Involvement of the C‐terminal end of the prostrate‐specific antigen in a conformational epitope: characterization by proteolytic degradation of monoclonal antibody‐bound antigen and mass spectrometry

Abstract

Prostate-specific antigen (PSA), a 237-amino acid glycoprotein, encoded by the hKLK3 gene, is widely used as a serum marker for the diagnosis and management of prostate cancer. We report here the localization of a conformational epitope recognized by the anti-total PSA monoclonal antibody (mAb) 11E5C6, by proteolytic degradation of mAb-bound antigen followed by mass spectrometric analyses of the peptides generated. These two technologies, combined with molecular display, allowed the identification of amino acid residues contained within three different peptides distant on the PSA sequence, but close in the PSA three-dimensional structure, that may be part of the mAb 11E5C6 epitope. The last four C-terminal amino acid residues are included in this epitope, as well as certain other C-terminal residues between Y225 and T232. The involvement of the PSA C-terminal end in the mAb 11E5C6 epitope was confirmed by western blotting experiments with the recombinant protein proPSA-RP1, resulting from the cloning of an alternative transcript of the hKLK3 gene, in which the PSA C-terminal end was deleted and replaced by another sequence. Although the anti-total PSA mAb 5D5A5 used as a control bound proPSA-RP1, mAb 11E5C6 did not. The requirement of the C-terminal end for the recognition by mAb 11E5C6 may be useful for the discrimination of PSA-related forms.

Characterization of prostate‐specific antigen binding peptides selected by phage display technology

“…Anti-total PSA mAbs 11E5C6, 8G8F5, 5D5A5 and 16H9A12 were produced by bioMérieux (Marcy l'Etoile, France) and were described previously (Michel et al, , 2001a(Michel et al, , 2001b(Michel et al, , 2005 TM is a combinatorial library of random peptide 12-mers already described (JolivetReynaud et al, 2004). Ph.D.-C7C TM is a combinatorial peptide 7-mers flanked by a pair of cysteine residues and also fused to the minor coat protein (pIII) of M13 phage.…”