A MONOCLONAL-ANTIBODY TO A MULTIPHOSPHORYLATED, CONFORMATIONAL EPITOPE AT THE CARBOXY-TERMINUS OF P53

Mutations of the gene encoding the tumor suppressor protein p53 are th e most common molecular alterations of cancer cells found in about hal f of all human tumors. Mutations which cluster in well-defined hot spo ts change the structure of the protein thus affecting its ability to b ind to DNA. Post-translational modifications, primarily phosphorylatio n, might also influence how p53 binds to DNA or folds to its active te trameric: form. However, the lack of appropriate biochemical markers t o characterize the status of phosphorylation in different cell types a nd in cells at different stages of tumor progression has prohibited su ch investigations. To generate a sensitive and phosphorylation-specifi c monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. Th e peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immuno dominant T-helper cell epitope in mice of the H-2(k) haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensiti ve reagent. By enzyme-linked immunosorbent assay,:p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Esch erichia coil. Moreover, murine p53 from insect cells could be immune p urified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments ve rified the presence of phosphate groups at both Ser375 and Ser389. Fro m the corresponding human protein fragments, mAb p53-18 bound to the i mmunizing peptide phosphorylated on Ser378 and on Ser392, but failed t o cross-react with the unphosphorylated peptide, or peptides phosphory lated individually on either Ser378 or Ser392. The binding to the unph osphorylated peptide could be restored, however, if the peptide confor mation was stabilized to that of an alpha-helix. The immunogenic natur e of the multiphosphorylated C-terminus of p53 is indicated by the fin ding that human sera, mostly fi-om cancer patients, preferentially rec ognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly us eful biochemical marker to detect low levels of p53 protein in differe nt tissues, and to be a key tool to characterize the phosphorylation s tatus of the C-terminus of p53 protein originated from various sources . 0167-4889/98/$ - see front matter (C) 1998 Elsevier Science B.V. All rights reserved.