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PubMed1987

Limulus alpha 2-macroglobulin. First evidence in an invertebrate for a protein containing an internal thiol ester bond.

Intra-chain thiol ester bonds are present in a limited number of proteins. The thiol ester class of proteins includes vertebrate alpha 2-macroglobulin and the complement proteins C3 and C4. We report here the first instance of a thiol ester protein from an invertebrate, the alpha 2-macroglobulin proteinase-inhibitor homologue present in the plasma of the American horseshoe crab Limulus polyphemus. Our evidence is of three kinds: (1) the proteinase-binding activity of Limulus alpha 2-macroglobulin is inactivated by the low-molecular-mass primary amine methylamine; (2) the native protein is subject to autolytic fragmentation during mild thermal denaturation, yielding fragments of approx. 125 kDa and 55 kDa, whereas the methylamine-treated protein is stable under these conditions of thermal treatment; (3) new thiol groups are generated rapidly during reaction of the protein with trypsin. The demonstration of the thiol ester bond in a protein from an ancient invertebrate provides evolutionary evidence for the importance of this bond in the function of plasma forms of the alpha 2-macroglobulin-like proteinase inhibitors.

Animals↗

PubMed1991

Kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin as measured by small-angle neutron scattering.

The kinetics of the urea-induced dissociation of human plasma alpha 2-macroglobulin into two half-molecular fragments was investigated at 21.0 degrees C by using small-angle neutron scattering. The relative change in molecular mass that occurs upon dissociation was monitored by recording the forward scattering of neutrons as a function of time. All these kinetic data can be explained by a reaction that is first-order with respect to the concentration of undissociated alpha 2-macroglobulin. The velocity constant is a function of urea concentration and it varies within wide limits. For instance, the half-life of the reaction at the lowest concentration of [2H]urea studied (2.70 M) is 328 h, whereas the same value at the highest concentration of [2H]urea (6.24 M) is only 8 min. Measurements were made both with [1H]urea in 1H2O and with [2H]urea in 99% 2H2O, and it was found that there is a pronounced kinetic isotope effect, i.e. the dissociation is 4 times faster in the 1H-containing medium as compared with the 2H-containing medium at the same molar concentration of urea. From the angular dependence of the neutron scattering it can be concluded that the dissociation is associated with a drastic change in structure. This is directly shown by the radius of gyration, which increases from about 7.4 nm immediately after the addition of urea up to about 9.4 nm when the protein is fully dissociated. A structural analysis shows that the scattering curve of urea-dissociated alpha 2-macroglobulin can best be explained by that of a Gaussian coil with a radius of gyration equal to 9.44 nm. These data indicate that the so-called non-covalent interaction of alpha 2-macroglobulin probably is more complicated than just a pure hydrophobic interaction. Finally, it is also shown that the dissociation is accompanied by a loss in trypsin-binding activity, which is directly related to the fraction of dissociated protein.

Animals↗

PubMed1991

Recognition of chylomicron remnants and beta-migrating very-low-density lipoproteins by the remnant receptor of parenchymal liver cells is distinct from the liver alpha 2-macroglobulin-recognition site.

The uptake in vivo of chylomicrons and beta-migrating very-low-density lipoprotein (beta-VLDL) by rat liver, which is primarily carried out by parenchymal cells, is inhibited, 5 min after injection, to respectively 35 and 8% of the control values after preinjection of lactoferrin. The decrease in the uptake of lipoproteins by the liver caused by lactoferrin is a specific inhibition of uptake by parenchymal cells. Competition studies in vitro demonstrate that chylomicron remnants and beta-VLDL compete for the same recognition site on parenchymal cells. Data obtained in vivo together with the competition studies performed in vitro indicate that chylomicron remnants and beta-VLDL interact specifically with the same remnant receptor. Hepatic uptake of 125I-labelled-alpha 2-macroglobulin in vivo, mediated equally by parenchymal and endothelial cells, is not decreased by preinjection of lactoferrin and no effect on the parenchymal-cell-mediated uptake is found. In vitro, alpha 2-macroglobulin and chylomicron remnants or beta-VLDL show no cross-competition. Culturing of parenchymal cells for 24-48 h leads to a decrease in the cell association of alpha 2-macroglobulin to 26% of the initial value, while the cell association of beta-VLDL with the remnant receptor is not influenced. It is concluded that beta-VLDL and chylomicron remnants are recognized by a specific remnant receptor on parenchymal liver cells, while uptake of alpha 2-macroglobulin by liver is carried out by a specific receptor system (presumably involving the LDL-receptor-related protein) which shows properties that are distinct from those of the remnant receptor.

