alpha(2)-macroglobulin and alpha(1)-inhibitor-3 mRNA expression in the rat liver after slow interleukin-1 stimulation

In this study we have investigated total fiver RNA and the expression of mRNA in the rat fiver in vivo after a slow stimulation of interleukin-1. A total dose of 4 mug interleukin-1beta was administered via a subcutaneously implanted osmotic minipump over a period of 7 days. Plasma concentrations of alpha(2)-macroglobulin manifested a rapid increase, reaching a peak on day 2, while alpha(1)-inhibitor-3 manifested a marked initial decrease to 50% of the baseline level, followed by a tendency to increase again. For measurement of total RNA and specific mRNAs from the fiver, rats were sacrificed at different times during the experimental period. Total RNA peaked at 6 h, the level being approximately 60% higher than baseline value. Specific mRNA from the liver for alpha(2)-macroglobulin and alpha(1)-inhibitor-3 were quantified using laser densitometry on slot blots. The amounts measured during the experimental period agreed with the pattern of corresponding plasma protein levels. From barely detectable amounts at baseline, alpha(2)-macroglobulin mRNA peaked on day 1, and then declined. Levels of alpha(1)-inhibitor-3 mRNA manifested an initial increase at 3 h, but then declined and remained low until day 5 when there was a tendency towards an increase. It was concluded that the levels of plasma concentrations of alpha(2)-macroglobulin and alpha(1)-inhibitor-3 are mainly regulated at the protein synthesis level, and that long-term interleukin-1beta release could not override the initial acute phase protein counteracting mechanism triggered.     

Isolation and purification of rat acute-phase alpha 2-macroglobulin

Acute-phase alpha 2-macroglobulin was highly purified from the serum of rats in which this protein had been induced 48 h previously by the injection of croton oil, an inflammatory agent. The isolation protocol involved two non-denaturing steps; first, separation according to molecular weight by gel filtration on Ultrogel AcA 22 and second, negative affinity chromatography which bound contaminating proteins to the column while allowing acute-phase alpha 2-macroglobulin to pass through. Several criteria were used to assess the purity of acute-phase alpha 2-macroglobulin, after which the protein by mass determination and by two different protein assays. Pure rat acute-phase alpha 2-macroglobulin was used to produce a monospecific antiserum and to calibrate a secondary standard of rat acute-phase serum by developing and characterizing rocket immunoelectrophoresis assay.     

Limited proteolysis of the alpha-macroglobulin rat alpha 1-inhibitor-3. Implications for a domain structure.

Rat alpha 1-inhibitor-3 is a 180-kDa monomeric proteinase inhibitor found in high concentration in rat plasma. By several criteria it has been shown to be a member of the family of alpha-macroglobulin proteinase inhibitors often exemplified by the tetrameric human alpha 2-macroglobulin. We have used limited proteolysis of rat alpha 1-inhibitor-3 to probe the domain structure of this family of proteins. Proteinases of different specificities, including trypsin, chymotrypsin, thermolysin, and Staphylococcus aureus V8 proteinase, were employed and a common fragmentation pattern was observed when the reaction products were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These fragments were electrotransferred to polyvinylidene difluoride membranes and subjected to NH2-terminal amino acid sequence analysis in order to position them within the context of the primary structure. The fragmentation pattern may define the domain structure of alpha 1-inhibitor-3 and serve as a model for the domain organization of the family of alpha-macroglobulin proteinase inhibitors.     

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.     

