THIOL ESTER ROLE IN CORRECT FOLDING AND CONFORMATION OF HUMAN ALPHA(2)-MACROGLOBULIN - PROPERTIES OF RECOMBINANT C949S VARIANT

To determine the role of the thiol ester in the folding of human alpha(2)-macroglobulin (alpha(2)M) in the active conformation, we have characterized a recombinant variant of alpha(2)M, C949S, expressed in baby hamster kidney cells, that lacks the thiol ester-forming cysteine. C949S cr,M behaves like methylamine-treated plasma alpha(2)M, with correctly formed inter-subunit disulfide bridges, non-covalent association of covalent dimers to form tetramers, and exposure of the receptor binding domain, but an inability to inhibit proteinases, and inaccessibility of the bait regions to proteolysis. We concluded that correct folding of monomers or their association to give tetrameric alpha(2)M does not require a pre-formed thiol ester. Active alpha(2)M may form in vivo by a two-step process involving initial folding to give a structure resembling that of C949S alpha(2)M followed by thiol ester formation and a conformational change that gives the native active state.