to create a white suspension. 3. To prepare unilamellar vesicles, the suspension is passed 15 times through a 50‐nm pore polycarbonate filter using an extrusion apparatus per the manufacturer’s instructions. The turbid suspension clarifies after extrusion. To remove large vesicles, the extruded preparation may be centrifuged at 100,000g for 30 min at 15 . The supernatant, containing unilamellar vesicles, is stable for up to 3–4 days at 4 under nitrogen. Liposome Immobilization and SPR. The following procedure is for use in a Biacore 3000 (Biacore AB) or similar biosensor. 1. An L1 sensor chip is docked in the biosensor and cleaned for 10 min with washing solution at a flow rate of 10 l/min. The injection needle is cleaned with water, and the system primed with running buffer.
[15]
glycolipid–protein interactions
215
2. Control and glycolipid‐containing liposome preparations are injected into different channels (flow cells) of the sensor chip at a concentration of 500 M total lipid. Liposome preparations are injected at a flow rate of 2 l/min and stopped when mass adsorption reaches 2500 RU. 3. Lipid‐adsorbed chips are washed for 12 sec with regenerating solution at 100 l/min, then immediately with SPR blocking buffer for 5 min at 10 l/min. The cells are then washed again with regeneration solution for 12 sec followed immediately with running buffer. 4. When the baseline stabilizes, running buffer containing GBP is injected at a rate of 40 l/min. Typically, concentrations of GBP used range from 10 nM–1 M, depending on affinity. If nonspecific binding is unacceptably high, GBP can be diluted in blocking buffer. The GBP is diluted to produce a specific response of 100 RU (maximum) (see Note 17). 5. Binding curves are subsequently performed at multiple flow rates (e.g., 20–100 l/min) using the optimal GBP concentration. In the absence of mass transport or rebinding limitations, the binding and dissociation rates should be independent of the flow rate. If the binding or dissociation rates increase with increasing flow rate, subsequent studies should be performed using the lowest flow rate that results in the maximum binding and dissociation rates for a fixed concentration of GBP. 6. Using the optimal flow rate, binding determinations are performed using different concentrations of GBP to provide a range of binding and dissociation rates (see example in Fig. 3). Typically, binding curves include 3 min of association and up to 15 min of dissociation. For high‐affinity ligands, longer dissociation times may be required. Between runs, the cells are washed with regeneration solution (12 sec, 100 l/min) and running buffer to re‐establish the initial baseline (see Note 18). 7. Data are fitted to appropriate models of receptor‐ligand binding, using the biosensor software or independent curve fitting programs. Most such programs allow for consideration of multivalent binding, which may be appropriate for GBPs such as multivalent lectins and antibodies. Notes
1. The example given is for 96‐well microplates but can be applied to other microplate formats. Plain (untreated) polystyrene plates are required, because ‘‘tissue culture’’–treated plates are modified to increase the hydrophilic nature of the plastic surface, making them less suitable for immobilization of amphipathic molecules. 2. Aluminum‐backed plates are convenient, in that they can be easily cut to size with a scissors. For TLC blotting and centrifuge‐based techniques
216
glycolipid interaction
[15]
FIG. 3. SPR analysis of cholera toxin A subunit binding to ganglioside‐containing liposomes immobilized on sensor chips using the method described by MacKenzie et al. (1997) (see Note 16). (A) Effect of cholera toxin concentration on binding to immobilized GM1. Kinetic plots of binding of different toxin concentrations (as indicated) to GM1‐containing liposomes on sensor chips are shown; (B) Plots of binding of 100 nM toxin to liposomes containing different glycolipids (as indicated) are shown. Note the preferential binding to GM1. Modified from MacKenzie et al. (1997), with permission.
(eukaryotic cell adhesion), nonpliable glass‐backed plates (E. Merck 5635) are more typically used. 3. Volume ratios of chloroform‐methanol‐0.25% aqueous KCl (or CaCl2) may be altered to 70:30:5 to enhance separation of small or nonpolar glycolipids, or 45:45:10 for large and/or highly charged glycolipids. The efficacy of various solvent systems to separate neutral and charged glycolipids are published elsewhere (Schnaar and Needham, 1994).
[15]
glycolipid–protein interactions
217
4. PIBM working solutions should be prepared immediately before use to avoid changes in concentration because of solvent evaporation. Do not attempt to prepare PIBM working solution directly from powder into n‐hexane, because the plastic is difficult to dissolve in hexanes. 5. Glycolipid added at 100 pmol/well to 96‐well microplates typically provides ample glycolipid for binding studies. Adding more than 200 pmol/ well is not productive, because the surface area of each well limits maximum adsorption (Blackburn et al., 1986). Using lower concentrations, or concentration curves, can provide insights on relative binding affinities (Collins et al., 2000). For large‐scale screening (e.g., of sera or hybridoma supernatants), 25–50 pmol/well is usually sufficient. Inclusion of PC and cholesterol enhance the stability of adsorbed glycolipids and ensure a uniform background substratum over a large range of glycolipid concentrations. Some laboratories omit co‐adsorbed carrier lipids (Foxall et al., 1992). Adsorption efficiency is typically 40–70%, depending on the particular glycolipid used (Blackburn et al., 1986). 6. During the partial evaporation step, the gradual loss of ethanol results in partitioning of the lipids onto the plastic surface, where they remain stably adsorbed in aqueous solutions. Some laboratories use an alternate procedure, dissolving glycolipids in methanol or methanol‐water (1:1), adding to microwells, then allowing the solvent to evaporate completely (e.g., overnight) before water washing and use (Foxall et al., 1992; Karlsson and Stromberg, 1987). 7. Whereas binding of soluble GBPs is performed as described, binding of intact cells, especially larger eukaryotic cells, requires a different approach (Collins et al., 2000). Fluid shear inherent in standard microwell plate washing typically disrupts eukaryotic cell adhesion to immobilized glycolipids. Therefore, centrifugal force, which is adjustable and can be much milder than fluid sheer, is preferred. For this purpose, a custom Plexiglas box was developed, into which a fluid‐filled microwell plate can be inverted and sealed. The sealed box fits into a table‐top centrifuge carrier, where it is subjected to centrifugal forces sufficient to detach nonadherent cells but leave specifically adherent cells attached to the glycolipid‐adsorbed surface. After centrifugation, the plate is recovered, and adherent cells are quantified. Complete details are published elsewhere (Collins et al., 2000). 8. It is often valuable to compare chemical staining and protein binding patterns, or binding of different proteins, to the same glycolipid mixtures resolved on the same TLC plate (see Fig. 2). This is accomplished by spotting replicate samples spaced laterally on the plate, developing the TLC, then cutting the plate into sections that are subjected to different chemical or GBP overlay procedures.
