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Originally published In Press as doi:10.1074/jbc.M412378200 on November 24, 2004

J. Biol. Chem., Vol. 280, Issue 6, 4307-4312, February 11, 2005
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Glycan Array Screening Reveals a Candidate Ligand for Siglec-8*{boxs}

Bruce S. Bochner{ddagger}§, Richard A. Alvarez¶, Padmaja Mehta||, Nicolai V. Bovin**, Ola Blixt{ddagger}{ddagger}, John R. White§§, and Ronald L. Schnaar¶¶

From the {ddagger}Division of Allergy and Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, ||Cardiovascular Biology Research Program, The Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, **Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia, {ddagger}{ddagger}Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, §§GlaxoSmithKline, King of Prussia, Pennsylvania 19406, and ¶¶Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received for publication, November 2, 2004 , and in revised form, November 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is selectively expressed on human eosinophils, basophils, and mast cells, where it regulates their function and survival. Previous studies demonstrated sialic acid-dependent binding of Siglec-8 but failed to reveal significant substructure specificity or high affinity of that binding. To test a broader range of potential ligands, a Siglec-8-Ig chimeric protein was tested for binding to 172 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. Of these, ~40 structures were sialylated. Among these, avid binding was detected to a single defined glycan, NeuAc{alpha}2–3(6-O-sulfo)Gal{beta}1–4[Fuc{alpha}1–3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. Notably, neither unsulfated sLex (NeuAc{alpha}2–3Gal{beta}1–4[Fuc{alpha}1–3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, NeuAc{alpha}2–3Gal{beta}1–4[Fuc{alpha}1–3](6-O-sulfo)GlcNAc) supported detectable binding. Subsequent secondary screening was performed using surface plasmon resonance. Biotin glycosides immobilized on streptavidin biosensor chips were exposed to Siglec-8-Ig in solution. Whereas surfaces derivatized with sLex and 6-sulfo-sLex failed to support detectable Siglec-8 binding, 6'-sulfo-sLex supported significant binding with a Kd of 2.3 µM.In a separate test of binding specificity, aminopropyl glycosides were covalently immobilized at different concentrations on activated (N-hydroxysuccinimidyl) glass surfaces (Schott-Nexterion Slide H). Subsequent exposure to Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc resulted in fluorescent signals at immobilized concentrations of 6'-sulfo-sLex of <5 pmol/spot. In contrast, sLex and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot). We conclude that Siglec-8 binds preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sialic acid-binding immunoglobulin-like lectins (Siglecs)1 are a recently designated family of cell surface molecules that are a subset of the immunoglobulin (Ig) gene superfamily (1–3). Siglecs differ from traditional Ig superfamily members in several ways. Although their extracellular domains contain a variable number of C2-set Ig domains, unlike other Ig superfamily members, Siglecs possess an NH2-terminal V-set Ig domain that binds sialylated structures (4–8). In addition, Siglec cytoplasmic domains typically contain multiple tyrosine residues, including some with consensus immunoreceptor tyrosine-based inhibitory motifs (1). This suggests that Siglecs possess signal transduction activity. Direct evidence of signaling has already been shown for several human Siglecs (9–13).

Siglec-8 (alternatively named SAF-2 (sialoadhesin family-2)) was discovered by CD33 homology screening of expressed sequence tag sequences from a human eosinophil cDNA library (14, 15). The highest levels of homology are found between Siglec-8 and Siglec-3 (49%), Siglec-5 (42%), and Siglec-7 (68%), with virtually all of the homology due to similarities in the extracellular and transmembrane regions. Subsequently, a splice variant of Siglec-8, termed Siglec-8L, which contains an identical extracellular domain but a longer cytoplasmic tail possessing two tyrosine-based motifs, was discovered from human genomic DNA (16, 17). Additional experiments using monoclonal antibodies revealed that Siglec-8 is expressed not only on the surface of eosinophils but also on basophils and mast cells (14), and the existence of both the Siglec-8 and Siglec-8L isoforms was verified in human eosinophils, basophils, and mast cells (18, 19). Most recently it was demonstrated that antibody cross-linking of Siglec-8 on human eosinophils induces caspase-dependent apoptosis in vitro (20).

