Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.     

Major secretory product of the mesometrial decidua in the rat,a variant of alpha-2-macroglobulin,binds insulin-like growth factor I via a protease-dependent mechanism

Decidualization-associated protein (DAP), the quantitatively major secretory product of the mesometrial decidua in the rat, is a pl variant of the liver-derived acute-phase reactant, alpha-2-macroglobulin (α₂M). α₂M, a broad spectrum protease inhibitor, has been demonstrated in the human to bind a variety of cytokines and growth factors. In humans, the quantitatively major secretory product of decidual tissue is an insulin-like growth factor (IGF) binding protein. In this study, we have therefore tested the ability of liver- and decidual-derived α₂M in the rat to bind IGF-I. α₂M purified from acute-phase plasma and DAP purified from cytosolic extracts of decidual tissue and medium from tissue incubations both bound radiolabeled IGF-I. The binding of IGF-I was principally dependent upon the coincubation of the protein with a proteinase. Therefore, it occurred during the conversion of the “slow” to the “fast” form of α₂M. Pretreatment with proteinase to produce the fast form before addition of the IGF-I reduced the binding. Binding was enhanced at a ratio protein:proteinase of 1:1. Results from gel electrophoretic analysis were consistent with the covalent linkage of IGF-1 to α₂M during the cleavage of the “bait region.” A saturable displacement by increasing concentrations of unlabeled IGF-I suggested high affinity interaction. Under conditions of demonstrated binding to purified proteins binding in acute-phase plasma, decidual tissue extracts and tissue incubation medium were associated with a high molecular weight species which was confirmed to represent α₂M and DAP, respectively. Our studies demonstrate that IGF-I may now be added to the list of regulatory peptides which α₂M may bind and that, in rat decidua, DAP may represent the functional homolog of decidual IGFBP-1 in the human and regulate growth factor function during placental development. © 1996 Wiley-Liss, Inc.     

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.     

alpha 2-macroglobulin traps a proteinase in the midregion of its arms. An immunoelectron microscopic study

alpha 2-Macroglobulin, one of the major plasma proteinase inhibitors with Mr = 720,000, is known to inhibit proteinases of all four classes through the "trap mechanism" (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724), but the proteinase binding site of alpha 2-macroglobulin has not been identified precisely. We localized bound proteinase molecules on the electron microscopic images of alpha 2-macroglobulin, using anti-proteinase IgG. Serratial Mr = 56,000 proteinase produced by Serratia marcescens was chosen as the antigenic probe in this study because its affinity to specific antibodies was retained in its bound state to alpha 2-macroglobulin. Dimers of alpha 2-macroglobulin/Mr = 56,000 proteinase complexes cross-linked with anti-Mr = 56,000 proteinase IgG were prepared and subjected to electron microscopic observations. The electron microscopic image of alpha 2-macroglobulin complexed with Mr = 56,000 proteinase had four straight arms with an overall shape looking like the character "H." From the way anti-Mr = 56,000 proteinase IgG linked two alpha 2-macroglobulins, it was concluded that the proteinase existed in the midregion of one of the arms. This result helps us to form a more concrete view of the trap mechanism in that one of the arms of alpha 2-macroglobulin wraps the trapped proteinase and holds it isolated from high molecular weight substrates in the surrounding medium.     

Binding of zinc to alpha-2-macroglobulin and its role in enzyme binding activity.

Physicochemical studies performed on alpha-2-macroglobulin were correlated with the biological activities of this protein. Equilibrium dialysis of the binding of 65Zn by alpha-2-macroglobulin at pH 7.9 showed heterogeneous binding which could be attributed to two classes of binding sites. The site of greatest affinity for zinc had an apparent stoichiometry (n1 in gatoms/mol of alpha-2-macroglobulin monomer) of 12 and an apparent association constant (K1) of 3.06.10(7). The second binding site had an n2 of 60 and K2 of 1.32.10(5). The trypsin binding activity of alpha-2-macroglobulin did not depend on the presence of zinc in this protein since all but traces of this metal could be removed by EDTA without loss of trypsin binding activity. Saturation of site 1 with zinc did not affect the trypsin binding activity of alpha-2-macroglobulin, but binding of the metal by site 2 progressively decreased the trypsin binding activity by causing an irreversable association of the alpha-2-macroglobulin molecules. Removal of excess zinc from alpha-2-macroglobulin did not restore its trypsin binding activity. Our results also indicate that the high zinc content of alpha-2-macroglobulin (320--770 microgram/g protein) reported in the literature is an artifact and that native alpha-2-macroglobulin contains approximately 150--180 microgram Zn/g protein.     

Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy

The structural change that occurs in alpha-2-macroglobulin upon its interaction with methylamine or chymotrypsin was studied by high-performance gel chromatography and electron microscopy. The result enabled us to estimate the Stokes radius of the protein as 8.8 nm and 7.9 nm before and after binding with the proteinase, respectively. The methylamine-treated protein also had the Stokes radius of 7.9 nm. Similar studies on the chicken and crocodilian ovomacroglobulins showed that these homologues of alpha 2-macroglobulin had Stokes radii of 9.2-9.3 nm and 8.5-8.7 nm before and after binding with chymotrypsin. Their Stokes radii did not change as a result of the methylamine treatment. Electron micrographs of the native and altered forms of the three proteins are presented. This study introduces a simple and quantitative method to study the structural change of alpha 2-macroglobulin and its homologues.     

The identification of distinctive forms of human alpha 2-macroglobulin by using the numerical relationship between trypsin binding in alpha- and beta-modes.

Interactions between the serine proteinase trypsin and the protein proteinase inhibitors in human blood were expressed in terms of a coupled set of non-linear differential equations, which has been solved for each of 110 samples of serum obtained from colleagues and from a variety of hospital sources. Optimization of nine unknown theoretical parameters and 21 experimental rate measurements of the hydrolytic activity of trypsin in free and bound states after admixture with various amounts of a given serum was achieved by an iterative procedure using initial estimates of the parameters derived from the "four-straight-line" model described in the preceding paper [Topping & Seilman (1979) Biochem. J. 177, 493--499.] Such a procedure yielded the following information for each sample of serum examined: (a) the concentrations of alpha 1-antitrypsin and alpha 2-macroglobulin; (b) the unequivocal assignment of alpha 2-macroglobulin into one of seven categories on the basis of trypsin binding in two kinetically differentiated modes (alpha and beta); (c) the hydrolytic activities of trypsin (versus Bz-Arg-OEt) when bound to alpha 1-antitrypsin, and to alpha 2-macroglobulin in the alpha- and beta-modes. Molecular interpretations of the binding of trypsin to alpha 2-macroglobulin are discussed and the potential clinical value of recognizing the nature of such binding is reported.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Proteinase-proteinase inhibitor complex in rats under oxidative stress caused by administration of cobalt chloride

Mechanisms of proteinase-inhibitor proteinase system response was estimated following of cobalt chloride injection. The increase proteinase activity, which led to significant decrease of alpha-2-macroglobulin (alpha-2-MG) level was established that indicated to the removal of the proteinase in complex with alpha-2-MG from the organism. Increase of alpha-1-proteinase inhibitor (alpha-1-PI) trypsin-inhibitory activity in the kidneys testify about removal of oxidative alpha-1-PI.     

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.     

alpha-1-Anti-trypsin, an inhibitor of renin.

A naturally occurring competitive inhibitor of pig kidney renin has been identified in human plasma. The inhibitor was shown to be alpha-1 anti-trypsin and the effect in vitro on the renin activity was examined. The slope in the Hill plot is compatible with the assumption of one-site competitive inhibition. Other proteinase inhibitors, such as alpha-2-macroglobulin and C1 inactivator, however, have no inhibitory effect on the renin-angiotensinogen reaction.     

