Abstract
CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcβ1–2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.
Highlights
CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors
Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions
Following protocols that have been used for purification of other C-type CRDs, a fragment of Bos taurus CD23 encompassing residues Ala157–Cys290, corresponding to the globular domain, was expressed in Escherichia coli
Summary
Crystals obtained in the presence of the simple sugar ligands ␣-methyl mannoside and GlcNAc1–2Man showed that binding of mannose at the primary binding site results from coordination and hydrogen bonds with the 3- and 4-OH groups in the arrangement typically seen in C-type CRDs that bind mannose and related sugars (Fig. 5, F and H). Increased affinity was achieved by forming a tetrameric complex with streptavidin, which bound more tightly to the resin in the presence of Ca2ϩ and was eluted with EDTA, confirming that mouse CD23 binds sugars in a Ca2ϩ-dependent manner, with lower affinity than cow CD23 (Fig. 9B). Similar fragments corresponding to the CRD from human CD23 failed to show any binding activity by affinity chromatography, even when biotin-tagged CRDs were complexed with streptavidin to increase the affinity (data not shown) These results correlate with the absence of one of the key Ca2ϩ-ligating residues at the primary sugar-binding site (Fig. 1A). Once sugar-binding activity was lost, selective pressure to retain other residues that form the Ca2ϩ- and sugar-binding sites was absent, and additional mutations in key residues occurred
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