The search for Siglec ligands remains rather complex. Many of the Siglecs recognize {alpha}2–3- and {alpha}2–6-linked sialic acids (21, 22), whereas others bind to other sialylated structures. For example, Siglec-1 has been shown to bind the highly glycosylated surface protein CD43 (23), the epithelial mucin MUC-1 (24), and sialylated lipopolysaccharide (25). Among a panel of glycans tested, Siglec-3 showed enhanced binding to a multivalent form of sialyl-Tn (NeuAc{alpha}2–6GalNAc) disaccharides (26). Siglec-7 binds to GD3 ganglioside, LSTb oligosaccharide, sialyl Lewisa, and NeuAc{alpha}2–8NeuAc, whereas Siglec-9 preferentially binds GD1a ganglioside and LSTc oligosaccharide (5, 27, 28). For Siglec-8, it has been shown that red blood cell rosettes are formed with Siglec-8, and neuraminidase treatment alters rosette formation (14, 15). Specific structures shown to bind Siglec-8 include forms of sialic acid that are linked {alpha}2–3 or {alpha}2–6 to Gal{beta}1–4GlcNAc (15). In a more comprehensive evaluation of binding specificities, 10 Siglec-Ig chimeras were screened for binding to 28 different sialoside-streptavidin-alkaline phosphatase probes, and a wide range of binding patterns were observed, but there was no clear binding preference for Siglec-8 (29).

The Consortium for Functional Glycomics was funded as a large research initiative by the National Institutes of General Medical Sciences to facilitate research efforts focused on improving the understanding of the mechanisms by which glycan-binding proteins mediate cell communication. The Consortium is integrating the efforts of several scientific cores and participating investigators to achieve these aims. Among several cores is the protein-carbohydrate interaction core H and carbohydrate synthesis protein expression core D. These cores have developed a high throughput screening platform for identifying glycan-binding protein-ligand interactions using a streptavidin/biotin-based glycan array containing ~180 different glycan structures immobilized as biotinylated glycosides on a 384-well streptavidin-coated plate. This was developed as an expansion of previous efforts (29) and in conjunction with the carbohydrate synthesis core D to determine the glycan binding specificity of glycan-binding proteins. A secondary analysis method was also used to quantitate the relative binding affinities of candidate ligands by surface plasmon resonance. Here we report that these and other approaches have been used to determine that Siglec-8 is a highly specific lectin, binding preferentially to the sLex structure bearing an additional sulfate ester on the galactose 6-hydroxyl, namely NeuAc{alpha}2–3(6-O-sulfo)Gal{beta}1–4[Fuc{alpha}1–3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex, which is a structure closely related to 6-sulfo-sLex, a candidate ligand for L-selectin.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Siglec-8-Ig Chimera—The extracellular domain of Siglec-8 was inserted in-frame with a Factor Xa cleavage site and the Fc portion of human IgG1, expanded in electroporated CHOEA1 cells, and then purified from supernatants (final concentration, 2.4 mg/ml) using protein A-Sepharose as described previously (14).

Glycomics Consortium Screening for Siglec-8 Ligands—Biotinylated glycosides (30) were coated in replicates of n = 3 on streptavidin-coated microtiter plates (Pierce Reacti-BindTM NeutrAvidinTM coated high-binding capacity black 384-well plates, product number 15513). Each well was incubated overnight at 4 °C with 30 pmol/well of glycoside in 25 µl of phosphate-buffered saline (PBS), pH 7.4. The plates were washed three times with 100 µl of PBS using an automated plate washer (Molecular Devices, Sunnyvale, CA) and stored sealed at 4 °C in 25 µl/well PBS with 0.1% azide until use. Prior to assay, the plates were washed three times with 100 µl/well wash buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, and 0.05% Tween 20). A stock solution of Siglec-8-Ig (30 µg/ml) was added to each well in 25 µl of binding buffer (wash buffer plus 1% bovine serum albumin) and incubated at room temperature for 1 h. The plates were washed three times with 100 µl/well wash buffer and incubated for 1 h with 25 µl/well goat anti-human IgG-Alexa 488 (catalogue number A11013 [GenBank] , Molecular Probes, Eugene, OR) at 5 µg/ml in binding buffer. The plates were washed and read in 25 µl of wash buffer on a Victor 2TM 1420 Multilabel Counter (PerkinElmer Life Sciences) at excitation 485/emission 535. The glycosides probed are listed in Fig. S1 of the supplemental materials.