218
glycolipid interaction
[15]
9. Partially purified glycolipid extracts from tissue may be used, although contaminating phospholipids may compromise TLC resolution. Partially purified glycolipid extract from the equivalent of 10–20 mg (wet weight) of tissue per lane is applied. This amount can be increased or decreased empirically. Samples spotted too heavily will fail to resolve and will instead produce a smear. Samples spotted too lightly may not provide sufficient glycolipid for detection, especially for less abundant species. When interpreting GBP binding to glycolipid mixtures, the relative abundance of each species must be considered. Low‐affinity binding to highly abundant species will be as robust as high‐affinity binding to less abundant glycolipids (Suetake and Yu, 2003). 10. An industrial forced air dryer (e.g., product HG‐201A, Master Appliance Corp., Racine, WI) may be used, without heat, to speed drying of applied samples and aid in spotting larger volumes. Set the dryer so that the air flows over and parallel to the plate surface. 11. A 5‐cm long plate may provide ample resolution for many glycolipid mixtures. Running a longer plate may not improve separation, because development time will be increased, and diffusion of the samples may compromise further resolution. 12. PIBM coating results in lower background binding and greatly enhanced sensitivity. It has been postulated that the hydrophobic plastic coating reorients the glycolipids so that their polar glycans are oriented away from the sorbent layer and outwards into the solvent, where they may interact more efficiently with complementary binding proteins (Karlsson and Stromberg, 1987). Although the recommended PIBM concentration (0.1 % w/v) typically results in low background and high sensitivity for GBPs, it may be adjusted empirically to enhance signal/ noise, with lower concentrations optimal for intact cell adhesion. 13. For high‐affinity binding proteins, such as bacterial toxins, concentrations as low as 10 ng/ml may be sufficient. Low temperature incubation over longer periods (e.g., 4 , overnight) typically enhances sensitivity. The choice of binding buffer may be modified on the basis of the specific requirements and sensitivities of the GBP to ionic strength, divalent cations, and pH. 14. Higher sensitivity may be obtained using enhanced chemiluminescence for detection (Arnsmeier and Paller, 1995). The procedure described is suitable for soluble binding proteins (antibodies, lectins, toxins), as well as for viruses and bacteria, but not for eukaryotic cells that are susceptible to removal by fluid sheer. Intact eukaryotic cell adhesion to TLC‐resolved glycolipids is detected using mild centrifugation in a fluid‐ filled custom‐designed Plexiglas box to remove nonadherent cells (Schnaar, 1994).
[15]
glycolipid–protein interactions
219
15. A commercial device developed to enhance the reproducibility of TLC blotting is available (TLC Thermal Blotter Model AC‐5970, Atto Corp., Tokyo, Japan). 16. Another option is to add a lipid antigen unrelated to the binding interaction under study to the glycolipid/phospholipid mixture. The unrelated lipid antigen is then used as a handle to capture the resulting liposomes on an antibody‐coated biosensor chip (MacKenzie et al., 1997). Alternately, GBP can be bound to the chip surface and exposed to glycolipid‐containing liposomes in solution (Sandhoff et al., 2005). 17. Binding curves should be close to a plateau at the end of the association phase (3 min). If this is not the case, the ligand density on the sensor chip may be decreased to <2500 RU, and/or the GBP concentration may be increased. 18. If regeneration (return to baseline) is incomplete, the time of treatment with regenerating solution may be increased, or a more stringent regenerating solution (consistent with the stability of the immobilized glycolipid‐containing liposomes) may be used.
References Arnsmeier, S. L., and Paller, A. S. (1995). Chemiluminescence detection of gangliosides by thin‐layer chromatography. J. Lipid Res. 36, 911–915. Bashor, M. M. (1979). Dispersion and disruption of tissues. Methods Enzymol. 58, 119–131. Blackburn, C. C., Swank‐Hill, P., and Schnaar, R. L. (1986). Gangliosides support neural retina cell adhesion. J. Biol. Chem. 261, 2873–2881. Collins, B. E., Yang, L. J. S., and Schnaar, R. L. (2000). Lectin‐mediated cell adhesion to immobilized glycosphingolipids. Methods Enzymol. 312, 438–446. Feizi, T., and Chai, W. (2004). Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell. Biol. 5, 582–588. Foxall, C., Watson, S. R., Dowbenko, D., Fennie, C., Lasky, L. A., Kiso, M., Hasegawa, A., Asa, D., and Brandley, B. K. (1992). The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewisx oligosaccharide. J. Cell Biol. 117, 895–902. Hakomori, S. (2002). The glycosynapse. Proc. Natl. Acad. Sci. USA 99, 225–232. Karlsson, K.‐A., and Stromberg, N. (1987). Overlay and solid phase analysis of glycolipid receptors for bacteria and viruses. Methods Enzymol. 138, 220–232. Kuziemko, G. M., Stroh, M., and Stevens, R. C. (1996). Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. Biochemistry 35, 6375–6384. MacKenzie, C. R., Hirama, T., Lee, K. K., Altman, E., and Young, N. M. (1997). Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance. J. Biol. Chem. 272, 5533–5538. MacKenzie, C. R., and Hirama, T. (2000). Quantitative analyses of binding affinity and specificity for glycolipid receptors by surface plasmon resonance. Methods Enzymol. 312, 205–216.
