Kristine K. Kikly, PhDa, Bruce S. Bochner, MDg, Sylvie D

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Identification of SAF-2, a novel siglec expressed on eosinophils, mast cells, and basophils Kristine K. Kikly, PhDa, Bruce S. Bochner, MDg, Sylvie D. Freeman, PhDh, K.B. Tan, BSc, Kathleen T. Gallagher, BSb, Karla J. D’Alessio, BSd, Stephen D. Holmes, PhDe, Julie A. Abrahamson, BSe, Connie L. Erickson-Miller, PhDf, Paul R. Murdock, PhDc, Hiroshi Tachimoto, MD, PhDg, Robert P. Schleimer, PhDg, John R. White, PhDa Journal of Allergy and Clinical Immunology Volume 105, Issue 6, Pages 1093-1100 (June 2000) DOI: 10.1067/mai.2000.107127 Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 1 Predicted protein alignment of SAF-2 (GenBank No. AF223403), CD33 (GenBank No. M23197), siglec-5 (AF170484), siglec-6 (D86358), and siglec-7 (AF170485). Alignment was performed with Clustal analysis in MegAlign (Lasergene) and optimized by eye. Residues conserved in all sequences are boxed, and those conserved in 3 of the 5 are shaded. Strand predictions and domains were based on the work of Cornish et al.24 The predicted signal peptide, strand predictions, Ig domains, TM, and cytoplasmic regions are marked above the alignment. The N-linked glycosylation site that has been shown to modulate sialic acid binding for CD22 and CD33 but is not conserved in SAF-2 is marked with the first asterisk (amino acid 106). The conserved R in the F strand of the N-terminal Ig domain is marked with the second asterisk (amino acid 125). Journal of Allergy and Clinical Immunology 2000 105, 1093-1100DOI: (10.1067/mai.2000.107127) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 2 TaqMan analysis of SAF-2 and siglec-5. For each tissue, 4 different samples were measured for expression of SAF-2 and siglec-5 mRNA by using templates derived from 1 ng of polyA+ RNA. Data are shown as mean ± SEM. Expression of the housekeeping gene GAPDH is shown for comparison. Journal of Allergy and Clinical Immunology 2000 105, 1093-1100DOI: (10.1067/mai.2000.107127) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 3 Expression of SAF-2 on human peripheral blood eosinophils, basophils, and 16-week-old cord blood–derived cultured mast cells. Histograms shown are representative of 3 to 4 experiments with virtually identical results for each cell type. Monoclonal reagents used as positive and negative controls are also shown. Journal of Allergy and Clinical Immunology 2000 105, 1093-1100DOI: (10.1067/mai.2000.107127) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 4 Binding of human RBCs to SAF-2 expressed on COS cells. Binding assays were performed with COS cells mock-transfected or transiently expressing SAF-2, siglec-5, CD22, or CD33. Human RBCs were added to untreated (black bars) or sialidase-pretreated (white bars) COS cells and allowed to bind for 30 minutes at 4°C. After washing, cells were fixed and percentage of COS cells with RBC rosettes (defined as COS cell binding more than 20 RBCs) was scored by counting at least 200 COS cells per well. All constructs were expressed at comparable levels as determined by flow cytometric analysis before assays (data not shown). Results shown are mean ± SD of triplicate wells from a representative experiment. Journal of Allergy and Clinical Immunology 2000 105, 1093-1100DOI: (10.1067/mai.2000.107127) Copyright © 2000 Mosby, Inc. Terms and Conditions

Fig. 5 Schematic representation of siglec family. The most N-terminal Ig domain belongs to the V-set subtype and is represented by a dark blue structure . This is followed by varying numbers of C2-set Ig domains represented by red circles . Cytoplasmic domains are drawn to scale relative to each other. Tyrosines in the cytoplasmic domain are represented as black dots ; most tyrosines fall into ITIMs or ITAMs and are thought to participate in signaling. Journal of Allergy and Clinical Immunology 2000 105, 1093-1100DOI: (10.1067/mai.2000.107127) Copyright © 2000 Mosby, Inc. Terms and Conditions