Animals↗

PubMed1971

Tetrameric immune macroglobulins in three orders of bony fishes.

The immune IgM-like macroglobulins were isolated from three species of bony fishes (Ictalurus punctatus, Lepisosteus osseus, and Polyodon spathula) representing the three orders of the subclass Actinopterygii. These macroglobulins were found to have sedimentation coefficients of 14 S and molecular weights of 600,000-630,000. The carbohydrate compositions were determined and found to be different from that of human macroglobulins. After reduction and alkylation, the heavy and light chains could be separated by gel filtration and were found to have molecular weights of approximately 70,000 and 23,000 respectively. The amino acid compositions of these chains were similar to those of mammalian IgM heavy and light chains. The macroglobulins were found by electron microscopy to have a tetrameric structure, in contrast to the pentameric structure found in mammalian, chicken, and shark IgM, i.e. in species later on the evolutionary scale.

Amino Acids↗

PubMed1988

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Well-preserved structures of native and alpha-chymotrypsin-bound alpha 2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native alpha 2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the alpha 2-macroglobulin-chymotrypsin conjugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of alpha 2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex.

Chymotrypsin↗

PubMed1987

The elimination of trypsin--alpha-macroglobulin complexes from the blood circulation in pigs during acute pancreatitis and after massive intravenous trypsin infusion.

The elimination from plasma of intravenously injected 125I-labelled trypsin-alpha-macroglobulin complexes was studied in both healthy and diseased, anaesthetized pigs. The t1/2 for the 125I-labelled complexes was 5-10 min in healthy pigs as well as in two groups of pancreatitis pigs. Pre-loading of the eliminating capacity by massive intravenous trypsin infusion, raised t1/2 to 80-100 min. Thus, the eliminating capacity seems to be saturable. Elimination of the complexes from the blood circulation occurred within 4-5 h. Even massive intravenous amounts of trypsin-alpha-macroglobulin complexes gave no demonstrable complement or kinin activation. Neither were other deleterious effects seen during the experiment or during 7 days of follow-up. The results indicate that treatment of acute pancreatitis in humans with alpha 2-macroglobulin might be possible, without any danger arising from the protease-alpha 2-macroglobulin complexes formed.

Acute Disease↗

PubMed1997

Development of a competitive enzyme linked immunosorbent assay to measure alpha 2-macroglobulin in irradiated rat serum.

A competitive enzyme linked immunosorbent assay with antigen immobilized on the solid phase was developed to measure alpha 2-macroglobulin in rat serum. The cross reactivity with albumin and hemoglobin was eliminated by use of IgG fractions that were isolated after chromatography on Cibacron Blue F3-GA Sepharose immobilized rat whole serum and hemoglobin Sepharose. Blocking materials and pH of the coating buffer had no effect on the amount of alpha 2-macroglobulin that binds to the plate. When the coating amount of alpha 2-macroglobulin was 100 ng/well, at pH 7.4, 10 mM Tris-HCl containing 150 mM sodium chloride and the IgG amount added was 60 ng/well, then albumin and hemoglobin did not affect the assay at concentrations of 150 micrograms/ml or 200 micrograms/ml. This assay is useful for measuring the concentration of alpha 2-macroglobulin in normal and irradiated rat serum.

Animals↗

PubMed1989

Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2-macroglobulin inactive complex.

