Epitopic characterization of the human wild-type and mutant ras proteins using membrane-bound peptides.
Overlapping octapeptides encompassing the entire sequences of the human oncogene products Ha-ras, K-ras and N-ras protein were synthesized as spots on polypropylene membrane sheets. The binding of anti-ras protein monoclonal antibodies (mAbs) to the membrane-bound peptides was assessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 18 mAbs to the human ras proteins were mapped and identified by this procedure. The epitopes of nine of the mAbs are within residues 28-39 in the constant domain common to the three ras proteins, whereas the epitope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The minimal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb binding of mutated epitopes was determined by systematically replacing each residue in the epitope with each of the 20 common amino acids. The results show that most of these mAbs have essentially the same binding specificity, namely for the sequence YDPT (residues 32-35) or for slightly longer sequences containing these residues. This site is in the switch 1 region (residues 32-38) in the ras effector loop, indicating that some of the same residues important for the interaction of ras with other proteins (GTPase-activating protein, neurofibromin or raf) are highly antigenic. In addition, we investigated epitopes and specificity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful both for study of the complicated functions of ras proteins and clinical detection of ras oncogenes in human tumor cells.
Wang
Z
Z
Department of Chemistry, Boston University, Massachusetts, USA.
Carney
W P
WP
Laursen
R A
RA
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D000939:Epitopes / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D006801:Humans; D008567:Membranes, Artificial; D009154:Mutation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008567:Membranes, Artificial; D010446:Peptide Fragments; D018631:ras Proteins