Measurement of Siglec-8-Ig Binding Affinity Using Surface Plasmon Resonance—All surface plasmon resonance (SPR) experiments were performed at 25 °C on a Biacore 3000 instrument (Biacore Inc., Piscataway, NJ). Biotinylated glycosides were captured on research grade streptavidin-coated sensor chips (Sensor Chip SA, Biacore Inc.) that were pretreated according to the manufacturer's instructions. A solution of each biotinylated glycoside (10 fmol/µl) was injected at 2 µl/min in PBS, pH 7.4, containing 0.005% Tween 20 (running buffer) for varying lengths of time (3–7 min) until an optimal amount of glycan was captured on each independent surface. Three related glycosides were studied using one streptavidin sensor chip. A control (non-binding) glycan, LacNAc (Gal{beta}1–4GlcNAc), was also captured on the same sensor chip, and the specific binding of Siglec-8-Ig for the test glycans was measured using the in-line reference subtraction feature of the Biacore 3000 instrument. Increasing concentrations of Siglec-8-Ig (0.1–18 µM) were injected at a flow rate of 60 µl/min over all four surfaces of the sensor chip. Bound Siglec-8-Ig was found to elute with normal buffer flow after the injection was complete. The equilibrium binding data of Siglec-8-Ig were analyzed by non-linear curve fitting using the BIAevaluation software (Biacore Inc.).

Binding of Siglec-8-Ig to Aminoalkyl Glycosides Immobilized on Activated (N-hydroxysuccinimidyl) Glass Surfaces—The N-succinimidyl-activated glass slides (kindly provided by Schott-North America, Duryea, PA) were found to be compatible with the consortium library by coupling amino-terminated glycans and are currently being evaluated for a printed version of the consortium glycan array. A description of the printing and evaluation of the full array in this format has just been published elsewhere.2 Fluorescein isothiocyanate-labeled goat anti-human IgG (Fc-specific) was from Jackson Immunoresearch (West Grove, PA). Aminopropyl glycosides were prepared as described previously (29, 31). Amino hexylglucoside was synthesized as described (32).

Aminoalkyl glycosides were covalently immobilized on N-succinimidyl-activated glass (Schott-Nexterion Slide H) using the manufacturer's protocols. All procedures were performed at ambient temperature. Briefly, each aminoalkyl glycoside was prepared at a series of concentrations ranging from 0.5 µM to 1 mM in 300 mM sodium phosphate buffer, pH 8.5, 0.005% Tween 20 (spotting buffer). Drops (~0.3 µl) of each glycan at each concentration were hand-spotted in quadruplicate on the slide surface in a humid chamber. After spotting, the slide was maintained for an additional hour in the humid chamber and then transferred to a desiccated chamber for 16 h. The slide was then immersed in 50 mM ethanolamine, 50 mM sodium borate, pH 9.0, for 1 h, rinsed with water, dried under a stream of microfiltered air, and stored desiccated.

