Bait Region Cleavage Research Articles

The conformational change of the protease inhibitor α2-macroglobulin is triggered by the retraction of the cleaved bait region from a central channel

The protease inhibitor α2-macroglobulin (A2M) is a member of the ancient α2-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel’s subsequent collapse triggers the conformational change of A2M and other A2MF proteins.

Cryo-EM structures of human A2ML1 elucidate the protease-inhibitory mechanism of the A2M family

A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1’s thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation.

Structural Investigations of Human A2M Identify a Hollow Native Conformation That Underlies Its Distinctive Protease-Trapping Mechanism

Human α2-macroglobulin (A2M) is the most characterized protease inhibitor in the alpha-macroglobulin (αM) superfamily, but the structure of its native conformation has not been determined. Here, we combined negative stain electron microscopy (EM), small-angle X-ray scattering (SAXS), and cross-linking–mass spectrometry (XL-MS) to investigate native A2M and its collapsed conformations that are obtained through aminolysis of its thiol ester by methylamine or cleavage of its bait region by trypsin. The combined interpretation of these data resulted in a model of the native A2M tetramer and its conformational changes. Native A2M consists of two crescent-shaped disulfide-bridged subunit dimers, which face toward each other and surround a central hollow space. In native A2M, interactions across the disulfide-bridged dimers are minimal, with a single major interface between the linker (LNK) regions of oppositely positioned subunits. Bait region cleavage induces both intrasubunit domain repositioning and an altered configuration of the disulfide-bridged dimer. These changes collapse the tetramer into a more compact conformation, which encloses an interior protease-trapping cavity. A recombinant A2M with a modified bait region was used to map the bait region’s position in native A2M by XL-MS. A second recombinant A2M introduced an intersubunit disulfide into the LNK region, demonstrating the predicted interactions between these regions in native A2M. Altogether, our native A2M model provides a structural foundation for understanding A2M’s protease-trapping mechanism, its conformation-dependent receptor interactions, and the dissociation of native A2M into dimers due to inflammatory oxidative stress.

Probing the stability of native and activated forms of α 2-macroglobulin

α 2-Macroglobulin (α 2M) is a 718 kDa homotetrameric proteinase inhibitor which undergoes a large conformational change upon activation. This conformational change can occur either by proteolytic attack on an ∼40 amino acid stretch, the bait region, which results in the rupture of the four thioester bonds in α 2M, or by direct nucleophilic attack on these thioesters by primary amines. Amine activation circumvents both bait region cleavage and protein incorporation, which occurs by proteolytic activation. These different activation methods allow for examination of the roles bait region cleavage and thioester rupture play in α 2M stability. Differential scanning calorimetry and urea gel electrophoresis demonstrate that both bait region cleavage and covalent incorporation of protein ligands in the thioester pocket play critical roles in the stability of α 2M complexes.

New member of the trefoil factor family of proteins is an α-macroglobulin protease inhibitor

The amino acid sequence of the monomeric α-macroglobulin (αM) from the American bullfrog, Rana catesbiana, was determined. The mature protein consisted of 1469 amino acid residues and shared sequence identity with other members of the αM family of protein. The central portion of the frog monomeric αM contained Cys residues positioned analogously to the Cys residues in human α 2-macroglobulin (α 2M), known to be involved in disulfide bridges. Additionally, the frog monomeric αM contained six Cys residues in a ∼60 residue COOH-terminal extension not present in previously characterized αMs. The spacing of the Cys residues and the overall sequence identity of this COOH-terminal extension were consistent with a trefoil motif. This is the first time a member of the trefoil factor family has been identified in the circulatory system. The “bait region” was located between Arg 675–Lys 685 and contained mainly basic amino acid residues. The COOH-terminal receptor-binding domain was not exposed prior to proteolysis of this highly susceptible region. The proximity of the receptor-binding and trefoil domains implied that the trefoil domain is similarly concealed before bait region cleavage.

Bait region cleavage and complex formation of human alpha2M with a Porphyromonas gingivalis W50 protease is not accompanied by enzyme inhibition.

Three isoforms of extracellular Arg-specific proteases of P. gingivalis, W50, HRgpA, RgpAcat and mt-RgpAcat, which are all products of the same gene, show identical enzymatic properties toward small chromogenic substrates but have different subunit organisation and molecular size. In order to examine the potential inhibition of these proteases in vivo by host protease inhibitors, the interaction of HRgpA (approximately 110 kDa) and RgpAcat (approximately 55 kDa) with human (alpha2M and their cytotoxicity toward cultured fibroblasts were investigated. Both enzymes formed complexes with (alpha2M as shown by gel filtration chromatography and both cleaved the 'bait' region at Arg696-Leu697. However, whereas (alpha2M-RgpAcat) complex was unable to hydrolyse large substrates such as hide powder azure, (alpha2M-HRgpA) complex hydrolysed both small and large substrates. HRgpA was able to bind to alpha2M saturated with trypsin and also to methylamine-treated alpha2M. This suggested that HRgpA is able to bind to both 'slow' and 'fast' forms of alpha2M and formation of (alpha2M:HRgpA) complex does not trap HRgpA and cause inhibition of activity toward hide powder azure. However, the (alpha2M-HRgpA) complex is not able to cleave other alpha2M molecules, which suggests that the active site of HRgpA in the complex is constrained probably due to steric reasons. The (alpha2M-HRgpA) complex was cytotoxic to 3T3 cells, causing them to round up and detach from the surface with a reduction in metabolic activity.

