Journal of Biological Chemistry vol:263 issue:6 pages:2981-9
To study the three-dimensional organization of alpha 2-macroglobulin (alpha 2M) from human plasma, immunoelectron microscopy of negatively stained specimens was used. A panel of monoclonal antibodies (mAb) with specificities typical for the two major conformers of alpha 2M (native and protease-transformed) was explored. The mAb have been selected and were classified biochemically as specific for either native or transformed alpha 2M or as reactive with both conformers. Furthermore, among the mAb that were specific for the proteinase-transformed form of alpha 2M, those reacting with the 20-kDa receptor-binding domain were considered a fourth category. Immunoelectron microscopy with these 20-kDa receptor-binding domain-specific mAb yielded the most typical result: predominantly, individual H-like alpha 2M-chymotrypsin molecules were complexed with two IgG molecules, each one bound to the extremities of two arms of the H-like figure. The resulting planar complex has the appearance of a dumbbell. Since this was observed with eight different mAb of this specificity, the result is interpreted to mean that the 20-kDa receptor-binding domain is compact and constitutes the outermost domain at the extremes of the arms of the H-like transformed alpha 2M. The mAb which are specific for the transformed state of alpha 2M but which do not react with the 20-kDa receptor-binding domain, also bound at the arms of the H-like figure, but at nonterminal positions. Moreover, these mAb produced mostly linear, chain-like immune complexes of numerous H-like alpha 2M molecules cross-linked by the IgG. The large category of mAb that reacted with both conformers of alpha 2M (native and proteinase complex) were observed to make various types of immune complexes with intra- and intermolecular cross-linking by the IgG. The observations of reaction of these mAb with Cd2+-induced dimers (half-molecules of alpha 2M), either native or transformed, proved helpful and, for certain mAb, essential to understand the organization of the alpha 2M-IgG complexes. Combined, the observations allow us to propose new models for the three-dimensional organization of native and chymotrypsin-transformed dimeric and tetrameric human alpha 2M.