The epitopes of two complex-specific monoclonal antibodies, related to the receptor recognition site, map to the COOH-terminal end of human alpha 2-macroglobulin.
The elucidation of the molecular structure of the receptor recognition site of human alpha 2-macroglobulin (alpha 2M) was approached by mapping the epitopes of two monoclonal antibodies (F2B2 and F12A3). These antibodies were shown to be complex-specific, defining neo-antigenic sites not detectable on native alpha 2M and thereby mimicking the specificity of the receptor expressed on macrophages and fibroblasts. The antibodies inhibited binding of alpha 2M-trypsin complexes to the receptor. The epitopes of both monoclonal antibodies are shown here to be located on the Mr 60,000 heat-induced fragment of partially reduced alpha 2M. Limited proteolysis of alpha 2M-methylamine with lysine-specific bacterial endoproteinase was examined by rate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to correlate the loss of the epitopes with the generation of defined fragments. 14C-Labeled alpha 2M-methylamine was used as an internal marker for the position of the thioesters. Finally, the epitopes were protected toward proteolysis by subjecting immune complexes of alpha 2M-methylamine with the monoclonal antibodies to proteolysis under the same conditions as uncomplexed alpha 2M-methylamine. The results obtained allowed us to map the epitopes of both the monoclonal antibodies to within a distance of about Mr 20,000 from the COOH-terminal end of human alpha 2M.
F
F
Van Leuven
P
P
Marynen
J J
JJ
Cassiman
H
H
Van den Berghe
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes / Q000032:analysis*; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D000511:alpha-Macroglobulins / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D010446:Peptide Fragments; D000511:alpha-Macroglobulins