Characterisation of the binding sites of monoclonal antibodies reacting with the Plasmodium falciparum rhoptry protein RhopH3.
Twenty one mouse monoclonal antibodies reacting or cross-reacting with the Plasmodium falciparum RhopH3 protein reacted with Ag44, a recombinant antigen expressing the 134 C-terminal RhopH3 residues. Using overlapping peptides scanning this region, two major binding sites were identified. The first one, recognised by eight anti-RhopH3 and seven cross-reacting mAbs, was mapped to the sequence Thr Asp Asn Thr Tyr or Thr Asp Asn Thr Tyr Lys (aa 823-828), depending on the support used for synthesis. Binding specificity and affinity were investigated for a subset of four mAbs reacting with this epitope, including one growth inhibitory mAb. Systematic replacements showed that the various mAbs had similar requirements. The inhibitory mAb presented a higher affinity for this sequence and bound to the adjacent sequence, Tyr Lys Glu Met Glu Leu (aa 827-832). A 2nd binding site, located around residue 850, was recognised by two anti-RhopH3 mAbs, which reacted exclusively with the 110 kDa RhopH3 polypeptide, unlike the other mAbs, which reacted with the 110 and 105 kDa RhopH3 antigens. This suggested that the 105 kDa RhopH3 polypeptide derives from the 110 kDa by C-terminal processing. Experimental evidence substantiating this conclusion was provided by the observation that antisera raised to peptides located upstream of the putative cleavage site reacted with both the 110 kDa and 105 kDa polypeptides, whereas antisera raised to the 45 C-terminal amino acids of RhopH3 reacted exclusively with the larger, 110 kDa product. The biological significance of this processing is discussed.
Doury
J C
JC
Institut de Médecine Tropicale, Service de Santé des Armées, Marseille Armées, France.
Goasdoue
J L
JL
Tolou
H
H
Martelloni
M
M
Bonnefoy
S
S
Mercereau-Puijalon
O
O
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000913:Antibodies, Protozoan / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D001666:Binding Sites, Antibody; D018604:Epitope Mapping; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D010963:Plasmodium falciparum / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D015800:Protozoan Proteins / Q000276:immunology* / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D010446:Peptide Fragments; D015800:Protozoan Proteins; C065352:rhoptry associated protein, Plasmodium