220
glycolipid interaction
[15]
Magnani, J. L., Smith, D. F., and Ginsburg, V. (1980). Detection of gangliosides that bind cholera toxin: Direct binding of 125I‐labeled toxin to thin‐layer chromatography plates. Anal. Biochem. 109, 399–402. Nakajima, H., Kiyokawa, N., Katagiri, Y. U., Taguchi, T., Suzuki, T., Sekino, T., Mimori, K., Ebata, T., Saito, M., Nakao, H., Takeda, T., and Fujimoto, J. (2001). Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance. J. Biol. Chem. 276, 42915–42922. Sandhoff, R., Grieshaber, H., Djafarzadeh, R., Sijmonsma, T. P., Proudfoot, A. E., Handel, T. M., Wiegandt, H., Nelson, P. J., and Grone, H. J. (2005). Chemokines bind to sulfatides as revealed by surface plasmon resonance. Biochim. Biophys. Acta 1687, 52–63. Schnaar, R. L. (1994). Isolation of glycosphingolipids. Methods Enzymol. 230, 348–370. Schnaar, R. L. (1994). Immobilized Glycoconjugates for Cell Recognition Studies. In ‘‘Neoglycoconjugates: Preparation and Application’’ (Y. C. Lee and R. T. Lee, eds.), pp. 425–443. Academic Press, San Diego. Schnaar, R. L. (2000). Glycobiology of the Nervous System. In ‘‘Carbohydrates in Chemistry and Biology, Part II:Biology of Saccharides’’ (B. Ernst, G. W. Hart, and P. Sinay¨, eds.), pp. 1013–1027. Wiley‐VCH, Weinheim, Germany. Schnaar, R. L. (2004). Glycolipid‐mediated cell‐cell recognition in inflammation and nerve regeneration. Arch. Biochem. Biophys. 426, 163–172. Schnaar, R. L., Fromholt, S. E., Gong, Y., Vyas, A. A., Laroy, W., Wayman, D. M., Heffer‐Lauc, M., Ito, H., Ishida, H., Kiso, M., Griffin, J. W., and Sheikh, K. A. (2002). Immunoglobulin G‐class mouse monoclonal antibodies to major brain gangliosides. Anal. Biochem. 302, 276–284. Schnaar, R. L., and Needham, L. K. (1994). Thin‐layer chromatography of glycosphingolipids. Methods Enzymol. 230, 371–389. Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., and Reinhold, V. N. (1996). Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 1. Separation of E‐selectin binding from nonbinding gangliosides, and absence of sialosyl‐Lex having tetraosyl to octaosyl core. Biochemistry 35, 758–769. Stults, C. L. M., Sweeley, C. C., and Macher, B. A. (1989). Glycosphingolipids: Structure, biological source, and properties. Methods Enzymol. 179, 167–214. Suetake, K., and Yu, R. K. (2003). Thin‐layer chromatography; immunostaining of glycolipid antigens; and interpretation of false‐positive findings with acidic lipids. Methods Enzymol. 363, 312–319. Taki, T., Handa, S., and Ishikawa, D. (1994). Blotting of glycolipids and phospholipids from a high‐performance thin‐layer chromatogram to a polyvinylidene difluoride membrane. Anal. Biochem. 221, 312–316. Willison, H. J. (2005). The immunobiology of Guillain‐Barre syndromes. J. Peripher. Nerv. Syst. 10, 94–112.
glycolipid–protein interactions
205
[15] Determination of Glycolipid–Protein Interaction Specificity By PABLO H. H. LOPEZ and RONALD L. SCHNAAR Abstract
Glycolipids are found on all eukaryotic cells. Their expression varies among tissues, with the highest density found in the brain, where glycolipids are the most abundant of all glycoconjugate classes. In addition to playing roles in membrane structure, glycolipids also act as cell surface recognition molecules, mediating cell–cell interactions, as well as binding certain pathogens and toxins. Because of their amphipathic nature, underivatized glycolipids are amenable to immobilization on hydrophobic surfaces, where they can be probed with lectins, antibodies, pathogens, toxins, and intact cells to reveal their binding specificities and affinities. Three particularly useful methods to probe specific glycolipid‐mediated recognition events are microwell adsorption (ELISA), thin layer chromatography overlay, and surface plasmon resonance (SPR) spectroscopy.
Overview
Glycolipids are amphipathic molecules consisting of a hydrophilic oligosaccharide chain linked to a hydrophobic lipid that anchors the glycolipid in cell membranes (Hakomori, 2002). Two major classes of glycolipids, glycoglycerolipids and glycosphingolipids, are distinguished by their lipid moieties, diacylglycerol (or alkylacylglycerol) and ceramide, respectively. Whereas glycerol‐based glycolipids are most abundant in microbes and plants, glycosphingolipids are by far the most abundant and diverse class of glycolipids in animals. Animal glycosphingolipids are classified into families according to their core glycan sequences, on which many different terminal variations are elaborated (Stults et al., 1989). Glycosphingolipid families are expressed in tissue‐specific patterns. For example, ganglio‐series glycosphingolipids, based on the core structure Gal1‐3GalNAc1‐4Gal1‐4Glc1‐10 ceramide, predominate in the brain (Schnaar, 2000), whereas neolacto‐series glycolipids, based on the core structure Gal1‐4GlcNAc1‐3Gal1‐4Glc1‐10 ceramide are common on leukocytes (Stroud et al., 1996). Glycosphingolipids are further subclassified as neutral, sialylated (having one or more sialic acid residues), or sulfated (Stults et al., 1989). Sialylated glycosphingolipids are also known as gangliosides, which are widely distributed in vertebrates and METHODS IN ENZYMOLOGY, VOL. 417 Copyright 2006, Elsevier Inc. All rights reserved.
0076-6879/06 $35.00 DOI: 10.1016/S0076-6879(06)17015-9
206
glycolipid interaction
[15]
are the most abundant glycoconjugates on nerve cells. Hundreds of distinct glycolipid structures have been reported (Stults et al., 1989), and their structural diversity underlies glycosphingolipid functions, being responsible for specific cell–cell interactions, pathogen/toxin tropism, and autoimmune pathologies (Hakomori, 2002; Schnaar, 2004; Willison, 2005). Whereas this chapter describes methods to determine glycolipid binding specificities using purified or partially purified natural glycolipids, the methods can also be applied to synthetic neoglycolipids (Feizi and Chai, 2004). Although the amphipathic nature of glycolipids poses challenges for their isolation and purification, their lipid moieties provide the means to readily immobilize and orient them on surfaces for probing with complementary binding molecules. In this chapter, methods to probe binding specificities of purified glycolipids by means of microplate adsorption and surface plasmon resonance spectroscopy (SPR) are described, as well as methods to identify binding components of glycolipid mixtures using thin layer chromatography (TLC) overlay.