A solution of 100 µg/ml Siglec-8-Ig and 50 µg/ml fluorescein isothiocyanate-labeled goat anti-human IgG (Fc-specific) in Dulbecco's PBS containing 0.005% Tween 20 was incubated for 30 min at 37 °C to allow complexes to form. The glycan-derivatized slide was immersed in PBS containing 0.01% Tween, drained, and then overlaid with the Siglec-8-Ig-fluorescein isothiocyanate antibody conjugate. After 2 h in a dark humid chamber, the slide was washed by successive immersion in PBS/0.01% Tween (three times) and water/0.01% Tween (twice). The slide was briefly rinsed with distilled water and dried under microfiltered air. An image of the bound fluorescence was obtaining using a microarray scanner (ScanArray Express, PerkinElmer Life Sciences). The integrated spot intensities were determined using Metamorph software (Universal Imaging, Downingtown, PA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Glycan Array Screening—Using the glycan array available through the Consortium for Functional Glycomics, experiments were initiated to identify compounds that selectively and specifically bind to Siglec-8. Using Siglec-8-Ig fusion protein, a panel of 172 carbohydrate-based structures (see Fig. S1 of the supplemental materials) was screened for specific Siglec-8 binding. As shown in Fig. 1, Siglec-8-Ig fusion protein had high affinity (12:1 signal-to-noise binding) for structure 181, also know as 6'-sulfo-sLex or NeuAc{alpha}2–3(6-O-Su)Gal{beta}1–4(Fuc{alpha}1–3)GlcNAc{beta}1-O(CH2)3NH-CO(CH2)5NH-biotin (Fig. 2). Of additional relevance was that closely related structures to 181, such as structure 182, also known as 6-sulfo-sLex NeuAc{alpha}2–3Gal{beta}1–4(Fuc{alpha}1–3)(6-O-Su)GlcNAc{beta}1-O(CH2)3NHCO(CH2)5NH-biotin, which only differs from structure 181 by the location of the 6-O-Su (Fig. 2), and structure 108, also known as sLex or NeuAc{alpha}2–3Gal{beta}1–4(Fuc{alpha}1–3)GlcNAc{beta}1-O(CH2)2NHCO(CH2)5NHCO(CH2)5NH-biotin, had minimally increased binding affinity (1.2:1 and 0.8:1 signal-to-noise binding, respectively) for Siglec-8. The spacer arm itself, -O(CH2)3NH-CO(CH2)5NH-biotin, found on many of the other non-binding structures, cannot explain the binding activity for Siglec-8. Taken together, this information demonstrates that the 6-position O-linked sulfate on galactose is key to the specificity and affinity of this structure for Siglec-8 (Fig. 2).



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  		FIG. 1.
Results from the Siglec-8-Ig glycan binding array analysis. A total of 172 glycans were screened for binding along with positive and negative controls, as described under "Experimental Procedures." Error bars represent mean ± S.D. replicate determinations from a single experiment. Note that ligand 181 (6'-sulfo-sLex) stands out among all the others for binding activity. RFU, relative fluorescence units.

 



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  		FIG. 2.
Structures of glycans used to verify specificity of Siglec-8-Ig binding. These included 6-sulfo-sLex,6'-sulfo-sLex, and sLex. Sia, sialic acid.

 
Validation of Glycan Array Results by SPR—The binding affinities of Siglec-8 for structures 108, 182, and 181 (Fig. 3, a–c, respectively) were determined on a Biacore 3000 instrument using a streptavidin-coated sensor chip. LacNAc was captured on one surface of the sensor chip and used as negative control (data not shown). As shown in Fig. 3, Siglec-8 showed specific binding, with very fast association and dissociation profiles only to the ligand 181. The sensorgrams showed that upon injection of the Siglec, binding rapidly reached equilibrium in the first few seconds, and the bound Siglec fell off rapidly after the injection was stopped. The apparent Kd for binding of Siglec-8 to ligand 181 was between 2 and 2.5 µM (Fig. 4).



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  		FIG. 3.
Specificity of Siglec-8 binding as assessed using SPR. No binding of Siglec-8-Ig was detected to sLex (a) or 6-sulfo-sLex (b), whereas reproducible binding was detected to 6'-sulfo-sLex (c). Individual plots of SPR response versus time are shown for concentrations of Siglec-8-Ig ranging from 0.1–18 µM. SPR response increases with increasing Siglec-8-Ig concentration in panel c only.

