Studies on the detection, expression, glycosylation, dimerization, and ligand binding properties of mouse Siglec-E

Shoib Siddiqui, Flavio Schwarz, Stevan Springer, Zahra Khedri, Hai Yu, Lingquan Deng, Andrea Verhagen, Yuko Naito-Matsui, Weiping Jiang, Daniel Kim, Jie Zhou, Beibei Ding, Xi Chen, Nissi Varki, Ajit Varki

Research output: Contribution to journalArticle

9 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1029-1037
Number of pages9
JournalJournal of Biological Chemistry
Volume292
Issue number3
DOIs
StatePublished - Jan 20 2017

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Sialic Acid Binding Immunoglobulin-like Lectins
Glycosylation
Dimerization
Ligands
Disulfides
Polysaccharides
Antibodies
Neutrophils
Spleen

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Studies on the detection, expression, glycosylation, dimerization, and ligand binding properties of mouse Siglec-E. / Siddiqui, Shoib; Schwarz, Flavio; Springer, Stevan; Khedri, Zahra; Yu, Hai; Deng, Lingquan; Verhagen, Andrea; Naito-Matsui, Yuko; Jiang, Weiping; Kim, Daniel; Zhou, Jie; Ding, Beibei; Chen, Xi; Varki, Nissi; Varki, Ajit.

In: Journal of Biological Chemistry, Vol. 292, No. 3, 20.01.2017, p. 1029-1037.

Research output: Contribution to journalArticle

Siddiqui, S, Schwarz, F, Springer, S, Khedri, Z, Yu, H, Deng, L, Verhagen, A, Naito-Matsui, Y, Jiang, W, Kim, D, Zhou, J, Ding, B, Chen, X, Varki, N & Varki, A 2017, 'Studies on the detection, expression, glycosylation, dimerization, and ligand binding properties of mouse Siglec-E', Journal of Biological Chemistry, vol. 292, no. 3, pp. 1029-1037. https://doi.org/10.1074/jbc.M116.738351
Siddiqui, Shoib ; Schwarz, Flavio ; Springer, Stevan ; Khedri, Zahra ; Yu, Hai ; Deng, Lingquan ; Verhagen, Andrea ; Naito-Matsui, Yuko ; Jiang, Weiping ; Kim, Daniel ; Zhou, Jie ; Ding, Beibei ; Chen, Xi ; Varki, Nissi ; Varki, Ajit. / Studies on the detection, expression, glycosylation, dimerization, and ligand binding properties of mouse Siglec-E. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 3. pp. 1029-1037.
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AU - Khedri, Zahra

AU - Yu, Hai

AU - Deng, Lingquan

AU - Verhagen, Andrea

AU - Naito-Matsui, Yuko

AU - Jiang, Weiping

AU - Kim, Daniel

AU - Zhou, Jie

AU - Ding, Beibei

AU - Chen, Xi

AU - Varki, Nissi

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