Fluorescence monitoring of the conformational change in alpha 2-macroglobulin induced by trypsin under second-order conditions: the macroglobulin acts both as a substrate and a competitive inhibitor of the protease.

The reaction of bovine pancreatic trypsin with human plasma alpha(2)-macroglobulin (alpha(2)M) was studied at 25 degrees C, using equimolar mixtures of E and I in 50 mM potassium phosphate buffer, pH 7. The conformational change in alpha(2)M was monitored through the increase in protein fluorescence at 320 nm (exc lambda, 280 nm). At [alpha(2)M](0) =[E](0) =11.5-200 nM, the fluorescence change data fit the integrated second-order rate equation, (F(infinity) -F(0) )/(F(infinity) -F(t) )=1+k(i,obsd) [alpha(2)M](0) t, indicating that cleavage of the bait region in alpha(2)M was the rate-determining step. The apparent rate constant (k(i,obsd)) was found to be inversely related to reactant concentration. The kinetic behavior of the system was compatible with a model involving reversible, nonbait region binding of E to alpha(2)M, competitively limiting the concentration of E available for bait region cleavage. The intrinsic value of k(i) was (1.7+/-0.24) x 10(7) M(-1) s(-1).K(p), the inhibitory constant associated with peripheral binding, was estimated to be in the submicromolar range. The results of the present study point to a potential problem in interpreting kinetic data relating to protease-induced structural changes in macromolecular substrates. If there is nonproductive binding, as in the case of trypsin and alpha(2)M, and the reactions are monitored under pseudo first-order conditions ([S](0) >>[E](0) ), an intrinsically second-order process (such as the rate-limiting bait region cleavage in alpha(2)M) may become kinetically indistinguishable from an intrinsically first-order process (e.g. rate-limiting conformational change). Hence an excess of one component over the other should be avoided in kinetic studies addressing such systems.

Inhibition of a snake venom hemorrhagic metalloproteinase by human and rat α-macroglobulins

Jararafibrase I is a hemorrhagic metalloproteinase purified from Bothrops jararaca venom, which induces local hemorrhage by degrading the basement membrane components. The present study was undertaken to investigate the inhibition of jararafibrase I by human and rat serum proteinase inhibitors. The proteolytic activity of jararafibrase I was completely inhibited by human and rat sera. In particular, rat serum displayed a greater inhibitory capacity. The inhibitory capacities of both sera were dependent on α-macroglobulins. SDS-PAGE analysis revealed that jararafibrase I formed complexes with α-macroglobulins that were present in normal sera. The proteolytic activity of jararafibrase I was completely inhibited by α1-macroglobulin and murinoglobulin in rat serum, and by human α2-macroglobulin. The inhibition molar ratios of α-macroglobulin/jararafibrase I were 1.5 for rat α1-macroglobulin and human α2-macroglobulin, and 2.4 for rat murinoglobulin. SDS-PAGE under reducing conditions demonstrated that the bait region of human α2-macroglobulin and rat murinoglobulin was cleaved by jararafibrase I. The bait region cleavage sites were identified as being situated at the 696Arg– 697Leu peptide bond in human α2-macroglobulin, and at the 686Ala– 687Val peptide bond in rat murinoglobulin.

Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes.

Chagas' disease patients (CDP) develop both humoral and cellular immune responses against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Here we demonstrate that complexes formed by cruzipain and alpha 2-macroglobulin (alpha 2M) are efficiently internalized by human monocytes, and that this process results in enhanced presentation of cruzipain peptides to CD4+ T cells from CDP. Purified or serum alpha 2M binds to polymorphic cruzipains, but only a fraction of the proteinases become covalently linked. Once bound to alpha 2M, fluorescein-labeled cruzipain (FITC-cruzipain) or [125I]cruzipain were more efficiently internalized by normal peripheral blood mononuclear cells (PBMC) or monocytes; this effect was abolished by (I) pre-treating the cells with receptor-associated protein (rRAP), a known antagonist the of alpha 2M receptor (alpha 2MR/LRP), and (II) inactivating [125I]cruzipain's active site prior to the reaction with alpha 2M, indicating that the exposure of receptor binding sites on alpha 2M complexes required bait region cleavage. We then sought to determine if the alpha 2MR/LRP-dependent uptake of alpha 2M:cruzipain by monocytes resulted in increased CD4+ T cell responses of PBMC-CDP (n = 13). These effects were only revealed after depletion of CD19+ B lymphocytes from PBMC-CDP; the threshold of T cell stimulation was far lower in cultures stimulated with alpha 2M:cruzipain, as compared to antigen alone. Myocardial specimens from CDP with chronic myocardiopathy (three necropsies) were analyzed by immunohistochemistry with mAb anti-cruzipain or anti-alpha 2MR/LRP (CD81+). Extracellular depots of cruzipain were localized amidst inflammatory mononuclear infiltrates, part of which contained CD91+ macrophage-like cells. Ongoing studies should clarify if T. cruzi cysteinyl proteinases play a role in the pathogenesis of Chagas' heart disease.

α2-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 2M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human alpha 2M 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 2M, 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 2M than native alpha 2M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type alpha 2M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric alpha 2M, 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 2M. 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 2M and suggest a role for the bait region in normal alpha 2M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping.

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

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.

Probing different conformational states of pregnancy-zone protein. Fluorescence studies utilizing the binding of 4,4'-bis(8-anilino-1-naphthalenesulphonate).

The binding of the fluorescence probe 4,4'-bis(8-anilino-1-naphthalenesulphonate) (bis-ANS) to the human proteinase inhibitor pregnancy-zone protein (PZP) and its complexes with methylamine and chymotrypsin were investigated. The existence of dimeric PZP-chymotrypsin complex was demonstrated and both the dimeric and the tetrameric PZP-chymotrypsin complexes could be studied separately. The fluorescence data indicate that bis-ANS binds to two different sites on PZP and its complexes. The values of the dissociation constant, Kd1, for the binding to the high-affinity site were determined to be 231 +/- 14, 220 +/- 28, 114 +/- 15 and 49 +/- 1 nM, for the binding to native PZP, PZP-methylamine and dimeric and tetrameric PZP-chymotrypsin, respectively. An 11-30-fold decrease was observed in the affinity for the second site, the corresponding values of the dissociation constant, Kd2, being 1.5-2.8 +/- 1.0 microM, which are not significantly different for PZP and its derivatives. The results suggest that the probe bis-ANS discriminates between the different conformational states of PZP and that while the conformation of the complex with methylamine does not differ much from that of the native protein, there is a significant change in conformation when chymotrypsin cleaves the bait region. This is substantiated by a 30%-45% decrease in the maximum enhancement of fluorescence intensity when PZP is treated with chymotrypsin. Although the dimeric and tetrameric forms of PZP-chymotrypsin complexes differ in Kd1 values, the difference in the maximum enhancement of the fluorescence of bis-ANS by the two forms is not significant. This indicates that dimer-dimer interaction in the tetrameric form does not involve hydrophobic sites. The necessity of bait-region cleavage for extensive conformational changes in PZP distinguishes it from alpha 2-macroglobulin, the other alpha-macroglobulin in human plasma.

α 2-Macroglobulin bait region integrity : Role in determining fast-form structure

To determine whether integrity of the bait region affects the structure of the remainder of human α 2-macroglobulin (α 2M), we have determined the separation between cysteine residues in a methylamine-reacted fast-form of α 2 M. From reduction in fluorescence intensity of covalently-bound donor fluorophore caused by proximity to an acceptor, a separation of 35 ± 8 Å was calculated, which is identical to a previously determined value for proteinase-treated fast-form α 2M. This indicates that although bait region cleavage is the physiological route to conformational change in α 2M, bait region integrity per se does not significantly affect the structure of fast-form α 2M.

Direct kinetics of bait region cleavage of α-2-macroglobulin by a rapid quenching method

The rate of bait region cleavage of human α-2-macroglobulin by chymotrypsin was determined by a rapid quenching method under conditions where the bimolecular encounter between the two reactants was not rate-limiting. α 2M was first mixed with a 30 molar excess of chymotrypsin in a sequential stopped-flow apparatus and after programmed time intervals the activity of chymotrypsin was quenched with 1 N HCl. The fraction of uncleaved subunits was quantitated by SDS-PAGE under reducing conditions. The result indicated that the bait region cleavage proceeded following a two-exponential decay curve with respective rate constants of k 1 = 40 s −1 and k 2 = 2 s −1.

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

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.

Kinetics and mechanism of proteinase-binding of pregnancy zone protein (PZP). Appearance of sulfhydryl groups in reactions with proteinases.

