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 the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k 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 sensitive 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 Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.
Otvos
L
L
The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. otvos@wista.wistar.upenn.edu
Hoffmann
R
R
Xiang
Z Q
ZQ
O
I
I
Deng
H
H
Wysocka
M
M
Pease
A M
AM
Rogers
M E
ME
Blaszczyk-Thurin
M
M
Ertl
H C
HC
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000276:immunology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D010766:Phosphorylation; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D014357:Trypsin; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D016159:Tumor Suppressor Protein p53; D012694:Serine; D014357:Trypsin