Effect of pentoxyphylline on certain indicators of the proteinase-proteinase inhibitor system in rats upon administration of cycloheximide

The pentoxifylline influence on neutral proteinase, alpha-2-macroglobulin, trypsin-alpha-1-proteinase inhibitor and elastaseinhibitory activity under cycloheximide injection has been investigated. Two hours after cycloheximide injection the activity of neutral proteinases increases in rats serum, lungs, heart, liver and kidneys. The preliminary injection of pentoxifylline prevents increase of neutral proteinases activity. Cycloheximide also decreases alpha-2-macroglobulin activity in serum and liver and trypsin-, elastaseinhibitory activity of alpha-1-proteinase inhibitor in all investigated organs. At using pentoxifylline the alpha-2-macroglobulin activity doesn't change in liver and increases in serum in comparison with only cycloheximide and there are no observed any alpha-1 inhibitor proteinase activity changes in rats serum and organs.     

Alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity in cerebrospinal fluid as indices of blood-cerebrospinal fluid barrier

Concentration of alpha-2-macroglobulin, albumin, and chymotrypsin inhibitory capacity representing mainly alpha-1-proteinase inhibitor were estimated in cerebrospinal fluid in disorders of the central nervous system. While chymotrypsin inhibitory capacity was elevated in all cases with derangement of the blood-cerebrospinal fluid barrier, in 30% of the cases alpha-2-macroglobulin levels were in the normal range. The difference can be attributed to the much larger size of the latter. Better correlation between albumin concentration and chymotrypsin inhibitory capacity (r = 0.84) than between albumin and alpha-2-macroglobulin (r = 0.62) supports the view that the rate of entry of proteins from blood into cerebrospinal fluid is inversely related to their size.     

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.     

Interaction of alpha 2-macroglobulin with trypsin, chymotrypsin, plasmin, and papain

The interaction alpha 2-macroglobulin with four proteinases has been investigated by binding assays and by gel electrophoresis. At pH 7.65 the binding ratios of the proteinase-alpha 2-macroglobulin complexes were found to be 2:1 (trypsin and papain), 1.4:1 (chymotrypsin), and 1:1 (plasmin). The progressive decrease in the stoichiometry of the three seryl proteinase complexes was paralleled by a concomitant decrease in the proteinase-dependent specific cleavage of the alpha 2-macroglobulin peptide chains. Rate studies have shown that the relative rates of reaction of the proteinases with alpha 2-macroglobulin also varied greatly: papain greater than trypsin greater than chymotrypsin greater than plasmin. The data suggest that the ability of a proteinase to saturate the second proteinase binding site is a reflection of its ability to bind to alpha 2-macroglobulin and cleave the second pair of scissile alpha 2-macroglobulin peptide bonds before the alpha 2-macroglobulin has undergone the conformational change initiated by the formation of the 1:1 proteinase alpha 2-macroglobulin complex.     

The effect of zinc and other divalent cations on the structure and function of human alpha 2-macroglobulin

Zinc binding to human alpha 2-macroglobulin was studied to assess its involvement in the structure and function alpha 2-macroglobulin. Equilibrium dialysis experiments indicated multiple classes of zinc-binding sites, the one of highest affinity having a site number of 20 and a Kd value of 8 X 10(-7) M. Native alpha 2-macroglobulin and alpha 2-macroglobulin-trypsin complexes bound comparable amount of zinc. The proteinase inhibitory activity of alpha 2-macroglobulin was not affected by zinc binding at physiological concentrations nor by the removal of zinc by EDTA. Above 25 microM zinc, alpha 2-macroglobulin activity decreased, although binding of [125I]trypsin was not affected. When nondenaturing gel electrophoresis was performed, the preparation of alpha 2-macroglobulin migrated as half-molecules at increasing zinc concentration. Experiments with other divalent cations correlated decreases in alpha 2-macroglobulin activity with apparent dissociation of the alpha 2-macroglobulin tetramer in the presence of copper and mercury, but not barium, cadmium or nickel. While zinc binding to alpha 2-macroglobulin does not function in proteinase inhibition, it might be involved in zinc transport in vivo. At nonphysiological concentrations, zinc and other divalent cations are useful as probes of protein quaternary structure.     

Mannose-specific lectins bind alpha-2-macroglobulin and an unknown protein from human plasma.