Materials
Glycolipids Organic solvents are used to solubilize endogenous glycolipids from tissues and cells, where they are typically expressed on plasma membranes. Extraction procedures have been optimized, often using defined chloroform‐methanol‐water mixtures added in specific solvent sequence and ratio, to maximize precipitation and removal of proteins and nucleic acids while maximizing solubilization of glycosphingolipids along with other lipids (Schnaar, 1994). Because glycosphingolipids aggregate with each other and other lipids in aqueous solution, organic solvents are used throughout subsequent purification steps, which typically involve solvent partition, ion exchange chromatography, and/or silicic acid chromatography. Details for glycolipid purifications, which vary depending on the source and specific nature of the target molecules, have been published elsewhere (Schnaar, 1994), and are beyond the scope of this chapter. Purified glycolipids dissolved in organic solvents (e.g., chloroform‐methanol‐water [4:8:3]) are typically stable when stored at 20 for years or even decades. Microplate (ELISA)‐Based Recognition 1. 96‐Well EIA/RIA clear flat‐bottom untreated polystyrene microplate (Corning Life Sciences, Acton, MA, product #9017) (see Note 1) 2. Reagent grade butanol (for prewashing microplates)
[15]
glycolipid–protein interactions
207
3. Reagent grade 100% ethanol 4. A 100‐fold stock of a lipid mixture consisting of 0.1 mM phosphatidylcholine (PC) and 0.4 mM cholesterol is prepared in ethanol. To 4.8 ml of 100% ethanol add 0.15 ml of a 3.3 mM stock of phosphatidylcholine in chloroform (Avanti Polar Lipids, Alabaster, AL, product 840053) and 50 l of 40 mM stock of cholesterol in chloroform (prepared from dry powder, Avanti product 700000). Store the lipid stock tightly capped in a glass tube with Teflon‐lined seal at 20 . 5. Dulbecco’s phosphate‐buffered saline (PBS) contains (g/l): NaCl (8.0), KCl (0.20), CaCl2 (0.10), MgCl26H2O (0.10), Na2HPO4 (2.3), and KH2PO4 (0.20) (Bashor, 1979). 6. Microplate blocking buffer is prepared by dissolving bovine serum albumin (BSA, e.g., Sigma‐Aldrich, St. Louis, MO, product A‐7030) in PBS at 1 mg/ml TLC Overlay 1. Aluminum‐backed silica gel high‐performance HPTLC plates (20 20 cm, E. Merck, Darmstadt, Germany, product 5547) (see Note 2) 2. TLC running solvent. A wide‐spectrum running solvent suitable for many applications is chloroform‐methanol‐aqueous 0.25% w/v KCl (60:35:8) (see Note 3) 3. Glass TLC chromatography developing tank (e.g., 12 5 12 cm, rectangular, Camag Scientific, Muttenz, Switzerland, product 022.5515). 4. Microliter TLC spotting syringe (Hamilton Co., Reno, NV, product 701N) 5. Glass petri dish 6. Poly(isobutyl methacrylate) (PIBM) solution. A stock solution of 5% w/v of PIBM (e.g., Sigma‐Aldrich, product 181544) dissolved in chloroform is prepared. The stock solution is diluted to working concentration (e.g., 0.1% w/v) in n‐hexane immediately before use (see Note 4) 7. Binding buffer is prepared by adding 1 mg/ml BSA and 0.05% (w/v) Tween‐20 (e.g., Pierce Chemical Co., Rockford, IL, product 28320) to PBS Surface Plasmon Resonance Spectroscopy 1. SPR‐based biosensor (e.g., Biacore 3000, Biacore AB, Uppsala, Sweden). 2. Lipid vesicle extrusion apparatus (Avanti Polar Lipids, product 610000) and 50‐nm polycarbonate filters (product 610003)
208
glycolipid interaction
[15]
3. 1,2‐Dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC, Avanti Polar Lipids product 850345) 4. SPR blocking buffer is prepared by dissolving 0.1 mg/ml BSA in 10 mM sodium phosphate, 150 mM sodium chloride, and adjusting to pH 7.4 (degassed and filtered daily) 5. Running buffer consists of 10 mM phosphate buffer, pH 7.4, 150 mM sodium chloride, pH 7.4 (degassed and filtered daily) 6. Sensor chip L1 (product BR‐1005‐58), Biacore AB 7. Washing solution consists of 20 mM CHAPS detergent (e.g., Sigma C‐5070) in water 8. Regenerating solution consists of 20 mM NaOH in water (degassed and filtered daily) Methods
The three methods for determining glycolipid–protein interactions described here—microplate, TLC, and SPR—provide a wide range of capabilities applicable to different experimental goals. In each case, glycolipids are non‐covalently adsorbed to a solid support (microwell, TLC plate or SPR chip), where they remain stably attached in aqueous solutions. Exposure of the adsorbed glycolipids to potential binding proteins (lectins, antibodies, toxins) or biological entities (viruses, bacteria, intact cells) results in specific interactions that are detected using direct or indirect methods. Microplate adsorbed purified glycolipids can be used to determine relative binding specificities of soluble lectins, antibodies, toxins, pathogens, or cells, or for high‐throughput screening, for example, of sera or hybridoma supernatants. TLC overlay is most useful for detecting and identifying binding species within mixtures of partially purified glycolipids from natural sources. SPR using purified glycolipids and unlabeled soluble glycan binding proteins (GBPs) provides real‐time binding kinetics for determination of affinities and specificities of glycolipid–protein interactions. Microplate (ELISA)‐Based Recognition Because of their amphipathic nature, glycolipids spontaneously and stably adsorb to hydrophobic surfaces under the appropriate conditions. Commercially available polystyrene microplates are excellent substrates for glycolipid immobilization and are amenable to subsequent incubation with GBPs and their detection by direct or indirect methods (Collins et al., 2000; Foxall et al., 1992; Schnaar et al., 2002). 1. To remove mold release agents used in their manufacture, microplates are prewashed (batchwise) with butanol and ethanol and then stored for
[15]
glycolipid–protein interactions
209
subsequent use. Microplates are immersed upright in a shallow glass dish (e.g., a glass baking dish) of butanol and allowed to incubate 15 min. Plates are drained and immersed in three sequential dishes of ethanol, draining between transfers. Finally, plates are drained, allowed to air dry, and are stored in sealed plastic bags. 2. Desired concentrations of glycolipid (typically 2–200 pmol/well) are adsorbed, along with a constant amount of PC (25 pmol/well) and cholesterol (100 pmol/well), as follows. PC/cholesterol working solution is prepared by diluting the stock 100‐fold in 100% ethanol (final concentration 1 M PC, 4 M cholesterol). The total amount of glycolipid required is calculated, and an aliquot containing a 10–20% excess (to account for handling losses) is transferred to a glass test tube and evaporated to dryness under a stream of nitrogen or in an evaporator (e.g., SpeedVac Concentrator, Thermo Electron Corp., Waltham, MA). The dried glycolipid is redissolved at the desired concentration(s) in the PC/cholesterol working solution. A bath sonicator (brief sonication) and/or vigorous vortex mixing are used to ensure that the glycolipids are efficiently redissolved. Glycolipid solutions are stored in screw‐capped glass tubes until ready for addition to microplate wells (see Note 5). 3. Immediately before addition to microplate wells, glycolipid solutions (from Step 2) are diluted with an equal volume of water and mixed vigorously. A 50‐l aliquot is then pipetted into each well of a 96‐well microplate using standard single or multichannel micropipettors with standard plastic disposable tips. The plate is then placed on a well‐ventilated bench or chemical fume hood and left uncovered at ambient temperature for 90 min to allow partial evaporation. After incubation, any remaining solvent is removed by inversion and vigorous shaking of the microplate, and the wells are washed three times by repeated immersion in water, inversion, and shaking. The plate is righted and water (200 l/well) added. Glycolipid‐adsorbed plates may be used immediately or stored in water for several hours (e.g., overnight) at ambient temperature without measurable loss of adsorbed lipids (see Note 6). 4. Water is removed from the glycolipid‐adsorbed wells by inverting and shaking the microplate, then is replaced with 200 l/well of microplate blocking buffer. The plate is incubated covered for 30 min at 37 , then the blocking buffer is removed by inversion and shaking, and the wells washed three times with PBS. 5. Binding and detection. Microplate‐adsorbed glycolipids are amenable to probing with lectins, proteins, and other biological entities that are either directly labeled or that can be detected with secondary reagents. Any standard ELISA‐type protocol can be applied to the microplates. One protocol for determining anti‐glycolipid antibody binding specificity follows (Schnaar et al., 2002). Primary antibodies (antisera, hybridoma
