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 huma n alpha(2)-macroglobulin (alpha(2)M) can be removed without adversely affecting the protein's structural and functional properties, we expre ssed two human alpha(2)M variants with truncated bait regions and exam ined whether these variants folded normally and functioned as proteina se 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 res idues, respectively, and lack the C-terminal portion as well as differ ent parts of the N-terminal section of the bait region, We found that such bait region truncation permitted normal folding of the monomers a s well as formation of the thiol ester and dimerization by disulfide c ross-linking, although the resulting species bound 6-(p-toluidino)-2-n aphthalenesulfonic acid in a manner more like thiol ester-cleaved alph a(2)M than native alpha(2)M. The variants' thiol esters reacted with n ucleophiles at rates identical to wild-type alpha(2)M. Surprisingly, h owever, the truncations prevented the noncovalent association of the c ovalent 360-kDa dimers that normally gives tetrameric alpha(2)M, decou pled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to ''trap'' proteinase, This was de spite apparent cleavage of the bait region by proteinase, albeit at ve ry much reduced rates relative to wild-type tetrameric alpha(2)M. The kinetics of thiol ester cleavage-dependent protein conformational chan ges also changed from sigmoidal to exponential, These findings indicat e that residues in the bait region appear to be necessary for noncoval ent 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 i n coupling bait region cleavage to the sequence of conformational chan ges that result in thiol ester activation and ultimately proteinase tr apping.