 



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  		FIG. 4.
Affinity of Siglec-8-Ig binding to 6'-sulfo-sLex as measured by SPR. Based on three replicate analyses, the dissociation constant (Kd) was 2.2–2.3 µM. The equilibrium SPR response (see Fig. 3c) is plotted versus the Siglec-8-Ig concentration.

 
Further Verification of the Glycan Array Results—Binding of Siglec-8-Ig precomplexed with fluorescein isothiocyanate anti-human Fc to aminopropyl glycosides immobilized on activated (N-hydroxysuccinimidyl) glass surfaces was also used to further verify the specificity of 6'-sulfo-sLex as a Siglec-8 ligand. As shown in Fig. 5, fluorescent signals were detected at immobilized concentrations of 6'-sulfo-sLex as low as 5 pmol/spot. In contrast, sLex and 6-sulfo-sLex did not support any Siglec-8 binding at the highest concentration tested (300 pmol/spot).



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  		FIG. 5.
Further verification of Siglec-8-Ig binding specificity using immobilized glycans on glass slides. Aminoalkyl glycans (as indicated; AH-Glc, amino hexylglucoside) were covalently spotted (as indicated, 0.15–300 pmol/spot) on an activated glass slide. Binding was visualized using Siglec-8-Ig fusion protein precomplexed with fluorescent anti-human Fc antibody as described under "Experimental Procedures." Binding was only seen with 6'-sulfo-sLex (a) at concentrations of 4.7 pmol/spot and above. b, replicate spot fluorescence intensities were averaged and displayed with error bars representing means ± S.D. from a single experiment using quadruplicate spots.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using a glycan array assembled by the Consortium for Functional Glycomics, 172 structures were screened for their ability to bind to Siglec-8-Ig fusion protein. By far the greatest binding was seen with ligand 181, which is 6'-sulfo-sLex (Figs. 1 and 2). Specificity was subsequently verified using SPR and a separate assay using immobilized glycans (Figs. 3, 4, 5). These three separate methods confirmed the specificity to be 6'-sulfo-sLex and not closely related structures because Siglec-8-Ig fusion protein failed to bind to unsulfated sLex as well as the closely related structure 6-sulfo-sLex. In addition, the specificity of binding, with reasonably high affinity (Kd 2.3 µM) to only one of 172 ligands tested, suggests that 6'-sulfo-sLex is a strong candidate for being a Siglec-8 ligand. Because candidate ligands for most other Siglecs remain virtually unknown, it is difficult to compare the Kd value for 6'-sulfo-sLex-Siglec-8 binding (2.3 µM) with Kd values for other Siglec-ligand interactions. Perhaps the best comparisons are provided by Blixt et al. (29), who tested a panel of 28 monovalent sialosides as inhibitors of Siglec binding. For the eight human Siglecs tested, the Ki of the best sialoside for each ranged from 120 to 4700 µM (average ~2mM). A "potent" sialoside inhibitor of mouse Siglec-4 had a Ki of 0.3 µM. The only other structure in the glycan array to show binding to Siglec-8 above background was ligand 188, which is a mixture of biantennary N-glycans from Wehi-3 cell supernatants. It is not known whether this supernatant contains 6'-sulfo-sLex. Although the specificity of binding of 6'-sulfo-sLex for Siglec-8 versus other human Siglecs has not yet been explored, this is the first time this ligand has been suspected to bind to any of the Siglecs. The likely mouse ortholog for Siglec-8 is mSiglec-F (33), but it is currently unknown whether 6'-sulfo-sLex is a ligand. However, additional experiments (data not shown) using an mSiglec-G-Ig fusion protein failed to show specific binding to 6'-sulfo-sLex, providing some degree of selectivity of binding for Siglec-8. Additionally, it remains to be determined what role various components of 6'-sulfo-sLex, such as fucose, play in binding. Finally, there are no known receptors for the structure, although initial studies suggested that 6'-sulfo-sLex might serve as an L-selectin ligand (34). However, subsequent studies found that 6-sulfo-sLex rather than 6'-sulfo-sLex is a preferred ligand (35–38).

