Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of .alpha.2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

Roche, Paul A.; Moncino, Mark D.; Pizzo, Salvatore V.

doi:10.1021/bi00445a019

Cited by 12 publications

(4 citation statements)

References 47 publications

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“…agreement with this hypothesis is the finding that the human dimeric a-macroglobulin homologuc pregnancy zone protein associates to form tetramers when reacted with proteinase but not with methylamine (Christensen et al, 1989). These studies are consistent with our previous studies of the independent and separable nature of the bait region cleavage dependent and thiolester bond cleavage dependent conformational change in a2M (Roche et al, 1989).…”

Section: Discussionsupporting

confidence: 91%

“…Proteins. Native a2M (Roche et al, 1989), rat apinhibitor 3 (o113) (Enghild et al, 1989), and arproteinase inhibitor («|PI) (Panned et al, 1974) were purified as previously described. Porcine pancreatic elastase (type IV) (PPE) and bovine trypsin (type VIII) were obtained from Sigma Chemical Co., St. Louis, MO. Human neutrophil elastase (HNE) was a kind gift of Dr. Weislaw Watorek, University of Georgia, Athens, GA.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of human .alpha.2-macroglobulin monomers obtained by reduction with dithiothreitol

Moncino

Roche²,

Pizzo³

1991

Biochemistry

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We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.

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Section: Discussionsupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Characterization of human .alpha.2-macroglobulin monomers obtained by reduction with dithiothreitol

Moncino

Roche²,

Pizzo³

1991

Biochemistry

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“…Purification of a2-Macroglobulin. Human a2M was purified from recently expired plasma (American Red Cross, Nashville Region) by zinc-chelate chromatography, as described previously (Roche et al, 1989). The purity of each preparation was checked by electrophoresis under nondenaturing conditions and also in the presence of SDS.…”

Section: Methodsmentioning

confidence: 99%

Separation and localization of the four cysteine-949 residues in human .alpha.2-macroglobulin using fluorescence energy transfer

Gettins

Beechem²,

Crews³

et al. 1990

Biochemistry

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To localize the pair of thiol-ester-derived cysteine-949 residues within a half-molecule of a,M and estimate the internal diameter of this a2-macroglobulin trap, we have measured the separation between these cysteines. We unexpectedly found that the two cysteines of intermediate form a,M had different reactivity, which permitted selective modification of one cysteine with dansyl, and the other with fluorescein fluorophores. From fluorescence energy transfer measurements, we calculated a separation of 41 + 10 8, between these fluorophores. This indicates that the Cys-949 residues are probably located at the perimeter of the trap with an internal diameter at least as large as this separation. a,-Macroglobulin; Fluorescence resonance energy transfer; Thiol ester: Structure

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“…Introduction of cross-links across this interface, either by disulfide formation, as described here, or by chemical cross-linking, as suggested elsewhere (25,26), slows or prevents this reorientation with consequent reduction in rate of conformational change and in efficiency of proteinase trapping.…”

Section: Ability Of Variants To Inhibit Trypsin By Trapping-mentioning

confidence: 99%

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Bowen¹,

Gettins

1998

Journal of Biological Chemistry

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We have characterized four human ␣ 2 -macroglobulin (␣ 2 M) bait region variants (G679C, M690C, V700C, and T705C) to test the hypothesis that the bait regions are involved in the interface between noncovalently associated dimers. All four variants folded correctly as judged by many normal properties. However, the presence of a cysteine resulted in disulfide formation between otherwise noncovalently associated dimers in all four variants. The extent of disulfide cross-linking varied with the location of the cysteine and gave a mixture of species that probably contained two, one, or zero interdimer disulfides in the tetramer. This was reflected in heterogeneity of conformational change upon thiol ester cleavage by methylamine, with the presence of crosslinks correlating with blockage of conformational change. The stoichiometry of trypsin inhibition was less in all cases than for wild-type ␣ 2 M. The M690C variant also showed evidence of some species with an intramolecular disulfide between bait regions of monomers within the same dimer. Taken together, the results are consistent with a location of the four bait regions in contact with, or in very close proximity to, one another. This suggests that they form all or part of the "cavity body" seen in the low resolution x-ray structure of transformed ␣ 2 M. Inhibition of proteinases by humanresults from a series of conformational changes that are initiated by the proteinase and that result in the physical sequestration of the proteinase within the closed cage-like structure of the conformationally altered ␣ 2 M (1, 2). The initiating event is a proteolytic cleavage by the attacking proteinase near the center of the ␣ 2 M polypeptide in a region termed the bait region (3, 4). Cleavage anywhere within the bait region also results in activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the ␣ 2 M, it has been shown that the conformational change occurs cooperatively after both thiol esters within one half of the ␣ 2 M tetramer have been cleaved (5, 6).Knowledge of the structural relationship between the thiol ester and the bait region and hence of the details of the activation mechanism is limited by the absence of a high resolution x-ray structure of either native or conformationally altered human ␣ 2 M. From fluorescence resonance energy transfer measurements, it was shown that the four cysteines that form the four thiol esters of the human ␣ 2 M tetramer are centrally located and are about 35 Å apart (7). This was subsequently confirmed by a low resolution (ϳ10 Å) x-ray structure of conformationally altered ␣ 2 M (8). NMR measurements on ␣ 2 M showed that the bait region of each monomer was unusually flexible (9, 10) and that it lies close (10 -17 Å) to the cysteine of the thiol ester in the transformed protein (11). However, the location of the four bait regions is still not known. Studies from this laboratory on ␣ 2 M varian...

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Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of .alpha.2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

Cited by 12 publications

References 47 publications

Characterization of human .alpha.2-macroglobulin monomers obtained by reduction with dithiothreitol

Characterization of human .alpha.2-macroglobulin monomers obtained by reduction with dithiothreitol

Separation and localization of the four cysteine-949 residues in human .alpha.2-macroglobulin using fluorescence energy transfer

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

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