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. 2010;478:343-63.
doi: 10.1016/S0076-6879(10)78017-4.

Multivalent Ligands for Siglecs

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Free PMC article

Multivalent Ligands for Siglecs

Mary K O'Reilly et al. Methods Enzymol. .
Free PMC article

Abstract

Siglecs have emerged as an important family of immunomodulatory glycan-binding proteins that can bind sialoside ligands both on the same cell surface, in cis, and on other cells, in trans. Expression of siglecs varies among a variety of immune cells, and tools to probe siglecs on these cells are crucial to understanding their function. In designing synthetic ligands, competition by cis ligands requires the use of multivalency to achieve sufficient avidity to stably bind siglecs on native cells. This chapter describes the use of multivalent ligands to probe cell surfaces, as well as to investigate ligand binding to recombinant siglecs.

Figures

Figure 1
Figure 1. Schematic of competition between trans ligands and cis ligands of siglecs
Using CD22 as an example, cis ligand binding leads to masking of the ligand-binding site. Only with sufficient avidity (or removal of sialic acids) can trans ligands compete with cis ligands to achieve stable binding at the cell surface.
Figure 2
Figure 2. PAA probe binding to siglec-expressing cells reveals importance of valency
6′-sulfo-sialyl-Lewisx-PAA of low molecular weight (30 kDa, approximately 15-mer) or high molecular weight (1000 kDa, approximately 500-mer) was incubated with eosinophils, which express Siglec-F, or CHO cells transfected with Siglec-F. Asialo cells were prepared by pre-treatment of native cells with sialidase. Reproduced with permission.
Figure 3
Figure 3. High-affinity PAA probe binding to siglec-expressing cells shows the importance of intrinsic affinity in overcoming cis ligands
A human BJAB B cell line (A) or murine B cells (B) were probed with native (NeuAc(Gc)α2,6-LacNAc-) or high-affinity BPC(BPA)NeuAc(Gc)α2,6-LacNAc- ligands appended to PAA. Reproduced with permission.
Figure 4
Figure 4. Siglec-Fc beads enable binding at elevated temperature without endocytosis to reveal improved binding
Human CD22-Fc beads were probed with high molecular weight LacNAc-PAA or BPCNeuAcα2,6-LacNAc-PAA at 4 °C or 37 °C for 16 hours.
Figure 5
Figure 5. Pre-complexation of Siglec-Fc is key to engaging with ligand-PAA-coated beads
Siglec-7-Fc from culture supernatant was incubated with streptavidin beads coated with unsubstituted PAA or NeuAcα2,8-NeuAcα2,3-Galβ1,4-GlcNAc-PAA, either in the absense (top, 2-step) or presence (botton, 1-step) of the detection antibody, FITC-anti-human IgG. For the 2-step method (top), unbound probe was washed away prior to adding the detection antibody.
Figure 6
Figure 6. Tuning the density of biotinylated ligand-coated beads to modulate avidity of siglec-Fc binding
Streptavidin beads were coated with biotinylated NeuAcα2,8-NeuAcα2,3-Galβ1,4-GlcNAc after pre-treatment of beads with varying concentrations of free biotin to adjust the density of carbohydrate ligands. Beads were then probed with siglec-E-Fc from culture supernatant in the presence of the detection antibody, FITC-anti-human IgG. Control beads had neither biotin nor ligand.
Figure 7
Figure 7. High-affinity ligand is required for adhesion between-PAA-coated beads and CD22-expressing cells
Streptavidin beads coated with 30 kDa or 1000 kDa versions of NeuGca2,6-LacNAc-PAA or BPANeuGca2,6-LacNAc-PAA were exposed to primary murine B cells. Reproduced with permission.

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