Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of human plasmin with human alpha 2-macroglobulin

The steady-state kinetic parameters of plasmin and the alpha 2-macroglobulin (alpha 2M)-plasmin complex toward the chromogenic substrate Val-Leu-Lys-p-nitroanilide (S-2251), in the presence and absence of plasmin competitive inhibitors, have been determined. At pH 7.4 and 22 degrees C, the Km values for plasmin and alpha 2M-plasmin for S-2251 were 0.13 +/- 0.02 mM and 0.3 +/- 0.03 mM. The kcat of this reaction, when catalyzed by alpha 2M-plasmin, was 6.0 +/- 0.5 s-1, a value significantly decreased from the kcat of 11.0 +/- 1.0 s-1, determined when free plasmin was the enzyme. KI values for benzamidine of 0.50 +/- 0.05 mM and 0.23 +/- 0.02 mM were obtained for S-2251 hydrolysis, as catalyzed by alpha 2M-plasmin and plasmin, respectively. When leupeptin was the competitive inhibitor, KI values of 5.0 +/- 0.65 microM and 1.0 +/- 0.1 microM were obtained when alpha 2M-plasmin and plasmin, respectively, were the enzymes employed for catalysis of S-2251 hydrolysis. The comparative rates of reaction of the peptide inhibitor Trasylol (Kunitz basic pancreatic inhibitor) with plasmin and alpha 2M-plasmin were also determined. A concentration of Trasylol of at least 3 orders of magnitude greater for alpha 2M-plasmin than for free plasmin was required to observe inhibition rates on comparable time scales.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.     

Subunit structure of human alpha 2-macroglobulin (alpha 2-MG) with respect to its interaction with trypsin.

SDS-polyacrylamide gel electrophoresis of a recently prepared alpha 2-macroglobulin solution showed only the polypeptide chains of 190,000 molecular weight. Reduction-alkylation of this preparation followed by gel-filtration on a Sephadex G-200 column in 5.2 M guanidine hydrochloride was unable to separate a fraction of 83,000 molecular weight as previously described. Nevertheless, after incubation of a mixture alpha 2-macroglobulin-trypsin during 45 minutes at 37 degrees C, approximately 60 per cent of the preparation were converted in a component with 83,000 molecular weight as detected in SDS polyacrylamide gel. That component was isolated on Sephadex G-200 in guanidine hydrochloride and corresponds to the subunit, fraction II. According to the results of the present work together with those of previous studies, it can be assumed that alpha 2-MG is a 780,000 molecular weight protein (19S) formed of two half-molecules of equal weight (11-12S). The half-molecule contains two polypeptide chains of 180,000-190,000 molecular weight, each of them having, in its middle, a specific region particularly susceptible to attack by proteases.     

Computer averaging of single molecules of alpha 2-macroglobulin and the alpha 2-macroglobulin/trypsin complex

From electron micrographs single molecules of alpha 2-macroglobulin in the "closed" form, the "open" form and as the trypsin complex have been computer averaged. The molecular images are discussed. Molecules of the electrophoretically fast migrating "F-form" have the "closed" form. In the case of the alpha 2-macroglobulin/trypsin complex the two attached trypsin molecules are located very near to each other and in the central part of the alpha 2-macroglobulin molecule.     

Interaction of human plasma kallikrein and its light chain with alpha 2-macroglobulin

Human plasma kallikrein participates in the contact activation system of plasma. The light chain of kallikrein contains the enzymatic active site; the heavy chain is required for binding to high molecular weight kininogen and for surface-dependent activation of coagulation. This study has examined the functional contributions of the heavy chain of kallikrein and of high molecular weight kininogen in the inactivation of kallikrein and of its isolated light chain by alpha 2-macroglobulin (alpha 2M). Irreversible inhibition was observed for both kallikrein and its light chain, with the initial formation of a reversible enzyme-inhibitor complex. The second-order rate constants for these reactions were 3.5 X 10(5) and 4.8 X 10(5) M-1 min-1 for kallikrein and its light chain, respectively. When present in excess, high molecular weight kininogen decreased the rate of kallikrein inactivation by alpha 2M, whereas the rate of inactivation of the light chain was unaffected by high molecular weight kininogen. Although at a drastically reduced rate, high molecular weight kininogen was cleaved by alpha 2M-bound kallikrein. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and kallikrein or its light chain. Under reducing conditions, four kallikrein-alpha 2M complexes were observed. Three of these complexes consisted of alpha 2M and the light chain of kallikrein (Mr 123 000, 235 000, and 330 000). Two alpha 2M-kallikrein light chain complexes incorporated [3H]diisopropyl fluorophosphate ( [3H]DFP) whereas the Mr 330 000 complex did not react with [3H]DFP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human pregnancy zone protein. Comparison with human alpha 2-macroglobulin

