ALPHA(2)-MACROGLOBULIN BAIT REGION VARIANTS - A ROLE FOR THE BAIT REGION IN TETRAMER FORMATION

To test the hypothesis that a large portion of the bait region of human alpha(2)-macroglobulin (alpha(2)M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human alpha(2)M variants with truncated bait regions and examined whether these variants folded normally and functioned as proteinase inhibitors, Each variant contains sites that are normal bait region cleavage sites in wild-type alpha(2)M, including the primary trypsin cleavage site, The truncated bait regions are shorter by 23 and 27 residues, respectively, and lack the C-terminal portion as well as different parts of the N-terminal section of the bait region, We found that such bait region truncation permitted normal folding of the monomers as well as formation of the thiol ester and dimerization by disulfide cross-linking, although the resulting species bound 6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more like thiol ester-cleaved alpha(2)M than native alpha(2)M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type alpha(2)M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric alpha(2)M, decoupled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to ''trap'' proteinase, This was despite apparent cleavage of the bait region by proteinase, albeit at very much reduced rates relative to wild-type tetrameric alpha(2)M. The kinetics of thiol ester cleavage-dependent protein conformational changes also changed from sigmoidal to exponential, These findings indicate that residues in the bait region appear to be necessary for noncovalent association of 360-kDa disulfide-linked dimers to give tetrameric alpha(2)M and suggest a role for the bait region in normal alpha(2)M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping.