Pentapeptide identified as a monoclonal antibody binding site in the serine-protease domain of t-PA.
The first defined sequential epitope of the tissue plasminogen activator (t-PA) was determined by a monoclonal antibody against a synthetic peptide segment corresponding to peptide sequence 341-354 of t-PA. This segment was selected by computer assisted epitope prediction. Balb/c mice were immunized with catalase-peptide and tripalmitoyl-S-glyceryl-cys-teinyl-seryl-peptide conjugates. A monoclonal antibody derived from this immunization was reactive with native recombinant t-PA (rt-PA) and reduced carboxymethylated recombinant t-PA (RCM rt-PA). The sequential epitope was detected by Pepscan method using overlapping octa- and nonapeptides. By fine epitope mapping with tetra-, penta-, hexa- and heptapeptides the epitope was minimized to the pentapeptide EEEQK (347-351). Replacement set analysis confirmed the importance of this amino acid sequence, especially of the amino acid E348, for antibody binding. Functional assays of rt-PA were not affected by this antibody indicating that the epitope has no influence on the enzymatic center and the binding site of the inhibitor. The analysis demonstrates that the predicted recognition site of the monoclonal antibody 17-134/11 is exposed on the surface of the native rt-PA molecule.
Gombert
F O
FO
Institut für Organische Chemie, Universität Tübingen.
Werz
W
W
Schlüter
M
M
Bayer
A
A
Werner
R G
RG
Berthold
W
W
Jung
G
G
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001666:Binding Sites, Antibody; D002352:Carrier Proteins; D000939:Epitopes / Q000276:immunology; D006241:Haptens; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology* / Q000378:metabolism; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D006241:Haptens; D009842:Oligopeptides; D012697:Serine Endopeptidases; D010959:Tissue Plasminogen Activator