Biosynthesis of rat alpha 1-macroglobulin. Identification of an intracellular precursor

Alpha 1-macroglobulin was purified from rat plasma by gel filtration (Sephacryl S-300) and ion exchange chromatography (DE52). Analysis of the purified alpha 1-macroglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two polypeptides: a light chain which could be resolved into a double band (36/38 kDa) and a heavy chain (160 kDa). Under non-reducing conditions complexes of 200 and 400 kDa could be demonstrated. Antibodies were raised against both chains of alpha 1-macroglobulin which did not cross-react with either rat alpha 2-macroglobulin or rat alpha 1-inhibitor 3. It was shown that in the medium of [35S]methionine-labeled hepatocytes the two subunits of alpha 1-macroglobulin are linked by disulfide bridges. Intracellularly, however, a high molecular mass polypeptide (185 kDa) could be immunoprecipitated with either the antiserum to the heavy or the light chain of alpha 1-macroglobulin, indicating the existence of a polyprotein precursor. Also in a cell-free translation system alpha 1-macroglobulin was synthesized as a polyprotein consisting of heavy and light chains (162 kDa). In a pulse-chase experiment using tunicamycin to block N-glycosylation, alpha 1-macroglobulin secretion was totally inhibited. This finding reflects the importance of the oligosaccharide side chains for the proteolytic processing to the two subunits and/or secretion of alpha 1-macroglobulin.     

Proteinase binding and inhibition by the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3

The inhibitory capacity of the alpha-macroglobulins resides in their ability to entrap proteinase molecules and thereby hinder the access of high molecular weight substrates to the proteinase active site. This ability is thought to require at least two alpha-macroglobulin subunits, yet the monomeric alpha-macroglobulin rat alpha 1-inhibitor-3 (alpha 1I3) also inhibits proteinases. We have compared the inhibitory activity of alpha 1I3 with the tetrameric human homolog alpha 2-macroglobulin (alpha 2M), the best known alpha-macroglobulin, in order to determine whether these inhibitors share a common mechanism. alpha 1I3, like human alpha 2M, prevented a wide variety of proteinases from hydrolyzing a high molecular weight substrate but allowed hydrolysis of small substrates. In contrast to human alpha 2M, however, the binding and inhibition of proteinases was dependent on the ability of alpha 1I3 to form covalent cross-links to proteinase lysine residues. Low concentrations of proteinase caused a small amount of dimerization of alpha 1I3, but no difference in inhibition or receptor binding was detected between purified dimers or monomers. Kininogen domains of 22 and 64 kDa were allowed to react with alpha 1I3- or alpha 2M-bound papain to probe the accessibility of the active site of this proteinase. alpha 2M-bound papain was completely protected from reaction with these domains, whereas alpha 1I3-bound papain reacted with them but with affinities several times weaker than uncomplexed papain. Cathepsin G and papain antisera reacted very poorly with the enzymes when they were bound by alpha 1I3, but the protection provided by human alpha 2M was slightly better than the protection offered by the monomeric rat alpha 1I3. Our data indicate that the inhibitory unit of alpha 1I3 is a monomer and that this protein, like the multimeric alpha-macroglobulins, inhibits proteinases by steric hindrance. However, binding of proteinases by alpha 1I3 is dependent on covalent crosslinks, and bound proteinases are more accessible, and therefore less well inhibited, than when bound by the tetrameric homolog alpha 2M. Oligomerization of alpha-macroglobulin subunits during the evolution of this protein family has seemingly resulted in a more efficient inhibitor, and we speculate that alpha 1I3 is analogous to an evolutionary precursor of the tetrameric members of the family exemplified by human alpha 2M.     

Isolation of active subforms of alpha 2-macroglobulin.

Anion-exchange chromatography is shown to permit resolution and separation of subforms of the serum glycoprotein alpha 2-macroglobulin. The subforms differ dramatically in their stability as judged by differential scanning calorimetry, undergoing thermally induced unfolding at temperatures of 61 and 69 degrees C respectively. In addition, the proteinase-binding stoichiometry of the subforms differs by a factor of 2, with the more- and less-stable forms binding 2 and 1 mol of proteinase per mol of tetramer respectively. The calorimetric stability of the two forms is differentially affected on treatment with neuraminidase, suggesting that the nature of glycosylation may in part account for the observed differences in physical and functional properties.     

