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

FASSOLARI MARISOL ; MARÍA LAURA CERUTTI; CLARA SMAL; GONZALO DE PRAT GAY.

Los Cocos, Proviencia de Córdoba

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

Sociedad Argentina de Biofísica

Persistent infections by high-risk human papillomaviruses (HPV) are the main etiologic factors for cervical cancer. Tha major cell transforming activity of HPVs is the E7 oncoprotein. HPV16 E7 is a 98-amino acid acidic protein. It is an extended dimer with a conserved N-terminal intrinsically disordered domain and a globular C-terminal dimerization domain with two CXXC zinc-binding motifs. Besides its relevance as a diagnostic marker, E7 is an interesting model antigen to investigate the recognition mechanism of intrinsically disordered antigens by antibodies. We here present a characterization of the interaction of HPV16 E7 with the specific M1 monoclonal antibody developed in our laboratory. We determine a 1:1 Fab M1:E7 stoichiometry of interaction and spectroscopic solution binding experiments indicates high binding affinity (KD 20 nM). Despite E7 has a lower pI, the M1-E7 interaction resulted to have a weak electrostatic component. Preliminary kinetic experiments suggest a slow association rate compared to other protein-protein interactions (kon ~ 104 s-1 M-1). ELISA experiments show that M1 is highly specific for the HPV16 E7 antigen, displaying low cross-reactivity against the homologous HPV18 E7. Fragmentation analysis of E7 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 for this peptide is 6-fold higher than that measured for full length E7, suggesting that the eptitope is linear only in part. The epitope is located within the N-terminal intrinsically disordered domain, but close to the boundary with the C-terminal domain. This strongly suggests that this region is in a dynamic conformation that requires the C-terminal globular domain full high affinity recognition of the epitope. This ?intermediate? behavior between linear and conformational could be a characteristic of intrinsically disordered antigens or domains, of which no models are available in the literature. Finally, the E7 protein is frequently found in cervical carcinomas and HPV positives cell lines therefore, the specificity and the high affinity of the M1 MAb may be a useful diagnostic tool.