210
glycolipid interaction
[15]
supernatants, monoclonal antibodies, etc.) are diluted (typically 1 g/ml antibody) in microplate blocking buffer and 50 l is added to each glycolipid‐adsorbed well. After 90 min at ambient temperature, the microplate is washed three times by immersion in PBS, inversion, and shaking. Secondary antibody (e.g., alkaline phosphatase (AP)–conjugated anti‐human or anti‐mouse IgG, as appropriate) diluted (typically to 1 g/ml) in microplate blocking buffer, is then added at 50 l/well and the microplate incubated 45 min at ambient temperature. Finally, the microplate is washed by immersion in PBS, then water (twice) and AP substrate, consisting of 2 mg/ml p‐nitrophenylphosphate in developing buffer (100 mM Tris, 100 mM NaCl, 5 mM MgCl2) is added at 100 l/well. Color development is determined with a microplate reader. An example of the use of microplate‐adsorbed gangliosides to determine binding specificities of antiganglioside monoclonal antibodies is shown in Fig. 1 (Schnaar et al., 2002) (see Note 7). TLC Overlay TLC overlay is performed in two steps (Karlsson and Stromberg, 1987; Magnani et al., 1980). First, a glycolipid sample is applied to a silica gel TLC plate and developed to resolve the different species. Second, the developed plate is coated with a thin plastic film and overlain with aqueous solution containing soluble GBPs or other biological binding entities to be analyzed. After washing to remove unbound material, bound material is detected either directly or using secondary reagents. When combined with enzymatic and/or chemical modification in situ, TLC overlay is a powerful tool for the analysis of glycolipid–protein interactions (Schnaar and Needham, 1994). Thin Layer Chromatography 1. The chromatography tank is pre‐equilibrated by adding developing solvent (typically chloroform‐methanol‐aqueous mixtures, see ‘‘Materials’’) to a depth of 0.5 cm in the bottom of the chromatography developing tank, which is then covered with a grease‐free lid (glass or metal) and incubated for at least 30 min before introducing the spotted TLC plate. The tank is placed in an area of uniform temperature protected from drafts or heat sources. 2. TLC plates are prepared for spotting. Aluminum‐backed HPTLC plates are cut to desired sizes with a scissors, using care to avoid folding or flaking of the silica gel sorbent. The width of the plate should be 3 cm plus 1 cm per sample to be spotted. The silica gel sorbent along cut edges is smoothed using the dry tip of a gloved finger. Using a ruler and pencil,
[15]
glycolipid–protein interactions
211
FIG. 1. Specific binding of four different antiganglioside monoclonal antibodies1 to major brain gangliosides (Schnaar et al., 2002). The four major brain gangliosides GM1 (——), GD1a (—□—), GD1b (——), and GT1b (—▾—) were adsorbed to microplate wells, along with phosphatidylcholine and cholesterol, at the indicated ganglioside concentrations (pmol/well added). Highly specific binding of four different IgG1‐class antibodies (one for each of the gangliosides, as indicated) was demonstrated using the methods described. Data are presented as the mean and range for duplicate determinations. Sugar structures are represented using the symbol nomenclature of the Consortium for Functional Glycomics (http://functionalglycomics. org). The four major brain gangliosides shown have the same neutral glycan backbone (Gal1‐ 3GalNAc1‐4Gal1‐4Glc1‐10 ceramide) with different numbers and positions of sialic acids (diamonds) as shown. Modified from Schnaar et al. (2002), with permission.
light marks for sample application are drawn along a line 1 cm above and parallel to the bottom of the TLC plate. Each sample application line should be 0.5 cm long, leaving 1.5 cm unused on the lateral edges of the plate (where distortion may occur during development) and 0.5 cm between samples (see Note 8). 3. Glycolipid samples are dissolved in organic solvents for spotting. For pure samples, 100 pmol/lane (or less for high‐affinity binding proteins) is 1
Under a licensing agreement between Seikagaku America and the Johns Hopkins University, Dr. Schnaar is entitled to a share of royalty received by the University on sales of the monoclonal antibodies described in Figs. 1 and 2. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.
212
glycolipid interaction
[15]
sufficient. For mixtures, 1 nmol (or more) may be spotted per lane (see Note 9). Optimally, each sample should be spotted in a total volume of 1 l, although larger volumes (up to 10 l) may be spotted with sufficient care and patience. Aliquots of glycolipid sample in organic storage solutions are evaporated to dryness under a nitrogen stream or in an evaporator then redissolved in a small volume of methanol or chloroform‐methanol‐water (4:8:3) for spotting. 4. Using a Hamilton syringe with the beveled edge of the needle closely apposed but not touching the TLC plate surface, samples are applied along the premarked pencil lines. For each lane, 0.5 l is applied, allowed to dry, then application and drying are repeated until the total desired volume has been spotted. Sufficient time is allowed after the last sample is applied for the spotting solvent to completely evaporate (see Note 10). 5. The TLC plate, with samples applied, is placed in the pre‐equilibrated developing tank, with the top of the plate resting against the tank wall. The tank is kept covered at uniform temperature away from drafts or heat sources until the solvent moves up the plate by capillary action to within a few millimeters from the top of the plate. To ensure reproducibility of TLC runs, used developing solvent is discarded after each run (see Note 11). TLC Overlay 1. After the solvent has reached the top of the TLC plate, it is removed from the developing tank and allowed to dry completely. Drying may be aided by unheated forced air (see Note 10). One corner of the aluminum‐ backed TLC plate is bent toward the sorbent‐adsorbed surface using a forceps to facilitate easier handling during subsequent steps. A glass petri dish is filled to 0.5‐cm depth with 0.1% PIBM in hexane. The TLC plate is slowly inserted, at an angle into the solution until it is completely immersed. The petri dish is covered and incubated for 90 sec. The TLC plate is removed and held vertically over adsorbent paper to allow excess PIBM solution to drain away from the surface. The treated TLC plate is dried completely, using unheated forced air to speed drying if desired (see Note 12). 2. A dilution of GBP (typically 1 g/ml) in binding buffer is placed in a small plastic tray whose size is close to that of the TLC plate. The TLC plate is immersed in the solution and pressed to the bottom of the container with a forceps to remove bubbles. The plate is incubated with GBP solution overnight at 4 with gentle shaking in a humidified atmosphere (see Note 13). 3. Bound proteins may be detected directly or with secondary reagents. For antibody binding (using sera or purified antibodies) after incubation of the TLC plate in GBP solution, the plate is washed three times for 1 min by immersion in PBS, then the plate is overlain with 0.5 g/ml of horseradish peroxidase–conjugated secondary antibody in binding buffer for 90 min at
[15]
glycolipid–protein interactions
213
FIG. 2. TLC immuno‐overlay of major brain gangliosides with antiganglioside monoclonal antibodies1 (Schnaar et al., 2002). A mixture of the four major brain gangliosides (GM1, GD1a, GD1b, GT1b) was spotted at the origin of replicate TLC lanes. After development, the plate was cut into sections, which were either stained with a chemical reagent to reveal the gangliosides (‘‘Std’’ lanes, top to bottom: GM1, GD1a, GD1b, GT1b) or immunostained with the indicated anti‐ganglioside monoclonal antibody, designated by its ganglioside binding specificity and IgG subclass (IgG1). Microplate binding data for the same four monoclonal antibodies is shown in Fig. 1. Modified from Schnaar et al. (2002), with permission.
