EPITOPIC CHARACTERIZATION OF THE HUMAN WILD-TYPE AND MUTANT RAS PROTEINS USING MEMBRANE-BOUND PEPTIDES

Overlapping octapeptides encompassing the entire sequences of the huma n oncogene products Ha-ras, K-ras and N-ras protein were synthesized a s spots on polypropylene membrane sheets, The binding of anti-ras prot ein monoclonal antibodies (mAbs) to the membrane-bound peptides was as sessed using an enzyme-linked immunosorbent assay. Epitopes of 10 of 1 8 mAbs to the human I as proteins were mapped and identified by this p rocedure. The epitopes of nine of the mAbs are within residues 28-39 i n the constant domain common to the three ras proteins, whereas the ep itope of the tenth (mAb 21) spans residues 136-144 in Ha-ras. The mini mal lengths of epitopes of all ten of the mAbs were further precisely mapped using peptides of varying length, and the tolerance for mAb bin ding of mutated epitopes was determined by systematically replacing ea ch 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 sli ghtly 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 wi th other proteins (GTPase-activating protein, neurofibromin or raf) ar e highly antigenic. In addition, we investigated epitopes and specific ity of five mAbs against the activated human ras proteins by the same procedure. The information gained from this study should be useful bot h for study of the complicated functions of ras proteins and for clini cal detection of ras oncogenes in human tumor cells. (C) Munksgaard 19 97.