Antigenicity and conformational analysis of the Zn2+-binding sites of two Zn2+-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11

The antigenic properties of the Zn2+-binding region of two Zn2+-metalloproteases, Leishmania surface protease gp63 and mammalian endopeptidase-24.11 (E-24.11), possessing in their active site the characteristic amino acid sequence HEXXH, were investigated by using oligoclonal antibodies raised against two synthetic peptides, V1VTHEMAHALG11 (pepgp63) and V1IGHEITHGFD11 (pepE-24.11), containing the respective Zn2+-binding sites of the cognate protein. The affinity-purified antibodies, tested on synthetic peptides modelled from the active sites of ten different Zn2+-metalloproteases, showed high selectivity for their respective peptides. However, cross-reactivity was revealed when the antibodies were tested against the gp63 and E-24.11 molecules. A panel of synthetic peptide analogues and peptides of various size was synthesized and used for the fine antigenic characterization of pepgp63 and pepE-24.11. The shortest peptides capable of significant antibody binding were the pentapeptides V1VTHE5 and E5ITHG9 for pepgp63 and pepE-24.11 respectively. His4 and Glu5 were found to be indispensable for anti-pepgp63 binding to pepgp63, whereas in the case of pepE-24.11, Glu5 and His8 were found to be critical. The conformational characteristics of the two peptides correlate well with the observed differences in their antigenicity. 1H-NMR studies showed that pepgp63 adopts a folded structure whereas pepE-24.11 takes up a rather flexible conformation. Moreover, the antigenically critical His4 of pepgp63 contributes to the structural stabilization of the peptide. Similarly, the antigenically critical His8 of pepE-24.11 is involved in partial structural stabilization of its C-terminal region. The generated antibodies may be useful tools for identifying and classifying proteins possessing similar Zn2+-binding motifs and/or environments.