ambient temperature. The plate is then washed three times for 1 min in PBS. Bound secondary antibody is detected by dipping the plate in substrate solution containing 2.8 mM 4‐chloro‐1‐naphthol, 0.01% H2O2 and 3.3% methanol in 20 mM Tris–HCl buffer, pH 7.4. A positive reaction results in a purple band. The reaction is stopped by washing the plate in PBS; then the plate is allowed to air dry. An example of TLC immunooverlay is shown in Fig. 2 (see Note 14). TLC Blotting. An alternative to direct TLC overlay is TLC blotting, in which TLC‐resolved glycolipids are transferred quantitatively to polyvinylidene fluoride (PVDF) membranes (Taki et al., 1994). Once transferred, glycolipids remain stably adsorbed to the hydrophobic membrane and are amenable to the same procedures used in Western blotting of PVDF‐transferred proteins. 1. Glycolipids are resolved using glass‐backed silica gel HPTLC plates using the protocols described above. 2. After developing and drying, the plate is dipped in blotting solvent (isopropanol‐0.2% aqueous CaCl2‐methanol [40:20:7]) for 20 sec, placed sorbent face up, and covered with a PVDF membrane sheet (Immobilon‐P, Millipore, Billerica, MA) cut to the same size. A glass fiber filter sheet (type GF/A, Whatman PLC, Middlesex, UK) is placed on top of the PVDF. 3. An iron preheated to 180 is pressed with even pressure on top of the glass fiber filter for 30 sec. The PVDF membrane is removed, washed with PBS, and probed using standard Western‐blotting techniques (see Note 15).
214
glycolipid interaction
[15]
Surface Plasmon Resonance (SPR) Spectroscopy SPR is performed by immobilizing glycolipids on a commercial SPR sensor chip, followed by exposure to unlabeled soluble GBPs (Kuziemko et al., 1996; MacKenzie et al., 1997; MacKenzie and Hirama, 2000; Nakajima et al., 2001). Binding is performed in a biosensor designed to detect mass bound to the sensor chip surface. Two biosensor chip surface chemistries are typically used to study lipid‐mediated binding events: a gold chip derivatized with a long‐chain thioalkanes to generate a flat hydrophobic monolayer on which lipids are immobilized (e.g., HPA chip, Biacore AB) and a chip derivatized with alkane‐derivatized dextran (e.g., L1 chip, Biacore AB), to which preformed glycolipid‐containing liposomes (bilayer lipid vesicles) are anchored. The later method is described here because of its high capacity and design flexibility (see Note 16). Glycolipid‐Containing Liposomes 1. The glycolipid of interest and carrier phospholipid (DMPC) are combined in organic solvent in a glass tube and evaporated to dryness under a stream of nitrogen or using a rotary evaporator. For example, 0.04 mg of glycolipid in storage solvent (chloroform‐methanol‐water [4:8:3]) is added to a glass tube, dried under nitrogen, then redissolved in 200 l of chloroform‐methanol (2:1) containing 2 mg of DMPC. The lipid mixture is again dried under nitrogen, then placed under vacuum for 2 h. As a control, identical liposomes are prepared without added glycolipid. 2. Thoroughly dried lipids are rehydrated by suspension in running buffer prewarmed to 37 at a final lipid concentration of 10 mM (300 l for the previous example). The suspension is mixed vigorously for 30 min, avoiding the generation of bubbles and keeping the solution at 25 to create a white suspension. 3. To prepare unilamellar vesicles, the suspension is passed 15 times through a 50‐nm pore polycarbonate filter using an extrusion apparatus per the manufacturer’s instructions. The turbid suspension clarifies after extrusion. To remove large vesicles, the extruded preparation may be centrifuged at 100,000g for 30 min at 15 . The supernatant, containing unilamellar vesicles, is stable for up to 3–4 days at 4 under nitrogen. Liposome Immobilization and SPR. The following procedure is for use in a Biacore 3000 (Biacore AB) or similar biosensor. 1. An L1 sensor chip is docked in the biosensor and cleaned for 10 min with washing solution at a flow rate of 10 l/min. The injection needle is cleaned with water, and the system primed with running buffer.