Proteinase binding by pregnancy zone protein (PZP), an alpha-macroglobulin involves bait region cleavages, association of dimeric-PZP into tetrameric and reaction of internal gamma-glutamyl-beta-cysteinyl thiol esters of PZP with proteinase side chains. The product is an equimolar enzyme-PZP(tetramer) covalently linked complex with four free sulfhydryl groups. The kinetics of the appearances of sulfhydryl groups during the reaction of PZP with chymotrypsin has been investigated using stopped-flow and conventional mixing techniques over a broad concentration range. Thiol ester cleavages followed double exponential decays corresponding with two steps. The faster one resulted in the appearance of three sulfhydryl groups with an observed rate constant, k(obs) = k1.1 + k1.2 delta E, dependent on the excess concentration of chymotrypsin, delta E, and k1.1 = 0.03 s-1 and k1.2 = 4 x 10(4) M-1 s-1. The last sulfhydryl group appeared in a slower step, with similar concentration dependence and k2.1 approximately 0.003 s-1 and k2.2 approximately 5 x 10(3) M-1s-1. Covalent binding of the enzyme apparently was simultaneous with the faster thiol ester cleavage step. Based on these and previous results a model of the reaction mechanism of the proteinase binding reaction of PZP is proposed. It consists of four major steps: (i) Bait region cleavage of PZP-dimers by the enzyme, (ii) fast association of enzyme-PZP(dimer) species with native PZP or with another enzyme-PZP(dimer) compound resulting in release of one of the associated enzyme molecules (iii) reaction of an average of three thiol esters of the enzyme-PZP(tetramer) intermediate with the associated internal enzyme molecule or with an external one. In this step one enzyme molecule becomes covalently linked to the PZP-(tetramer), three sulfhydryl groups appear and the enzymic activity of the bound enzyme molecule decreases to the level of that of the final complex. (iv) Hydrolysis of the last thiol ester and in the presence of excess enzyme, degradation of enzyme-PZP(tetramer) complexes and formation of fragments some of which are the size of PZP(dimer) with enzyme bound.

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)

Treatment of the human plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) with proteinase results in conformational changes in the inhibitor and subsequent activation and cleavage of the internal thiolester bonds of alpha 2M. Previous studies from this laboratory have shown that cross-linking the alpha 2M subunits with cis-dichlorodiammineplatinum(II) (cis-DDP) prevents the proteinase-induced conformational changes which lead to the activation and cleavage of the internal thiolester bonds of alpha 2M. In addition, cis-DDP treatment prevents the proteinase- or CH3NH2-induced conformational changes in alpha 2M which lead to a "slow" to "fast" change in nondenaturing polyacrylamide gel electrophoresis. In this paper, we demonstrate that treatment of alpha 2M with dithiobis(succinimidyl propionate) (DSP) also results in cross-linking of the subunits of alpha 2M with concomitant loss of proteinase inhibitory activity. Although proteinase is not inhibited by DSP-treated alpha 2M, bait region specific proteolysis of the alpha 2M subunits still occurs. Unlike cis-DDP-treated alpha 2M, however, incubation of DSP-treated alpha 2M with proteinase does not prevent the bait region cleavage dependent conformational changes which lead to activation and cleavage of the internal thiolester bonds in alpha 2M. On the other hand, cross-linking of alpha 2M with DSP does prevent the conformational changes which trigger receptor recognition site exposure following cleavage of the alpha 2M thiolester bonds by CH3NH2. These conformational changes, however, occur following incubation of the CH3NH2-treated protein with proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)

The α-Macroglobulin Bait Region: Sequence diversity and localization of cleavage sites for proteinases in five mammalian α-macroglobulins

Abstract The amino acid sequence of a 90-residue segment of human pregnancy zone protein containing its bait region has been determined. Human alpha 2-macroglobulin, human pregnancy zone protein, and rat alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor 3 variants 1 and 2 constitute a group of homologous proteins; but the sequences of their bait regions are not related, and they differ in length (32-53 residues). The alpha-macroglobulin bait region is located equivalently with residues 666-706 in human alpha 2-macroglobulin. In view of the extreme sequence variation of the bait regions, the evolutionary constraints for these regions are likely to differ from those of the remainder of the alpha-macroglobulin structure. The sites of specific limited proteolysis in the bait regions of human pregnancy zone protein and rat alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor 3 variants 1 and 2 by a variety of proteinases differing in specificity have been determined and compared with those identified earlier in human alpha 2-macroglobulin. The sites of cleavage generally conform to the substrate specificity of the proteinase in question, but the positions and nature of the P4-P4' sites differ. Most cleavages occur in two relatively small segments spaced by 6-10 residues; and in each case, bait region cleavage leads to alpha-macroglobulin-proteinase complex formation. The rate at which a given proteinase cleaves alpha-macroglobulin bait regions is likely to show great variation. Possible structural features of the widely different bait regions and their role in the mechanism of activation are discussed.