GNA, the mannose-specific lectin from Galanthus nivalis was confirmed to bind alpha-2-macroglobulin (A2M) but another protein was copurified with A2M from total human plasma. A total of 23 other lectins with diverse specificities were tested for reaction with human A2M and with three other members of the A2M family. NPA, a mannose-specific lectin isolated from Narcissus pseudonarcissus bulbs, and RSA, the Rhizoctonia solani agglutinin, were selected for further testing. For isolation of A2M, immobilized NPA was superior to GNA because its binding capacity was an order of magnitude higher. The specificity of these lectins must be very similar however, because the same unknown plasma protein was also bound by NPA. A2M and the unknown protein must share a unique mannose carbohydrate structure not present in any other human plasma protein. The copurified protein subunit size of 185 kDa is very similar to that of A2M, but the native molecular mass of 350 kDa indicated a noncovalent homodimer structure. Together with the acid isoelectric point this is not typical for any known plasma protein nor for any unidentified spot on the two-dimensional map of human plasma proteins. No immunological reaction with available antisera was evident. A specific antiserum raised to the unknown protein demonstrated its presence in all human plasma samples examined. The N-terminal residue was blocked, whereas internal protein sequences obtained after CNBr fragmentation and proteolysis were not homologous to any known protein sequence. These data demonstrate that this protein is unknown and not a proteinase inhibitor of the A2M family.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: effects of substrate opsonization

Inflammatory cells are capable of degrading extracellular matrix macromolecules in vivo in the presence of proteinase inhibitors. We and others have hypothesized that such proteolysis is permitted in large part by mechanisms operative in the immediate pericellular environment, especially at zones of contact between inflammatory cells and insoluble matrix components. To further test this hypothesis in vitro, we have used a model system in which viable polymorphonuclear neutrophils (PMN) are allowed to contact a surface coated with proteinase-sensitive substrate, and in which PMN interaction with the surface can be modulated. We have evaluated proteolysis of the surface-bound protein in the presence and absence of proteinase inhibitors. Our results were: (a) In the presence (but not in the absence) of proteinase inhibitors, proteolysis was confined to sharply marginated zones subjacent to the cells; (b) opsonization of the surface enhanced spreading of the PMN, (c) opsonization diminished the effectiveness of alpha-1-proteinase inhibitor (alpha-1-PI) and alpha-2-macroglobulin as inhibitors of proteolysis of surface-bound protein; (d) anti-oxidants did not alter the effectiveness of alpha-1-PI in inhibiting proteolysis of opsonized substrate by PMN; and (e) PMN could restrict entry of alpha-1-PI into zones of contact with opsonized surfaces. We conclude that: (a) In the presence of proteinase inhibitors, PMN can express sharply marginated and exclusively pericellular proteolytic activity; (b) locally high proteinase concentrations and/or exclusion of proteinase inhibitors from pericellular microenvironments may be important mechanisms for pericellular matrix degradation by PMN; and (c) these observations may have general relevance to extracellular matrix remodeling by a variety of inflammatory and other cell types.     

The effect of endogeneous proteinase inhibitors on the prophenoloxidase activating enzyme,a serine proteinase from crayfish haemocytes

Three proteinase inhibitors have so far been isolated and purified from crayfish haemolymph. One of these, isolated from crayfish plasma, namely a trypsin inhibitor with a molecular mass of 155 kDa was found to inhibit a serine proteinase, ppA, which is involved in the activation of prophenoloxidase, and is localized in the haemocytes. Another high molecular mass proteinase inhibitor, an α₂-macroglobulin from crayfish plasma, which is a dimer of 190 kDa-subunits, was only inhibitory towards ppA to a lesser extent. A 23 kDa subtilisin inhibitor, purified from haemocytes, did not have any effect on the serine proteinase.We suggest that mainly the trypsin inhibitor, but to some extent also the α₂-macroglobulin, are important in the regulation of the prophenoloxidase activating cascade, as they both inhibit ppA, which in its active form has been shown to mediate prophenoloxidase activation.