[15]
glycolipid–protein interactions
215
2. Control and glycolipid‐containing liposome preparations are injected into different channels (flow cells) of the sensor chip at a concentration of 500 M total lipid. Liposome preparations are injected at a flow rate of 2 l/min and stopped when mass adsorption reaches 2500 RU. 3. Lipid‐adsorbed chips are washed for 12 sec with regenerating solution at 100 l/min, then immediately with SPR blocking buffer for 5 min at 10 l/min. The cells are then washed again with regeneration solution for 12 sec followed immediately with running buffer. 4. When the baseline stabilizes, running buffer containing GBP is injected at a rate of 40 l/min. Typically, concentrations of GBP used range from 10 nM–1 M, depending on affinity. If nonspecific binding is unacceptably high, GBP can be diluted in blocking buffer. The GBP is diluted to produce a specific response of 100 RU (maximum) (see Note 17). 5. Binding curves are subsequently performed at multiple flow rates (e.g., 20–100 l/min) using the optimal GBP concentration. In the absence of mass transport or rebinding limitations, the binding and dissociation rates should be independent of the flow rate. If the binding or dissociation rates increase with increasing flow rate, subsequent studies should be performed using the lowest flow rate that results in the maximum binding and dissociation rates for a fixed concentration of GBP. 6. Using the optimal flow rate, binding determinations are performed using different concentrations of GBP to provide a range of binding and dissociation rates (see example in Fig. 3). Typically, binding curves include 3 min of association and up to 15 min of dissociation. For high‐affinity ligands, longer dissociation times may be required. Between runs, the cells are washed with regeneration solution (12 sec, 100 l/min) and running buffer to re‐establish the initial baseline (see Note 18). 7. Data are fitted to appropriate models of receptor‐ligand binding, using the biosensor software or independent curve fitting programs. Most such programs allow for consideration of multivalent binding, which may be appropriate for GBPs such as multivalent lectins and antibodies. Notes
1. The example given is for 96‐well microplates but can be applied to other microplate formats. Plain (untreated) polystyrene plates are required, because ‘‘tissue culture’’–treated plates are modified to increase the hydrophilic nature of the plastic surface, making them less suitable for immobilization of amphipathic molecules. 2. Aluminum‐backed plates are convenient, in that they can be easily cut to size with a scissors. For TLC blotting and centrifuge‐based techniques
216
glycolipid interaction
[15]
FIG. 3. SPR analysis of cholera toxin A subunit binding to ganglioside‐containing liposomes immobilized on sensor chips using the method described by MacKenzie et al. (1997) (see Note 16). (A) Effect of cholera toxin concentration on binding to immobilized GM1. Kinetic plots of binding of different toxin concentrations (as indicated) to GM1‐containing liposomes on sensor chips are shown; (B) Plots of binding of 100 nM toxin to liposomes containing different glycolipids (as indicated) are shown. Note the preferential binding to GM1. Modified from MacKenzie et al. (1997), with permission.
(eukaryotic cell adhesion), nonpliable glass‐backed plates (E. Merck 5635) are more typically used. 3. Volume ratios of chloroform‐methanol‐0.25% aqueous KCl (or CaCl2) may be altered to 70:30:5 to enhance separation of small or nonpolar glycolipids, or 45:45:10 for large and/or highly charged glycolipids. The efficacy of various solvent systems to separate neutral and charged glycolipids are published elsewhere (Schnaar and Needham, 1994).
[15]
glycolipid–protein interactions
217
4. PIBM working solutions should be prepared immediately before use to avoid changes in concentration because of solvent evaporation. Do not attempt to prepare PIBM working solution directly from powder into n‐hexane, because the plastic is difficult to dissolve in hexanes. 5. Glycolipid added at 100 pmol/well to 96‐well microplates typically provides ample glycolipid for binding studies. Adding more than 200 pmol/ well is not productive, because the surface area of each well limits maximum adsorption (Blackburn et al., 1986). Using lower concentrations, or concentration curves, can provide insights on relative binding affinities (Collins et al., 2000). For large‐scale screening (e.g., of sera or hybridoma supernatants), 25–50 pmol/well is usually sufficient. Inclusion of PC and cholesterol enhance the stability of adsorbed glycolipids and ensure a uniform background substratum over a large range of glycolipid concentrations. Some laboratories omit co‐adsorbed carrier lipids (Foxall et al., 1992). Adsorption efficiency is typically 40–70%, depending on the particular glycolipid used (Blackburn et al., 1986). 6. During the partial evaporation step, the gradual loss of ethanol results in partitioning of the lipids onto the plastic surface, where they remain stably adsorbed in aqueous solutions. Some laboratories use an alternate procedure, dissolving glycolipids in methanol or methanol‐water (1:1), adding to microwells, then allowing the solvent to evaporate completely (e.g., overnight) before water washing and use (Foxall et al., 1992; Karlsson and Stromberg, 1987). 7. Whereas binding of soluble GBPs is performed as described, binding of intact cells, especially larger eukaryotic cells, requires a different approach (Collins et al., 2000). Fluid shear inherent in standard microwell plate washing typically disrupts eukaryotic cell adhesion to immobilized glycolipids. Therefore, centrifugal force, which is adjustable and can be much milder than fluid sheer, is preferred. For this purpose, a custom Plexiglas box was developed, into which a fluid‐filled microwell plate can be inverted and sealed. The sealed box fits into a table‐top centrifuge carrier, where it is subjected to centrifugal forces sufficient to detach nonadherent cells but leave specifically adherent cells attached to the glycolipid‐adsorbed surface. After centrifugation, the plate is recovered, and adherent cells are quantified. Complete details are published elsewhere (Collins et al., 2000). 8. It is often valuable to compare chemical staining and protein binding patterns, or binding of different proteins, to the same glycolipid mixtures resolved on the same TLC plate (see Fig. 2). This is accomplished by spotting replicate samples spaced laterally on the plate, developing the TLC, then cutting the plate into sections that are subjected to different chemical or GBP overlay procedures.
218
glycolipid interaction
[15]
9. Partially purified glycolipid extracts from tissue may be used, although contaminating phospholipids may compromise TLC resolution. Partially purified glycolipid extract from the equivalent of 10–20 mg (wet weight) of tissue per lane is applied. This amount can be increased or decreased empirically. Samples spotted too heavily will fail to resolve and will instead produce a smear. Samples spotted too lightly may not provide sufficient glycolipid for detection, especially for less abundant species. When interpreting GBP binding to glycolipid mixtures, the relative abundance of each species must be considered. Low‐affinity binding to highly abundant species will be as robust as high‐affinity binding to less abundant glycolipids (Suetake and Yu, 2003). 10. An industrial forced air dryer (e.g., product HG‐201A, Master Appliance Corp., Racine, WI) may be used, without heat, to speed drying of applied samples and aid in spotting larger volumes. Set the dryer so that the air flows over and parallel to the plate surface. 11. A 5‐cm long plate may provide ample resolution for many glycolipid mixtures. Running a longer plate may not improve separation, because development time will be increased, and diffusion of the samples may compromise further resolution. 12. PIBM coating results in lower background binding and greatly enhanced sensitivity. It has been postulated that the hydrophobic plastic coating reorients the glycolipids so that their polar glycans are oriented away from the sorbent layer and outwards into the solvent, where they may interact more efficiently with complementary binding proteins (Karlsson and Stromberg, 1987). Although the recommended PIBM concentration (0.1 % w/v) typically results in low background and high sensitivity for GBPs, it may be adjusted empirically to enhance signal/ noise, with lower concentrations optimal for intact cell adhesion. 13. For high‐affinity binding proteins, such as bacterial toxins, concentrations as low as 10 ng/ml may be sufficient. Low temperature incubation over longer periods (e.g., 4 , overnight) typically enhances sensitivity. The choice of binding buffer may be modified on the basis of the specific requirements and sensitivities of the GBP to ionic strength, divalent cations, and pH. 14. Higher sensitivity may be obtained using enhanced chemiluminescence for detection (Arnsmeier and Paller, 1995). The procedure described is suitable for soluble binding proteins (antibodies, lectins, toxins), as well as for viruses and bacteria, but not for eukaryotic cells that are susceptible to removal by fluid sheer. Intact eukaryotic cell adhesion to TLC‐resolved glycolipids is detected using mild centrifugation in a fluid‐ filled custom‐designed Plexiglas box to remove nonadherent cells (Schnaar, 1994).
