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Epitope mapping of CD1d-specific mAbs.

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posted on 2010-09-30, 02:10 authored by Elisa Monzon-Casanova, Birte Steiniger, Stefanie Schweigle, Holger Clemen, Daniela Zdzieblo, Lisa Starick, Ingrid Müller, Chyung-Ru Wang, Sara Rhost, Susanna Cardell, Elwira Pyz, Thomas Herrmann

(A) Alignment of the amino acid sequences of the extracellular domains of the CD1d molecules used in this study. α-helical regions are illustrated in bold. The open triangle points out the localization of the rat CD1d allelism. Mutations in mouse CD1d1 are highlighted with closed triangles. The arrow indicates where mouse/human chimeras were joined. Numbers show rat CD1d amino acid positions. Accession numbers for the amino acid sequences can be found in GenBank: rCD1d (Rattus norvegicus CD1d), BAA82323; mCD1d1 (Mus musculus CD1d1), NP_031665; sCD1d1 (Mus spretus CD1d1), ACM45455; hCD1d1 (Homo sapiens CD1d), NP_001757. The mCD1d2 (Mus musculus CD1d2) amino acidic sequence differs in one amino acid in the signal peptide (tryptophan, position −13) from the sequence published under the accession number: P11610. (B) Binding capacity of mAbs to different CD1d molecules. Raji cells were transduced with CD1d molecules using a retroviral expression system. Bicistronic EGFP in the CD1d expression vectors served as reporter gene. Cells were stained with unconjugated WTH-1 or WTH-2 mAbs followed by PE-labeled donkey anti-mouse IgG or PE-labeled 1B1 mAb and were analyzed by flow cytometry. All primary antibodies were used at a final concentration of 250 ng/ml. (C) Localization of relevant amino acids in the CD1d tertiary structure. The model depicts part of the co-crystal of the PBS25 glycolipid and mouse CD1d (PDB: 3GMP) and it was visualized using PDB Swiss Viewer Deep View v4.0 [52] (http://www.expasy.org/spdbv/). The α1 and α2 domains are shown. Gray and green ribbon diagrams highlight the regions constituting the N- and C-terminal parts, respectively, of the chimeric molecules. Spheres visualize the amino acids discussed in the text: aspartic acid (D) 93 in red, methionine (M) 162 in green, valine (V) 118 in gray and arginine (R) 21 in blue.

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