Glycan Array Screening Reveals a Candidate Ligand for Siglec-8*

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, NeuAcα2–3(6-O-sulfo)Galβ1–4[Fucα1–3]GlcNAc, also referred to in the literature as 6′-sulfo-sLex. Notably, neither unsulfated sLex (NeuAcα2–3Galβ1–4[Fucα1–3]GlcNAc) nor an isomer with the sulfate on the 6-position of the GlcNAc residue (6-sulfo-sLex, NeuAcα2–3Galβ1–4[Fucα1–3](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.

[1]  L. Kjellén,et al.  Sulfotransferases in glycosaminoglycan biosynthesis. , 2003, Current opinion in structural biology.

[2]  S. Hemmerich,et al.  Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin , 1999, The Journal of cell biology.

[3]  K. Cheng,et al.  A new siglec family member, siglec-10, is expressed in cells of the immune system and has signaling properties similar to CD33. , 2001, European journal of biochemistry.

[4]  B. Bochner,et al.  Ligation of Siglec-8: a selective mechanism for induction of human eosinophil apoptosis. , 2003, Blood.

[5]  L. Moretta,et al.  p75/AIRM1 and CD33, two sialoadhesin receptors that regulate the proliferation or the survival of normal and leukemic myeloid cells , 2001, Immunological reviews.

[6]  B. Bochner,et al.  Human eosinophils express two Siglec-8 splice variants. , 2002, The Journal of allergy and clinical immunology.

[7]  E. Jones,et al.  Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin. , 2003, Structure.

[8]  A. May,et al.  Crystal structure of the N-terminal domain of sialoadhesin in complex with 3' sialyllactose at 1.85 A resolution. , 1998, Molecular cell.

[9]  S. Hemmerich,et al.  Identification of an N-acetylglucosamine-6-0-sulfotransferase activity specific to lymphoid tissue: an enzyme with a possible role in lymphocyte homing. , 1998, Chemistry & biology.

[10]  C. Bertozzi,et al.  Carbohydrate sulfotransferases of the GalNAc/Gal/GlcNAc6ST family. , 2002, Biochemistry.

[11]  T. Tedder,et al.  CHST1 and CHST2 sulfotransferases expressed by human vascular endothelial cells: cDNA cloning, expression, and chromosomal localization. , 1999, Genomics.

[12]  I. Tabas,et al.  Human chondroitin 6-sulfotransferase: cloning, gene structure, and chromosomal localization. , 1998, Biochimica et biophysica acta.

[13]  C. Bertozzi,et al.  L-selectin-carbohydrate interactions: relevant modifications of the Lewis x trisaccharide. , 1996, Biochemistry.

[14]  S. Hemmerich,et al.  Chromosomal localization and genomic organization for the galactose/ N-acetylgalactosamine/N-acetylglucosamine 6-O-sulfotransferase gene family. , 2001, Glycobiology.

[15]  James C Paulson,et al.  Negative Regulation of T Cell Receptor Signaling by Siglec-7 (p70/AIRM) and Siglec-9* , 2004, Journal of Biological Chemistry.

[16]  S. Hemmerich,et al.  Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes. , 2001, Immunity.

[17]  Structure of the saccharide-binding domain of the human natural killer cell inhibitory receptor p75/AIRM1. , 2004, Acta crystallographica. Section D, Biological crystallography.

[18]  T. K. van den Berg,et al.  Cutting Edge: CD43 Functions as a T Cell Counterreceptor for the Macrophage Adhesion Receptor Sialoadhesin (Siglec-1) , 2001, The Journal of Immunology.

[19]  E. Shimada,et al.  Molecular Cloning and Characterization of Human Keratan Sulfate Gal-6-Sulfotransferase* , 1997, The Journal of Biological Chemistry.

[20]  A. Varki,et al.  Siglecs, sialic acids and innate immunity. , 2001, Trends in immunology.

[21]  A. Varki,et al.  New Aspects of Siglec Binding Specificities, Including the Significance of Fucosylation and of the Sialyl-Tn Epitope* , 2000, The Journal of Biological Chemistry.

