title: Studies on the Detection, Expression, Glycosylation, Dimerization, and Ligand Binding Properties of Mouse Siglec-E.
abstract: 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.
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Shoib
authors.initials: S
authors.lastname: Siddiqui
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Flavio
authors.initials: F
authors.lastname: Schwarz
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Stevan
authors.initials: S
authors.lastname: Springer
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Khedri
authors.affiliation: the Department of Chemistry, University of California, Davis, California 95616, and.
authors.forename: Hai
authors.initials: H
authors.lastname: Yu
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Lingquan
authors.initials: L
authors.lastname: Deng
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Andrea
authors.initials: A
authors.lastname: Verhagen
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Naito-Matsui
authors.affiliation: BioLegend, Inc., San Diego, California 92121.
authors.forename: Weiping
authors.initials: W
authors.lastname: Jiang
authors.affiliation: BioLegend, Inc., San Diego, California 92121.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kim
authors.affiliation: BioLegend, Inc., San Diego, California 92121.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhou
authors.affiliation: BioLegend, Inc., San Diego, California 92121.
authors.forename: Beibei
authors.initials: B
authors.lastname: Ding
authors.affiliation: the Department of Chemistry, University of California, Davis, California 95616, and.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687.
authors.forename: Nissi
authors.initials: N
authors.lastname: Varki
authors.affiliation: From the Departments of Medicine and Cellular & Molecular Medicine, Glycobiology Research and Training Center (GRTC), University of California San Diego, La Jolla, California 92093-0687, a1varki@ucsd.edu.
authors.forename: Ajit
authors.initials: A
authors.lastname: Varki
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006031:Glycosylation; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D016296:Mutagenesis; D020125:Mutation, Missense; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D051381:Rats; D011917:Rats, Inbred Lew; D063265:Sialic Acid Binding Immunoglobulin-like Lectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C412227:SIGLEC9 protein, human; D063265:Sialic Acid Binding Immunoglobulin-like Lectins; C422908:Siglece protein, mouse; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6
keywords: Siglec-9; Siglec-E; cell signaling; dimerization; flow cytometry; monoclonal antibody; sialic acid