Native human pregnancy zone protein (PZP), a close homolog of alpha 2-macroglobulin (alpha 2M), can be obtained in approximately 20% yield from pooled late pregnancy plasma or serum by a combination of polyethylene glycol precipitation, euglobulin precipitation, DEAE-Sephacel chromatography, zinc-chelate affinity chromatography, and negative affinity chromatography on insolubilized antibodies against human serum proteins. Both proteins are similarly organized as disulfide-bridged dimers of 360 kDa containing 180-kDa subunits. These dimers constitute the proteinase-binding units of PZP, and in contrast to alpha 2M, they appear to be only loosely associated, indicating a subtle difference in the quaternary structure of these alpha-macroglobulins. The preparations contain functionally intact beta-cysteinyl-gamma-glutamyl thiol esters, located in the same nonapeptide sequence as found in alpha 2M, and form complexes with a variety of proteinases in which a large fraction of the proteinase is bound covalently. Proteinases bound to PZP are still active and poorly accessible to reaction with large inhibitors like alpha 1-proteinase inhibitor. The structural and functional features of PZP indicate that PZP and alpha 2M, although extremely similar, may have different yet overlapping sets of proteinases as targets. It is possible that PZP mainly controls the activity of cellular proteinases released under conditions of increased cellular turnover and that PZP could be the human equivalent to the acute phase alpha-macroglobulins known in other species.     

The three-dimensional structure of the human alpha 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin alpha 2-macroglobulin complexes

Three-dimensional electron microscopy reconstructions of the human alpha(2)-macroglobulin (alpha(2)M) dimer and chymotrypsin-transformed alpha(2)M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 A at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed alpha(2)M. During the transformation of the structure, the strands untwist to expose the alpha(2)M central cavity to the proteinase. This extraordinary change in the architecture of alpha(2)M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Kinetics of the reaction of streptokinase-plasmin complex with purified human and mouse alpha 2-macroglobulin. Implications for mechanism.

Streptokinase-human plasmin complex (Sk-hPm) reacted rapidly with purified mouse alpha 2-macroglobulin (m alpha 2M) in vitro at 37 degrees C. Approx. 98% of the plasmin in Sk-hPm bound covalently to at least one m alpha 2M subunit. Most of the streptokinase dissociated (95%). The rate of Sk-hPm inactivation clearly depended on the m alpha 2M concentration. With 1.2 microM-m alpha 2M, 50% of the Sk-hPm (0.02 microM) reacted in less than 50 s. A double-reciprocal plot comparing pseudo-first-order rate constants (kapp.) and m alpha 2M concentration yielded a second-order rate constant of 2.3 x 10(4) M-1.s-1 (r = 0.97). This value is an approximation, since Sk-hPm preparations are heterogeneous. Sk-hPm reacted with human alpha 2M (h alpha 2M), forming alpha 2M-plasmin complex (98% covalent). More than 99% of the streptokinase dissociated. The rate of reaction of Sk-hPm with h alpha 2M did not clearly depend on inhibitor concentration. The kapp. values determined with 0.6-1.2 microM-h alpha 2M were decreased 10-20-fold compared with m alpha 2M. In order to study the effect of Sk-hPm heterogeneity on the reaction with alpha 2M, the proteinase was incubated for various amounts of time at 37 degrees C before addition of inhibitor. The enzyme amidase activity was maximal within 5 min; however, reaction of Sk-hPm with m alpha 2M or h alpha 2M was most extensive after 20 min and 2 h respectively. After incubation for more than 1 h, Sk-hPm acquired fibrinogenolytic activity, suggesting plasmin dissociation. Therefore the enhanced reaction of h alpha 2M with 'older' Sk-hPm preparations may have resulted in part from dissociated plasmin or 'plasmin-like' species. By contrast, the reaction of Sk-hPm with m alpha 2M was most rapid when the proteinase preparation was free of plasmin, indicating direct reaction of Sk-hPm with m alpha 2M as the only major mechanism. Finally, streptokinase-cat plasminogen complex reacted more extensively with m alpha 2M than with h alpha 2M, suggesting that m alpha 2M may be a superior inhibitor with this class of plasminogen activators in general.     

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.     

Reaction of methylamine with human alpha 2-macroglobulin. Mechanism of inactivation