The control of smooth muscle cell (SMC) proliferation is determined by the combined actions of mitogens, such as platelet-derived growth factor, and the opposing action of growth inhibitory agents, such as heparin and transforming growth factor-beta (TGF-beta). The present studies identify an interaction between heparin and TGF-beta in which heparin potentiates the biological action of TGF-beta. Using a neutralizing antibody to TGF-beta, we observed that the short term antiproliferative effect of heparin depended upon the presence of biologically active TGF-beta. This effect was observed in rat and bovine aortic SMC and in CCL64 cells, but not in human saphenous vein SMC. Binding studies demonstrated that the addition of heparin (100 micrograms/ml) to medium containing 10% plasma-derived serum resulted in a 45% increase in the specific binding of 125I-TGF-beta to cells. Likewise, heparin induced a twofold increase in the growth inhibitory action of TGF-beta at concentrations of TGF-beta near its apparent dissociation constant. Using 125I-labeled TGF-beta, we demonstrated that TGF-beta complexes with the plasma component alpha 2-macroglobulin, but not with fibronectin. Heparin increases the electrophoretic mobility of TGF-beta apparently by freeing TGF-beta from its complex with alpha 2-macroglobulin. Dextran sulfate, another highly charged antiproliferative molecule, but not chondroitin sulfate or dermatan sulfate, similarly modified TGF-beta's mobility. Relatively high, antiproliferative concentrations of heparin (1-100 micrograms/ml) were required to dissociate the TGF-beta/alpha 2-macroglobulin complex. Thus, it appears that the antiproliferative effect of heparin may be partially attributed to its ability to potentiate the biological activity of TGF-beta by dissociating it from alpha 2-macroglobulin, which normally renders it inactive. We suggest that heparin-like agents may be important regulators of TGF-beta's biological activity.

Animals↗

PubMed1959

Macroglobulinemia. III. The effect of mercaptoethanol on the antigenic structure of macroglobulins.

The monomers obtained by treating gamma(1)-macroglobulins with mercaptoethanol have proved to be antigenically different from normal 7S 7gamma-globulin. The depolymerization of the macroglobulins resulted in the loss of several antigenic determinants, although the monomers still cross-reacted with antisera against macroglobulins. Reaggregation of the monomers occasionally resulted in the reconstitution of some or all of the antigenic determinants that were lost during depolymerization. Repeated freezing and thawing of pathological macroglobulins in iodoacetate resulted in their complete antigenic destruction. Repeated freezing and thawing of one of the monomers in excess iodoacetate resulted in the degradation to a protein antigenically indistinguishable from Bence Jones protein. The other two monomers studied were stable under these conditions.

Antigens↗

PubMed1985

Immunochemical similarities between monoclonal antibacterial Waldenstrom's macroglobulins and monoclonal anti-DNA lupus autoantibodies.

Six monoclonal IgM from patients with Waldenstrom's macroglobulinemia that react with Klebsiella polysaccharides were tested for their ability to bind to nucleic acid antigens. One of the macroglobulins bound to the polynucleotide poly(G), and one bound to poly(G), poly(I), and single-stranded DNA. The reaction with the polynucleotides was specifically inhibited by the Klebsiella polysaccharide K30. A monoclonal lupus anti-DNA antibody (16/6) was found to react weakly with the Klebsiella polysaccharides K30 and K21. Five of the Waldenstrom macroglobulins shared an idiotypic determinant with the 16/6 anti-DNA antibody. The reaction between the macroglobulins and the antiidiotype serum was specifically inhibited by Klebsiella polysaccharides, an indication that the idiotypic marker was in the antigen-binding site of the macroglobulins. These results indicate the existence of widely dispersed conserved variable region genes that encode idiotypically related immunoglobulins with the capacity to bind to both bacterial polysaccharides and nucleic acids. Such genes can be expressed by patients with either Waldenstrom's macroglobulinemia or systemic lupus erythematosus.

Antibodies, Bacterial↗

PubMed1989

Identification of the promoter sequences involved in the interleukin-6 dependent expression of the rat alpha 2-macroglobulin gene.

The 5'-region of the rat alpha 2-macroglobulin gene has been characterized. A 5.6 kb Sal I - Xba I fragment containing the first 4 exons of the alpha 2-macroglobulin gene and 1.3 kb of its 5'-flanking region was sequenced. The putative transcriptional start site was determined by RNase protection and primer extension analysis. TATA- and CAAT-box equivalent sequences were found. A potential glucocorticoid receptor binding site was located on the antisense strand. DNA sequences containing the 5'-flanking region of the rat alpha 2-macroglobulin gene were linked to the gene coding for the bacterial chloramphenicol acetyltransferase and introduced into Hep G2 cells. In these transfected Hep G2 cells CAT activity could be induced by recombinant human interleukin-6. Deletion analyses have shown that the sequences between -852 and -777 as well as between -404 and -165 relative to the cap site, contain regulatory elements involved in the interleukin-6 dependent induction of the alpha 2-macroglobulin gene.