Considering that the Siglec-8-Ig fusion protein is bivalent, the rapid nature of binding seen in Fig. 3 seems unexpected. Furthermore, because the reagent used for calculation of dissociation rates is bivalent, it is unclear whether the dissociation rate of Siglec-8 monomer, when expressed on the surface of a cell, would be similar or different. It is possible that the two binding sites on each arm of the chimera associate and dissociate from the glycan with the same binding parameters, but this remains unclear.

Synthesis of 6'-sulfo-sLex requires the presence of specific sulfotransferases in the Golgi apparatus of cells. Based on the known family of human Golgi-associated sulfotransferases (39–41), at least GST-1 (also known as keratin sulfate galactose 6-O sulfotransferase (KSGal6ST) and carbohydrate sulfotransferase 1 (CHST-1)), which is known to sulfate galactose residues linked to N-acetyl glucosamine, would likely be involved in the synthesis of 6'-sulfo-sLex (Fig. 2). These genes are located on chromosome 11p11.1–11.2, and studies report rather broad tissue distribution of CHST-1 (42, 43), although KSGal6ST reportedly is expressed predominantly in brain tissue and to a lesser degree in skeletal muscle (44). Methods such as immunohistochemistry would be useful in identifying tissue localization of 6'-sulfo-sLex expression, but to date, there are no antibody reagents that can distinguish 6'-sulfo-sLex from 6-sulfo-sLex (45). Previous studies with monoclonal antibodies suggest that cross-linking of Siglec-8 induces apoptosis on cells that express Siglec-8, such as human eosinophils (20), so it is tempting to speculate that expression of Siglec-8 ligand in the central nervous system may be a way to eliminate inflammatory cells that have infiltrated the central nervous system.

In summary, a Siglec-8-Ig chimeric protein was screened for binding to 172 different glycan structures immobilized as biotinylated glycosides. Among these, avid binding was detected to a single defined glycan, NeuAc{alpha}2–3(6-O-sulfo)Gal{beta}1–4[Fuc{alpha}1–3]GlcNAc, also referred to in the literature as 6'-sulfo-sLex. The data presented herein also demonstrate the utility and ease by which the Consortium for Functional Glycomics protein-carbohydrate interaction core H can readily be used to determine selective attachment of lectins, in this case a Siglec, to carbohydrate ligands. This rapid screening technique provides an initial method to define carbohydrate ligands for a wide range of structures and should prove useful for many such applications.


   FOOTNOTES
 
Note Added in Proof—Using a similar glycan array approach, it is now known that 6'-sulfo-sLex is also a ligand for langerin (Galustian, C., Park, G. G., Chai, W., Kiso, M., Bruening, S. A., Kang, Y. S., Steinman, R. M., and Feizi, T. (2004) Int. Immunol. 16, 853–866).

* This work was supported in part by the Consortium for Functional Glycomics under NIGMS, National Institutes of Health Grant GM62116. It was also supported by National Institutes of Health Grant AI41472 (to B. S. B.) and the Russian Academy of Sciences Physicochemical Biology Program (to N. V. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

§ To whom correspondence should be addressed: Division of Allergy & Clinical Immunology, Johns Hopkins Asthma & Allergy Center, 5501 Hopkins Bayview Circle, Rm. 2B.71, Baltimore, MD 21224-6821. Tel.: 410-550-2101; Fax: 410-550-1733; E-mail: bbochner{at}jhmi.edu.

1 The abbreviations used are: Siglec, sialic acid-binding immunoglobulin-like lectin; Fuc, fucose; sLex, sialyl Lewis X; PBS, phosphate-buffered saline; SPR, surface plasmon resonance. Back

2 Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., van Die, I., Burton, D., Wilson, I. A., Cummings, R., Bovin, N., Wong, C.-H., and Paulson, J. C. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 17033–17038. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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