The human protease inhibitor alpha 2-macroglobulin (alpha 2 M) is inactivated by reaction with methylamine. The site of reaction is a protein functional group having the properties of a thiol ester. To ascertain the relationship between thiol ester cleavage and protein inactivation, the rates of methylamine incorporation and thiol release were measured. As expected for a concerted reaction of a nucleophile with a thiol ester, the rates were identical. Furthermore, both rates were first order with respect to methylamine and second order overall. The methylamine inactivation of alpha 2M was determined by measuring the loss of total protease-binding capacity. This rate was slower than the thiol ester cleavage and had a substantial initial lag. However, the inactivation followed the same time course as a conformational change in alpha 2M that was measured by fluorescent dye binding, ultraviolet difference spectroscopy, and limited proteolysis. Thus, the methylamine inactivation of alpha 2M is a sequential two-step process where thiol ester cleavage is followed by a protein conformational change. It is the latter that results in the loss of total protease-binding capacity. A second assay was used to monitor the effect of methylamine on alpha 2M. The assay measures the fraction of alpha 2M-bound protease (less than 50%) that is resistant to inactivation by 100 microM soybean trypsin inhibitor. In contrast to the total protease-binding capacity, this subclass disappeared with a rate coincident with methylamine cleavage of the thiol ester. alpha 2M-bound protease that is resistant to a high soybean trypsin inhibitor concentration may reflect the fraction of the protease randomly cross-linked to alpha 2M. Both the thiol ester cleavage and the protein conformational change rates were dependent on methylamine concentration. However, the thiol ester cleavage depended on methylamine acting as a nucleophile, while the conformational change was accelerated by the ionic strength of methylamine. Other salts and buffers that do not cleave the thiol ester increased the rate of the conformational change. A detailed kinetic analysis and model of the methylamine reaction with alpha 2M is presented. The methylamine reaction was exploited to study the mechanism of protease binding by alpha 2M. At low ionic strength, the protein conformational change was considerably slower than thiol ester cleavage by methylamine. Thus, at some time points, a substantial fraction of the alpha 2M had all four thiol esters cleaved, yet had not undergone the conformational change. This fraction (approximately 50%) retained full protease-binding capacity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin

The biological latency of serum transforming growth factor-beta (TGF-beta) was shown to be due to the interaction of TGF-beta with a specific serum binding protein. This binding protein was affinity labeled with 125I-TGF-beta, and its Mr and subunit structure were determined using sodium dodecyl sulfate-gel electrophoresis and gel filtration chromatography. Its Mr is reminiscent of that of the serum protease inhibitor, alpha 2-macroglobulin (alpha 2M). Immunoprecipitation of the 125I-TGF-beta-binding protein complex by a specific anti-alpha 2M antibody, and the formation of identical complexes between 125I-TGF-beta and purified alpha 2M, confirmed that alpha 2M is the TGF-beta-binding protein in serum. Immunoblot analysis showed that endogenous serum TGF-beta is also bound to alpha 2M. However, in contrast to added 125I-TGF-beta, the majority of the endogenous TGF-beta is linked to alpha 2M covalently. Alpha 2M and acid-activated TGF-beta co-eluted from a Superose 6 fast protein liquid chromatography column, confirming that the interaction of TGF-beta with alpha 2M accounts for the latency of serum TGF-beta. It is proposed that alpha 2M may serve an important multifunctional role at sites of inflammation by scavenging both active peptides and proteases that are released by platelets at the site of injury.     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Evolution of alpha 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human alpha 2-macroglobulin.

The amino acid sequence of the Pronase-released heads of neuraminidase subtype N2 from the A/Tokyo/3/67 strain of influenza virus was determined by a combination of peptide and nucleic acid sequence analysis. The results show that the Pronase-released heads contain 396 amino acid residues and extend from residue 74 in the original protein to the C-terminus at residue 469. The heads contain five potential glycosylation sites at asparagine residues 86, 146, 200, 234 and 402, but only the first four are glycosylated. The sequence homology with the corresponding region of the previously published sequence of the neuraminidase subtype N1 [Fields, Winter & Brownlee (1981) Nature (London) 290, 213-217] is 45%. Detailed evidence for the sequence data has been deposited as Supplementary Publication SUP 50116 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1981) 193, 5.     

Stability and subunit structure of human alpha2-macroglobulin.

The molecular weights of alpha2-macroglobulin and its non-covalent subunits have been determined by equilibrium centrifugation. The secondary structure of the native and the thermally denatured molecules has been analyzed by circular dichroic measurements. In contrast to most proteins the thermally denatured form contains a slightly more highly organized polypeptide chain than the native form. The relaxation time of the native protein, as determined by fluorescence polarization measurements, indicates that alpha2-macroglobulin is composed of domains smaller than that of the two subunits. The transitions in acid, alkali, and at high temperatures have been explored in order to establish the pH and thermal range of stability of alpha-macroglobin.     

Purification of the human placental alpha 2-macroglobulin receptor

The alpha 2-macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross-linking of labelled ligand to intact membranes showed a receptor size compatible with 400-500 kDa. The membranes were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose-immobilized alpha 2-macroglobulin-methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS-PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70-90 kDa and 40 kDa. The yield was 400-800 micrograms receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the alpha 2-macroglobulin-trypsin complex with a dissociation constant of about 400 pM. 125I-iodinated receptor preparation bound almost quantitatively to Sepharose-immobilized alpha 2-macroglobulin-methylamine in the presence of CHAPS alone, and bound 70-80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS-PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.