Amino Acid Sequence↗

PubMed1996

Gene transfer mediated by alpha2-macroglobulin.

alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein.

Gene Transfer Techniques↗

PubMed1987

Dodecylsulfate-induced dissociation of human alpha 2-macroglobulin. An investigation using small-angle neutron scattering and the equilibrium dialysis technique.

The dodecylsulfate-induced dissociation of the tetrameric alpha 2-macroglobulin molecule from human plasma has been investigated by the small-angle neutron scattering (SANS) method. The great advantage with the SANS method is that, by using deuterated dodecylsulfate, and contrast variation by changing the D2O/H2O ratio of the solvent, we can selectively study just the protein part, or the dodecylsulfate part, of the protein-dodecylsulfate complex. More than a thousandfold excess of dodecylsulfate (on a molar basis) is needed in order to dissociate alpha 2-macroglobulin to particles with, on average, half the original molecular mass. By combining the SANS data with results obtained by the equilibrium dialysis technique it follows that, under these circumstances, approximately one thousand dodecylsulfate molecules are associated per alpha 2-macroglobulin molecule. From the significant increase in the radius of gyration, which accompanies the dissociation process, we can conclude that the dissociation is associated with a drastic change in conformation of the protein molecule. From measurements where the dodecylsulfate part of the complex dominates the SANS signal we also get an indication that the dodecylsulfate is randomly distributed along the polypeptide chain, rather than being arranged in large clusters at certain regions of the protein molecule. By fitting the parameters of a binding model to the experimental data we obtain the result that most of the more than one thousand bound dodecylsulfate molecules, necessary for dissociation, are involved in the change in conformation, and the dissociation process is, in fact, driven by the binding of a very few extra dodecylsulfate molecules to the dissociation products. These data indicate that the dodecylsulfate-induced dissociation of alpha 2-macroglobulin is probably more complicated than just breaking, for instance, a hydrophobic interaction.

Dialysis↗

PubMed1989

Differences in hydrophobic properties for human alpha 2-macroglobulin and pregnancy zone protein as studied by affinity phase partitioning.

Human alpha 2-macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines. The surface properties of the native, chymotrypsin-treated and methylamine-treated forms of alpha 2-macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two-phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of alpha 2-macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine-treated pregnancy zone protein, but the chymotrypsin-treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of alpha 2-macroglobulin and could be a signal for binding to cell-surface receptors, followed by internalization.

Centrifugation↗

PubMed1997

The contact zones in human alpha2-macroglobulin--functional domains important for the regulation of the trapping mechanism.

A functional domain termed the contact zone, which is the region of a subunit interacting with another non-covalently bound subunit, is suggested to play a decisive role in the trapping mechanism of human alpha2-macroglobulin. Tetrameric alpha2-macroglobulin can be dissociated into stable dimers with intact thiol esters by sodium thiocyanate, whereby the contact zones are disrupted. The dissociation leads to significant conformational changes, as studied by ultraviolet-difference spectroscopy, CD, fluorescence and affinity partitioning. The conformation of the dimers is similar to that of MeNH2-treated alpha2-macroglobulin, in which the thiol esters are cleaved, a conformational state with a closed trap occurs, and receptor-recognition sites are exposed. The receptor-binding domain is at least partly exposed in the dimer, as judged by binding of specific mAbs. The bait region in the dimers can be cleaved by proteases, and activation of the thiol esters ensues without binding of the protease. When the dimers were treated with MeNH2, no conformational changes could be detected by ultraviolet-difference spectroscopy or CD. The conformational changes occurring on dissociation into dimers are suggested to be related to trap closure and receptor-recognition-site exposure without cleavage of the thiol esters. The model presented here suggests that two separate conformational changes occur in alpha2-macroglobulin upon activation. The first involves changes at the contact zones as a result of the thiol-ester cleavage, and the second causes exposure of the receptor-recognition sites and closure of the trap.