[22]  T. Yamaji,et al.  A Small Region of the Natural Killer Cell Receptor, Siglec-7, Is Responsible for Its Preferred Binding to α2,8-Disialyl and Branched α2,6-Sialyl Residues , 2002, The Journal of Biological Chemistry.

[23]  P. Crocker,et al.  Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell-cell interactions and signalling. , 2002, Current opinion in structural biology.

[24]  K. Kumamoto,et al.  Loss of Disialyl Lewisa, the Ligand for Lymphocyte Inhibitory Receptor Sialic Acid-Binding Immunoglobulin-Like Lectin-7 (Siglec-7) Associated with Increased Sialyl Lewisa Expression on Human Colon Cancers , 2004, Cancer Research.

[25]  P. Murdock,et al.  Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils. , 2000, The Journal of allergy and clinical immunology.

[26]  J. Burchell,et al.  Macrophage–tumour cell interactions: identification of MUC1 on breast cancer cells as a potential counter‐receptor for the macrophage‐restricted receptor, sialoadhesin , 1999, Immunology.

[27]  P. Crocker,et al.  Characterization of CD33 as a new member of the sialoadhesin family of cellular interaction molecules. , 1995, Blood.

[28]  R. Hill,et al.  [29] Monosaccharides attached to agarose , 1974 .

[29]  P. Crocker,et al.  The murine inhibitory receptor mSiglec‐E is expressed broadly on cells of the innate immune system whereas mSiglec‐F is restricted to eosinophils , 2004, European journal of immunology.

[30]  A. Varki,et al.  CD33/Siglec-3 Binding Specificity, Expression Pattern, and Consequences of Gene Deletion in Mice , 2003, Molecular and Cellular Biology.

[31]  N. Bovin,et al.  Ganglioside GD3 expression on target cells can modulate NK cell cytotoxicity via siglec‐7‐dependent and ‐independent mechanisms , 2003, European journal of immunology.

[32]  P. Crocker,et al.  Recognition of sialylated meningococcal lipopolysaccharide by siglecs expressed on myeloid cells leads to enhanced bacterial uptake , 2003, Molecular Microbiology.

[33]  P. Crocker,et al.  Siglecs in the immune system , 2001, Immunology.

[34]  N. Bovin,et al.  Ganglioside binding pattern of CD33-related siglecs. , 2003, Bioorganic & medicinal chemistry letters.

[35]  R. Kannagi,et al.  Identification of a Major Carbohydrate Capping Group of the L-selectin Ligand on High Endothelial Venules in Human Lymph Nodes as 6-Sulfo Sialyl Lewis X* , 1998, The Journal of Biological Chemistry.

[36]  S. Tsuboi,et al.  6′-Sulfo Sialyl Lex but Not 6-Sulfo Sialyl Lex Expressed on the Cell Surface Supports L-selectin-mediated Adhesion* , 1996, The Journal of Biological Chemistry.

[37]  R. Biassoni,et al.  Identification and Molecular Cloning of P75/Airm1, a Novel Member of the Sialoadhesin Family That Functions as an Inhibitory Receptor in Human Natural Killer Cells , 1999, The Journal of experimental medicine.

[38]  G. Yousef,et al.  Molecular characterization of a Siglec8 variant containing cytoplasmic tyrosine-based motifs, and mapping of the Siglec8 gene. , 2000, Biochemical and biophysical research communications.

[39]  G. Yousef,et al.  Genomic organization of the siglec gene locus on chromosome 19q13.4 and cloning of two new siglec pseudogenes. , 2002, Gene.

[40]  High Resolution Crystal Structures of Siglec-7 , 2003, The Journal of Biological Chemistry.

[41]  James C. Paulson,et al.  Sialoside Specificity of the Siglec Family Assessed Using Novel Multivalent Probes , 2003, Journal of Biological Chemistry.

[42]  Jian Ni,et al.  Siglec-8 , 2000, The Journal of Biological Chemistry.