Abstract
The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.
Highlights
The human receptor designated as the dendritic cell-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN)4 has been identified both as an adhesion mol
Sequence Comparisons of Mouse SIGNs—Potential mouse orthologs of human DC-SIGN were previously identified by JULY 21, 2006
The genomic analysis presented here defines the full set of mouse homologs of human DC-SIGN, but the results shown in Figs. 3 and 5 indicate that sequence comparison alone is not sufficient to suggest that one of these proteins is a specific ortholog
Summary
Data Base Screening—The Ensembl annotation of the mouse genome was screened with InterPro profile IPR001304 to identify genes containing potential C-type CRDs. Additional binding assays with fluorescein-labeled, glycosylated polyacrylamide polymers (GlycoTech) were performed in the same format and read on a Victor plate reader from PerkinElmer Life Sciences In these studies, the buffer was 150 mM NaCl, 25 mM Tris-Cl, pH 7.4, 2 mM CaCl2, with 1% BSA present during ligand binding. For probing of the glycan array, tetramerized proteins were labeled with fluorescein isothiocyanate [9] following dialysis into 1.25 M NaCl, 25 mM Na-HEPES, pH 8.0, 25 mM CaCl2 or after repurification on a mannose-Sepharose column that was rinsed with buffer containing 1.25 M NaCl, 25 mM Bicine-Cl, pH 9.0, and 25 mM CaCl2 and eluted with 1.25 M NaCl, 25 mM Bicine-Cl, pH 9.0, and 2.5 mM EDTA. Protein expression was verified by Western blotting with antibodies to the bacterially expressed extracellular domain, raised in sheep
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