Molecular and functional characterization of porcine Siglec-3/CD33 and analysis of its expression in blood and tissues

• Published in: Developmental and comparative immunology Vol. 51; no. 2; pp. 238 - 250
• Main Authors: Álvarez, B., Escalona, Z., Uenishi, H., Toki, D., Revilla, C., Yuste, M., del Moral, M. Gómez, Alonso, F., Ezquerra, A., Domínguez, J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01-08-2015
• Subjects: Animals; Antibodies, Monoclonal - metabolism; Blood Cells - immunology; CD33; CHO Cells; Cricetulus; Gene Expression Profiling; Humans; Intestine, Small - physiology; Monoclonal antibody; Monocyte; Myeloid cells; Myeloid Cells - immunology; N-Acetylneuraminic Acid - metabolism; Protein Binding; Sequence Homology, Amino Acid; Sialic Acid Binding Ig-like Lectin 3 - genetics; Sialic Acid Binding Ig-like Lectin 3 - immunology; Sialic Acid Binding Ig-like Lectin 3 - metabolism; Siglec-3; Swine; Swine - immunology; Transgenes - genetics; Myeloid cells; Monocyte; HRP; ITSM; Sn4D-Fc; Siglec-3; Monoclonal antibody; CpG ODN; SHP-1; rp-GM-CSF; Siglec-3-Fc; GFP; PAA; RBC; Malp-2; ITIM motif; Swine; BMDC; pDCs; poly(I:C); rh M-CSF; Siglec-3-GFP; CD33
• ISSN: 0145-305X; 1879-0089
• DOI: 10.1016/j.dci.2015.04.002

•Cell lines expressing the porcine homologue of Siglec-3/CD33 have been generated.•Monoclonal antibodies to porcine Siglec-3 have been developed.•Porcine Siglec-3 is mainly expressed on myeloid cells and their precursors.
A cDNA clone encoding a 380 a-a type 1 transmembrane protein with homology to human Siglec-3/CD33 was obtained from a swine small intestine library. An analysis of protein sequence identified two immunoglobulin-like domains, a transmembrane region, and a carboxi-terminal tail with two tyrosine-based signalling motifs. Binding assays of Siglec-3 transfected CHO cells to polyacrylamide glycoconjugates showed a preference for α2–6-linked sialic acids. Using mAbs raised against a fragment containing the two Ig-like domains, porcine Siglec-3 was found to be expressed on monocytes and granulocytes, and their bone marrow precursors. It was also detected in lymph node, splenic and alveolar macrophages. MAbs immunoprecipitated, from granulocyte lysates, a protein of 51–60 kDa under both non-reducing and reducing conditions. MAbs were also used to analyse functional activity of Siglec-3 on bone marrow and blood cells. Engagement of Siglec-3 by mAb had no apparent effect on cell proliferation or cytokine production.