Antigenicity and conformational analysis of the Zn(2+)-binding sites of two Zn(2+)-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11.
The antigenic properties of the Zn(2+)-binding region of two Zn(2+)-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 Zn(2+)-binding sites of the cognate protein. The affinity-purified antibodies, tested on synthetic peptides modelled from the active sites of ten different Zn(2+)-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 Zn(2+)-binding motifs and/or environments.
Soteriadou
K P
KP
Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Tzinia
A K
AK
Panou-Pamonis
E
E
Tsikaris
V
V
Sakarellos-Daitsiotis
M
M
Sakarellos
C
C
Papapoulou
Y
Y
Matsas
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007891:Leishmania / Q000201:enzymology / Q000276:immunology / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D015032:Zinc / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D008666:Metalloendopeptidases; C055146:glycoprotein gp63, Leishmania; D015260:Neprilysin; D015032:Zinc