Dissecting the Interaction of the human papillomavirus oncoprotein E7 with a specific monoclonal antibody

FASSOLARI, M; CERUTTI, ML; SMAL, C; DE PRAT GAY, G

Los Cocos, Córdoba, Argentina

Congreso; XXXVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2009

Sociedad Argentina de Biofísica

  <!-- /* Font Definitions */ @font-face {font-family:"Times New Roman"; panose-1:0 2 2 6 3 5 4 5 2 3; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:Arial; panose-1:0 2 11 6 4 2 2 2 2 2; mso-font-charset:0; mso-generic-font-family:auto; mso-font-pitch:variable; mso-font-signature:50331648 0 0 0 1 0;} @font-face {font-family:Times-Roman; panose-1:0 0 0 0 0 0 0 0 0 0; mso-font-alt:Times; mso-font-charset:77; mso-generic-font-family:swiss; mso-font-format:other; mso-font-pitch:auto; mso-font-signature:50331648 0 0 0 1 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-ansi-language:EN-US;} em {font-weight:bold; font-style:normal;} table.MsoNormalTable {mso-style-parent:""; font-size:10.0pt; font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Persistent infections by high-risk human papillomaviruses (HPV) are the main etiologic factors for cervical cancer, the leading cause of cancer death among women in low-income countries.  Up to 90% of cervical carcinomas have HPV-16 and -18 DNA sequences integrated into chromosomal DNA.  Papillomaviruses are comprised of double stranded DNA genomes encoding for 8-10 proteins. It is well documented that cellular immortalization is mediated by the viral E6 and E7 oncoproteins, having E7 the major transforming activity.  The E7 protein is frequently found in cervical carcinomas and HPV positives cell lines.  HPV16 E7 is a 98-amino acid acidic protein.  Its conserved N-terminal region is intrinsically disordered and its C-terminal region is globular and contains two CXXC zinc-binding motifs responsible for the zinc-dependent dimerization.    The purpose of our study is to biochemically and biophysically characterize the interaction of HPV16 E7 with the specific M1 monoclonal antibody developed in our laboratory.  We determine the stoichiometry of the antibody:antigen interaction being one molecule of Fab per E7 monomer.  Solution binding experiments demonstrate that M1 has high affinity for E7 (KD XX) and that the interaction has a weak electrostatic component.  ELISA experiments demonstrate that M1 is highly specific for the E7 strain 16 antigen, displaying low cross-reactivity against the homologous HPV18 E7.  Epitope mapping analysis reveals that M1 recognizes an epitope comprising amino acids 36-48, which is located within a region of low homology between the two viral proteins.  The equilibrium dissociation constant of M1 towards this peptide is 5-fold higher than the calculated for E7 full length, suggesting the occurrence of a conformational rearrangement upon the interaction. The specificity and the high affinity we show in the present work suggest that the M1 mAb may be a useful tool to detect papillomavirus related malignancies and identify different high-risk groups.