Mapping of structure-function relationships in proteins with a panel of monoclonal antibodies: A study on human α2 macroglobulin

Abstract

Monoclonal antibody (Mab) F2B2, directed to the receptor-recognition site of human α 2 macroglobulin ( α 2M), has been instrumental in the characterization of that site and in the isolation of the receptor-binding domain. We have now prepared a panel of Mab to study the structure-function relationships in α 2M, and in particular the expression of the receptor-recognition site. Reversed dot-blotting was very effective to screen hybridoma supernatant for specificities to either the native or complex form of α 2M. Reaction with the isolated 20 kDa receptor-binding domain of α 2M and cross-reaction with pregnancy zone protein was detected by the same technique. Eventually, a panel of 45 Mab was constructed consisting of essentially five types of specificities, although in fact no two Mab reacted with complete identity in all assays. In addition to the assays already mentioned, the Mab were tested for interference with binding of α 2M-trypsin to the cellular receptor, for competition with F2B2 for α 2M-trypsin and for inhibition of trypsin by α 2M. Finally, Western blotting was used as a first approximate mapping of the epitope relative to the internal thiolesters by exploiting the heat-induced fragmentation of α 2M at this site. The five categories of Mab thus detected were: (i) five Mab that react with native α 2M and not with α 2M trypsin; (ii) 18 Mab that react with both native α 2M and with α 2M-trypsin; (iii) 12 Mab, including F2B2 and F12A3, that react with the receptor-binding domain, neo-antigenically expressed on α 2M-trypsin, (iv) six Mab that are also specific for α 2M-trypsin but map outside the receptor-binding domain; (v) three Mab that define hidden determinants, not expressed on undenatured α 2M. For completeness, the panel includes the Mab obtained against pregnancy zone protein.