Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E

Abstract

Edited by Amanda F. Fosang CD33-related Siglecs are a family of proteins widely expressed on innate immune cells. Binding of sialylated glycans or other ligands triggers signals that inhibit or activate inflammation.Immunomodulation by Siglecs has been extensively studied, but relationships between structure and functions are poorly explored. Here we present new data relating to the structure and function of Siglec-E, the major CD33-related Siglec expressed on mouse neutrophils, monocytes, macrophages, and dendritic cells. We generated nine new rat monoclonal antibodies specific to mouse Siglec-E, with no cross-reactivity to Siglec-F. Although all antibodies detected Siglec-E on transfected human HEK-293T cells, only two reacted with mouse bone marrow neutrophils by flow cytometry and on spleen sections by immunohistochemistry. Moreover, whereas all antibodies recognized Siglec-E-Fc on immunoblots, binding was dependent on intact disulfide bonds and N-glycans, and only two antibodies recognized native Siglec-E within spleen lysates. Thus, we further investigated the impact of Siglec-E homodimerization. Homology-based structural modeling predicted a cysteine residue (Cys-298) in position to form a disulfide bridge between two Siglec-E polypeptides. Mutagenesis of Cys-298 confirmed its role in dimerization. In keeping with the high level of 9-O-acetylation found in mice, sialoglycan array studies indicate that this modification has complex effects on recognition by Siglec-E, in relationship to the underlying structures. However, we found no differences in phosphorylation or SHP-1 recruitment between dimeric and monomeric Siglec-E expressed on HEK293A cells. Phylogenomic analyses predicted that only some human and mouse Siglecs form disulfide-linked dimers. Notably, Siglec-9, the functionally equivalent human paralog of Siglec-E, occurs as a monomer. Two broad groups of Siglecs are identified based on their patterns of evolution. Sialoadhesin, CD22, myelin-associated glycoprotein (MAG) and Siglec-15 are highly conserved and show few changes among species (2). In contrast, CD33-related Siglecs evolve rapidly in mammals (2, 9). As there are not clear functional orthologs between primates and rodent Siglecs, these receptors are assigned an alphabetic letter in rodents and a number in primates. Siglec-E, the main CD33-related Siglec in mouse, was first discovered by a yeast two-hybrid screen using SHP-1 as a bait (10). Studies using a sheep polyclonal Siglec-E antibody specific for the ectodomain indicated that Siglec-E is expressed on neutrophils, macrophages, monocytes, dendritic cells, and a subset of Natural killer cells (11). The organ with the * This work was supported, in whole or in part, by National Institutes of Health Grants P01HL107150 and R01GM32373 (to A. V.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. □ S Th...

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