Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6′-sulfo-sialyl Lewis X as a preferred glycan ligand

Abstract

Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6'-sulfo-sLe(X) (Neu5Acalpha2-3[6-SO4] Galbeta1-4[Fucalpha1-3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6'-sulfo-sLe X. Secondary analysis showed that related structures, sialyl-Lewis X (sLe X) and 6-sulfo-sLe X containing 6-GlcNAc-SO4 showed much lower binding avidity, indicating significant contribution of 6-Gal-SO4 on Siglec-F binding to 6'-sulfo-sLe x. The lectin activity of Siglec-F on mouse eosinophils was "masked" by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6'-sulfo-sLe X structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6'-sulfo-sLe X. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6'-sulfo-sLe X ligand in eosinophil biology.

Probing the cis interactions of the inhibitory receptor Siglec-7 with α2,8-disialylated ligands on natural killer cells and other leukocytes using glycan-specific antibodies and by analysis of α2,8-sialyltransferase gene expression

“…In previous studies, these antibodies have been shown to react similarly with b-series gangliosides GD1b, GT1b, GQ1b, and GD3 in a manner consistent with their minimal epitope being NeuAc␣2,8- [30,31]. As shown in Figure 3A, Ha1 antibody recognized sialic acid-containing glycosides exclusively and showed a strong preference for glycans containing NeuAc␣2,8-NeuAc␣2,3-Gal.…”

“…The sugar-binding specificities of Ha1 antibody were analyzed by the Consortium for Functional Glycomics (CFG; http://www.functionalglycomics.org/static/consortium) using Glycan Array v2.8 as described previously [30,31]. Briefly, streptavidin-coated microtiter plates (Reacti-Bind NeutrAvidin-coated high-binding capacity black 384-well plates, Pierce, Rockford, IL) were coated with various biotinylated glycosides (30 pmol/well) in PBS, pH 7.4, overnight at 4°C.…”

Eosinophils from Lineage-Ablated ΔdblGATA Bone Marrow Progenitors: The dblGATA Enhancer in the Promoter of GATA-1 Is Not Essential for Differentiation Ex Vivo

“…Eosinophils in culture were detected and enumerated by cell surface expression of Siglec F (12,13). In a previous study, we found no Siglec F ϩ eosinophils in the bone marrow of ⌬dblGATA mice when gating on the side scatter-high mature granulocyte population (8).…”

Expression and preliminary functional analysis of Siglec-F on mouse macrophages

“…Investigations of Siglec biology have focused on the mouse as a model organism due to the difficulties and limitations associated with this type of research in humans. Recent studies identified murine Siglec-F as the functional paralog of human Siglec-8 based on its expression on mouse eosinophils and its ability to adhere to the 6′-sulfo-sLex structure (Tateno et al, 2005;Bochner, 2009). Studies have demonstrated that cross-linking of Siglec-F with a specific antibody (Ab) induced mouse eosinophil apoptosis in vivo and in vitro (Zimmermann et al, 2008).…”