Oxidative dissociation of human alpha 2-macroglobulin tetramers into dysfunctional dimers.

V.Y Reddy,P.E Desorchers,S.V Pizzo,S.L Gonias,J.A Sahakian,R.L Levine,S.J Weiss

doi:10.1016/s0021-9258(17)41830-8

V.Y Reddy, P.E Desorchers + Show 5 more

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https://doi.org/10.1016/s0021-9258(17)41830-8

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Abstract

Human alpha 2-macroglobulin is a broad-spectrum, homotetrameric antiproteinase that can maximally bind up to two proteinase molecules in a ternary complex. Proteinases cleave the inhibitor within a peptide stretch termed the bait region and induce the emergence of internal thiol esters whose nucleophilic scission precede a major conformational change which entraps enzymes within molecular cages. In a previous study, leukocyte-generated hypohalous acids and N-haloamines were identified as the first examples of physiologically relevant inactivators of the antiproteolytic activity of alpha 2-macroglobulin (Reddy, V. Y., Pizzo, S. V., and Weiss, S. J. (1989) J. Biol. Chem. 264, 13801-13809), but the mechanisms whereby the oxidants damaged the inhibitor remained undefined. We now demonstrate that N-chloramines (RNCl) destroy the antiproteolytic activity of alpha 2-macroglobulin in an unusual biphasic process that results in the formation of inactive alpha 2-macroglobulin half-molecules. In the first phase, 8 eq of RNCl reacted with each alpha 2-macroglobulin subunit to generate a partially oxidized antiproteinase containing 8 methionyl sulfoxide residues/monomer. Structure-function analyses demonstrated that the oxidized inhibitor retained its homotetrameric structure as well as its ability to entrap proteinases. In marked contrast, the oxidation of an additional 6 methionyl residues and a single tryptophanyl residue fractured the alpha 2 M homotetramer across its non-covalent axis into two pairs of disulfide-linked dimers. Despite the fact that the oxidized dimers displayed normal bait regions whose cleavage by proteinases initiated thiol ester scission, all antiproteolytic activity was lost. Furthermore, the oxidized dimers were unable to undergo the critical conformational changes normally associated with bait region cleavage or thiol ester scission. Together, these results demonstrate that chlorinated oxidants destroy the antiproteolytic activity of alpha 2-macroglobulin by attacking a subset of methionyl and tryptophanyl residues whose oxidation mediates the dissociation of the native homotetramer into conformationally locked dimers.

Highlights

Human az-macroglobulinis a broad-spectrum, homo- The chlorinated oxidants and proteolytic enzymes released tetrameric antiproteinase that can maximally bindup to from triggered human neutrophils can compromise the functwo proteinase molecules in a ternary complex
We demonstrate that N-chlora- optimal conditions, bind a maximum of two proteinase molmines (RNCl)destroy the antiproteolytic activity of az- ecules [9, 10]. the organization of the a2M subunits macroglobulin inan unusual biphasic process that which allows for the formation of proteinase-binding sites reresults in the formation of inactive az-macroglobulin mains controversial
Together,these results demonstrate that chlorinated oxidants destroy the antiproteolytic activity of az-macroglobulinby attacking a subset of methionyl and tryptophanyl residues whose oxidation mediates the dissociation of the native homotetramer into tein) inhibits proteinasefrsom all four catalytic classesby trapping the targeted enzyme in a molecular cage from which its dissociation is prevented

Summary

RESULTS

Similar results were obtained when azM was inactivated by stimulated neutrophils (Fig. 5B).When the oxievents could upset theantiproteolytic potential of azM dimers, bait region hydrolysis, thiol ester scission, and proteinase endized azM sampleswere reduced and carboxymethylated prior to spectral analysis in order to minimize contributions from protein secondary structure, an identical loss of a single tryptophanyl residuelsubunit was detected (data not shown). IV, erated fragments again migrated in an identical fashion (data not shown) Because these results were consistent with the possibility that the primarycleavage site had occurred in an anomalous domain, samplesof native or oxidized azM that had been incubated with either porcine pancreatic elastase, human neutrophil elastase, or human fibroblastcollagenase were submitted for NHz-terminal sequence analysis.

Percent azM Inaclivated B

PPE PPE