Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173
Fig 1
A: Myc-tagged recombinant CN-1 fragments were expressed in COS7 cells by transfection. Cells were harvested and the recombinant proteins were detected by Westernblotting using RYSK173 or anti-Myc (Fig on the left). Three overlapping synthetic peptides (CN1-1, CN1-2 and CN1-3) corresponding to bp 313–471 were tested in dotblot analysis (Fig on the right). B: Two additional peptides (CN1-4 and CN1-5) corresponding to the overlapping sequence of CN1-2 and CN1-3 were tested in ELISA for recognition by RYSK173. C: Location of the dinuclear zinc center at the CN-1 homodimer interface. The monomer on the left is depicted as a ribbon drawing and the monomer on the right is rendered as solid surface. Zinc1 and zinc2 in the left monomer are shown as cyan and magenta colored spheres, respectively. The enlarged view details the position of the RYSK173 epitope, which is confined to the amino acid sequence H132 to D139 (both marked with an asterisk). The surface of the left subunit is rendered semi-transparent. The Fig was made with Protein Workshop. D: H132 was exchanged for glutamine (Q) by site directed mutagenesis. The recombinant proteins were detected in transfected cell lysates using the ATLAS and RYSK173 based ELISA. The results are expressed as RYSK173/ATLAS ratio x 100%. NC: negative control, WT: wild type.