[15]
glycolipid–protein interactions
219
15. A commercial device developed to enhance the reproducibility of TLC blotting is available (TLC Thermal Blotter Model AC‐5970, Atto Corp., Tokyo, Japan). 16. Another option is to add a lipid antigen unrelated to the binding interaction under study to the glycolipid/phospholipid mixture. The unrelated lipid antigen is then used as a handle to capture the resulting liposomes on an antibody‐coated biosensor chip (MacKenzie et al., 1997). Alternately, GBP can be bound to the chip surface and exposed to glycolipid‐containing liposomes in solution (Sandhoff et al., 2005). 17. Binding curves should be close to a plateau at the end of the association phase (3 min). If this is not the case, the ligand density on the sensor chip may be decreased to <2500 RU, and/or the GBP concentration may be increased. 18. If regeneration (return to baseline) is incomplete, the time of treatment with regenerating solution may be increased, or a more stringent regenerating solution (consistent with the stability of the immobilized glycolipid‐containing liposomes) may be used.
References Arnsmeier, S. L., and Paller, A. S. (1995). Chemiluminescence detection of gangliosides by thin‐layer chromatography. J. Lipid Res. 36, 911–915. Bashor, M. M. (1979). Dispersion and disruption of tissues. Methods Enzymol. 58, 119–131. Blackburn, C. C., Swank‐Hill, P., and Schnaar, R. L. (1986). Gangliosides support neural retina cell adhesion. J. Biol. Chem. 261, 2873–2881. Collins, B. E., Yang, L. J. S., and Schnaar, R. L. (2000). Lectin‐mediated cell adhesion to immobilized glycosphingolipids. Methods Enzymol. 312, 438–446. Feizi, T., and Chai, W. (2004). Oligosaccharide microarrays to decipher the glyco code. Nat. Rev. Mol. Cell. Biol. 5, 582–588. Foxall, C., Watson, S. R., Dowbenko, D., Fennie, C., Lasky, L. A., Kiso, M., Hasegawa, A., Asa, D., and Brandley, B. K. (1992). The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewisx oligosaccharide. J. Cell Biol. 117, 895–902. Hakomori, S. (2002). The glycosynapse. Proc. Natl. Acad. Sci. USA 99, 225–232. Karlsson, K.‐A., and Stromberg, N. (1987). Overlay and solid phase analysis of glycolipid receptors for bacteria and viruses. Methods Enzymol. 138, 220–232. Kuziemko, G. M., Stroh, M., and Stevens, R. C. (1996). Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance. Biochemistry 35, 6375–6384. MacKenzie, C. R., Hirama, T., Lee, K. K., Altman, E., and Young, N. M. (1997). Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance. J. Biol. Chem. 272, 5533–5538. MacKenzie, C. R., and Hirama, T. (2000). Quantitative analyses of binding affinity and specificity for glycolipid receptors by surface plasmon resonance. Methods Enzymol. 312, 205–216.
220
glycolipid interaction
[15]
Magnani, J. L., Smith, D. F., and Ginsburg, V. (1980). Detection of gangliosides that bind cholera toxin: Direct binding of 125I‐labeled toxin to thin‐layer chromatography plates. Anal. Biochem. 109, 399–402. Nakajima, H., Kiyokawa, N., Katagiri, Y. U., Taguchi, T., Suzuki, T., Sekino, T., Mimori, K., Ebata, T., Saito, M., Nakao, H., Takeda, T., and Fujimoto, J. (2001). Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance. J. Biol. Chem. 276, 42915–42922. Sandhoff, R., Grieshaber, H., Djafarzadeh, R., Sijmonsma, T. P., Proudfoot, A. E., Handel, T. M., Wiegandt, H., Nelson, P. J., and Grone, H. J. (2005). Chemokines bind to sulfatides as revealed by surface plasmon resonance. Biochim. Biophys. Acta 1687, 52–63. Schnaar, R. L. (1994). Isolation of glycosphingolipids. Methods Enzymol. 230, 348–370. Schnaar, R. L. (1994). Immobilized Glycoconjugates for Cell Recognition Studies. In ‘‘Neoglycoconjugates: Preparation and Application’’ (Y. C. Lee and R. T. Lee, eds.), pp. 425–443. Academic Press, San Diego. Schnaar, R. L. (2000). Glycobiology of the Nervous System. In ‘‘Carbohydrates in Chemistry and Biology, Part II:Biology of Saccharides’’ (B. Ernst, G. W. Hart, and P. Sinay¨, eds.), pp. 1013–1027. Wiley‐VCH, Weinheim, Germany. Schnaar, R. L. (2004). Glycolipid‐mediated cell‐cell recognition in inflammation and nerve regeneration. Arch. Biochem. Biophys. 426, 163–172. Schnaar, R. L., Fromholt, S. E., Gong, Y., Vyas, A. A., Laroy, W., Wayman, D. M., Heffer‐Lauc, M., Ito, H., Ishida, H., Kiso, M., Griffin, J. W., and Sheikh, K. A. (2002). Immunoglobulin G‐class mouse monoclonal antibodies to major brain gangliosides. Anal. Biochem. 302, 276–284. Schnaar, R. L., and Needham, L. K. (1994). Thin‐layer chromatography of glycosphingolipids. Methods Enzymol. 230, 371–389. Stroud, M. R., Handa, K., Salyan, M. E. K., Ito, K., Levery, S. B., Hakomori, S., Reinhold, B. B., and Reinhold, V. N. (1996). Monosialogangliosides of human myelogenous leukemia HL60 cells and normal human leukocytes. 1. Separation of E‐selectin binding from nonbinding gangliosides, and absence of sialosyl‐Lex having tetraosyl to octaosyl core. Biochemistry 35, 758–769. Stults, C. L. M., Sweeley, C. C., and Macher, B. A. (1989). Glycosphingolipids: Structure, biological source, and properties. Methods Enzymol. 179, 167–214. Suetake, K., and Yu, R. K. (2003). Thin‐layer chromatography; immunostaining of glycolipid antigens; and interpretation of false‐positive findings with acidic lipids. Methods Enzymol. 363, 312–319. Taki, T., Handa, S., and Ishikawa, D. (1994). Blotting of glycolipids and phospholipids from a high‐performance thin‐layer chromatogram to a polyvinylidene difluoride membrane. Anal. Biochem. 221, 312–316. Willison, H. J. (2005). The immunobiology of Guillain‐Barre syndromes. J. Peripher. Nerv. Syst. 10, 94–112.