Conformational state and receptor recognition of the C-terminal domain of human alpha(2)-macroglobulin after dissociation into half-molecules.
BACKGROUND
Dissociation of native human alpha(2)-macroglobulin (alpha(2)M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated alpha(2)-macroglobulin.


METHODS
The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of alpha(2)-macroglobulin.


RESULTS
The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed alpha(2)-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of alpha(2)-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric alpha(2)-macroglobulin in competition studies.


CONCLUSIONS
It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.
Jensen
P E
PE
The Neuroimmunology Laboratory, The Center for Neuroscience, Copenhagen University Hospital, Section 9202, Rigshospitalet, Juliane Mariesvej 24, 2100 Copenhagen, Denmark. pejensen@pet.rh.dk
Gunnarsson
M
M
Stigbrand
T
T
D000818:Animals; D000911:Antibodies, Monoclonal; D006801:Humans; D066298:In Vitro Techniques; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008657:Metabolic Clearance Rate; D008744:Methylamines; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011971:Receptors, Immunologic / Q000378:metabolism; D013861:Thiocyanates; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008744:Methylamines; D011971:Receptors, Immunologic; D013861:Thiocyanates; D000511:alpha-Macroglobulins; C024553:sodium thiocyanate; C027451:methylamine