Selection of mimotopes of the cell surface adhesion molecule Mel-CAM from a random pVIII-28aa phage peptide library.
The cell surface adhesion molecule Mel-CAM is highly expressed in advanced primary and metastatic melanoma. Mel-CAM was first described as an integral membrane glycoprotein of malignant melanoma cells. The murine monoclonal antibody MAd18-5D7 recognizes an epitope of the extracellular domain of Mel-CAM and is able to enhance Mel-CAM mediated adhesion of melanoma cells in aggregation assays. For the characterization of peptides that antigenically mimic surface-exposed areas of Mel-CAM we screened a newly constructed random pVIII-28aa bacteriophage peptide library against MAd18-5D7. After three panning rounds a population of phages binding to MAd18-5D7 was enriched. Peptides expressed on the surface of these phages were then tested for their specificity for the antibody's antigen binding site. DNA sequences coding for two specific peptide ligands were determined. One of the deduced amino acid sequences showed similarity to a portion of the sequence of the third immunoglobulin-like extracellular domain of Mel-CAM. Both peptides blocked the interaction of MAd18-5D7 with Mel-CAM present in a MelJuSo melanoma cell line lysate. Phage displayed as well as synthetic peptides inhibited in a dose-dependent manner the binding of MAd18-5D7 to recombinant Mel-CAM in enzyme-linked immunosorbent assay experiments. No such inhibition was observed using a panel of other anti-Mel-CAM antibodies. Our results clearly indicate that these 28mer peptides are structural equivalents of the MAd18-5D7 epitope of Mel-CAM and that they will be useful tools for further in vitro and in vivo studies of Mel-CAM mediated cell-cell interaction.
Hafner
Christine
C
Department of Dermatology, Division of General Dermatology, University of Vienna, Austria.
Samwald
Ursula
U
Wagner
Stefan
S
Felici
Franco
F
Heere-Ress
Elisabeth
E
Jensen-Jarolim
Erika
E
Wolff
Klaus
K
Scheiner
Otto
O
Pehamberger
Hubert
H
Breiteneder
Heimo
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD; D001666:Binding Sites, Antibody; D051929:CD146 Antigen; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D019006:Neural Cell Adhesion Molecules; D019151:Peptide Library; D011994:Recombinant Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D051929:CD146 Antigen; D000939:Epitopes; D008562:Membrane Glycoproteins; D019006:Neural Cell Adhesion Molecules; D019151:Peptide Library; D011994:Recombinant Proteins