Antibodies, Monoclonal↗

PubMed1998

Cathepsin B, alpha2-macroglobulin and cystatin levels in gingival crevicular fluid from chronic periodontitis patients.

Gingival crevicular fluid (GCF) was collected from 16 molar and premolar sites in each of 20 chronic periodontitis patients before and after periodontal therapy using filter paper strips. These were eluted individually into buffer for determination of cathepsin B and its endogenous inhibitors, alpha2-macroglobulin and cystatin. Cathepsin B activity was assayed with a fluorogenic peptide substrate, alpha2-macroglobulin by enzyme-linked immunosorbent assay and cystatin activity by inhibition of papain. Total amounts of enzyme and inhibitor per GCF sample decreased after treatment and correlated positively with pocket depth and gingival, bleeding and plaque indices. These comparisons were nearly always statistically significant for pooled site data and sometimes so for mean patient values. The amounts of alpha2-macroglobulin and cystatin were greater than those of cathepsin B and, surprisingly, enzyme and inhibitor levels correlated positively with each other. Experiments with purified reagents, however, demonstrated that the cathepsin B: alpha2-macroglobulin complex was still active against the low molecular weight substrate and that cystatin levels in GCF are probably insufficient to inhibit the enzyme substantially These factors may explain why GCF cathepsin B activity reflects the clinical status of periodontal lesions and has been identified in another study as a promising indicator of disease progression.

Adult↗

PubMed1997

The potential role of alpha 2-macroglobulin in the control of cysteine proteinases (gingipains) from Porphyromonas gingivalis.

Porphyromonas gingivalis is closely associated with the development of some forms of periodontitis. The major cysteine proteinases released by this bacterium hydrolyze peptide bonds only after arginyl (gingipain R) or lysyl residues (gingipain K). No target protein inhibitors have been identified for either enzyme, leading us to investigate their inhibition by human plasma alpha 2-macroglobulin (alpha 2M). Both 50- and 95 kDa gingipain R were efficiently inhibited by alpha 2M, whereas the catalytic activity of gingipain K could not be eliminated. All 3 enzymes were, however, inhibited by a homologous macroglobulin from rat plasma, alpha 1-inhibitor-3 (alpha 1I3). alpha-Macroglobulins must be cleaved in the so-called "bait region" in order to inhibit proteinases by a mechanism involving physical entrapment of the enzyme. A comparison of the amino acid sequences of the 2 macroglobulins indicates that the lack of lysyl residues within the bait region of alpha 2M protects Lys-specific proteinases from being trapped. On this basis, other highly specific proteinases might also not be inhibited by alpha 2M, possibly explaining the inability of the inhibitor to control proteolytic activity in some bacterially induced inflammatory states, despite its abundance (2-5 mg/ml) in vascular fluids.

Adhesins, Bacterial↗

PubMed1983

Structure of alpha 2-macroglobulin in solution and its interaction with proteases: an X-ray scattering study using the contrast variation method.

X-Ray scattering study of alpha 2-macroglobulin in solvents of variable electron densities (sucrose in water) shows that alpha 2-macroglobulin obeys the invariant volume hypothesis; thus, the structure of the particle is independent of the sucrose concentration of the solution. The particle structure is quantitatively described by a set of parameters such as the gyration radius, R = 8.0 nm, the volume, V = 1200 nm3, and the maximum distance within the particle, Dmax = 25 nm. The contrast dependence of the gyration radius indicates that in alpha 2-macroglobulin the regions of higher electron density are located closer to the center (core) than the regions of lower electron density. The core, which may be the place occupied by the carbohydrate, has a maximum dimension of 16 nm and it can be described as a flat cylinder. X-Ray scattering titrations indicate that alpha 2-macroglobulin forms a 1:2 complex as the main product with both trypsin and chymotrypsin simultaneously as the particle contracts. The formation of a ternary 1:1:1 complex with trypsin and chymotrypsin and the absence of higher complexes indicate that the sites for these proteases are closely related. This is further substantiated by the p(r) functions which are virtually identical for the 1:1:1 and 1:2 complexes.

Animals↗