Subunit and primary structure of a mouse alpha-macroglobulin, a human alpha 2-macroglobulin homologue

A mouse alpha-macroglobulin (AMG), a homologue of human alpha 2-macroglobulin (alpha 2 M), has been purified to homogeneity. In contrast to human and acute-phase rat alpha 2 M which contains subunits of about Mr 190 000, the mouse protein contains two major (Mr 163000 and 35000) and one minor (Mr 185000) subunits. Also unlike human alpha 2 M, which can be broken down into about 85000-dalton subunits when reacted with an endopeptidase, the native AMG is cleaved by trypsin into multiple components (Mr 86000, 63000, 61000 and 33000). Two-dimensional peptide map analysis of these various 125I-labeled subunit components reveals that the 185000- and 163000-dalton components are homologous proteins but only the 185000-dalton protein contains the 35000-dalton component. The 163000-dalton protein is cleaved by trypsin into 86000- and 63000-dalton components, and the 86-kDa component in turn can be broken down into 61000- and 33000-dalton fragments. Since the 35000-dalton component is serologically related to AMG but does not share any tryptic peptides with both the 163000- and 33000-dalton components, it is neither a copurified impurity nor a cleavage product of the major (163000-dalton) subunit. AMG, therefore, is composed of covalently linked subunits of Mr 163000 and 35000, and the 185000-dalton protein may be a variant subunit of AMG. Trypsin treatment of the [14C]methylamine-labeled AMG and alpha 2 M also sequentially generate subunit patterns indistinguishable from those of the unlabeled macroglobulins. The methylamine-sensitive site(s) of AMG is localized in the 63000-dalton peptide, which is rather resistant to trypsin digestion and to staining by Coomassie brillant blue. We conclude from this study that the mouse homologue has a subunit composition and primary structure distinctly different from those of human and rat alpha 2 M.     

Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

Binding of receptor-recognized forms of tetrameric human α₂-macroglobulin (α₂M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP₃) synthesis, and a concomitant rise in cytosolic free calcium ([Ca²⁺]_i). The α₂M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of α₂M* also occurs to the low density lipoprotein receptor-related protein/α₂M receptor (LRP/α₂MR), but this binding does not induce signal transduction. Rat α₁-inhibitor-3 (α₁I₃) is a monomeric member of the α-macroglobulin/complement superfamily. Like α₂M, it can react with proteinases or methylamine which induces a conformational change causing activated α₁I₃ to bind to LRP/α₂MR. We now report that α₁I₃-methylamine binds to the macrophage α₂M* signaling receptor inducing a rapid rise in the synthesis of IP₃ with a subsequent 1.5- to 3-fold rise in [Ca²⁺]_i. α₁I₃-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native α₁I₃, like α₂M, was unable to induce signal transduction. α₁I₃ forms a complex with α₁-microglobulin, which has a distinct conformation from α₁I₃ and is recognized by LRP/α₂MR. This complex also induces an increase in [Ca²⁺]_i comparable to the effect of α₁I₃-methylamine on macrophages. It is concluded that activation of α₁I₃ by methylamine or binding of α₁-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the α₂M* signaling receptor, as well as for LRP/α₂MR. © 1996 Wiley-Liss, Inc.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

Inhibition of beta2-microglobulin amyloid fibril formation by alpha2-macroglobulin

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α(2)-macroglobulin (α2M), an extracellular chaperone, forms a complex with β(2)-microglobulin (β2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the β2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and β2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured β2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with β2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native β2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured β2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling β2-m amyloid fibril formation.     

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

Primary structure of alpha 2-macroglobulin receptor-associated protein. Human homologue of a Heymann nephritis antigen.

The alpha 2-macroglobulin (alpha 2M) receptor complex as purified by affinity chromatography contains three polypeptides: a 515-kDa heavy chain, an 85-kDa light chain, and a 39-kDa associated protein. Previous studies have established that the 515/85-kDa components are derived from a 600-kDa precursor whose complete sequence has been determined by cDNA cloning (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gassepohl, H., and Stanley, K. (1988) EMBO J. 7,4119-4127). We have now determined the primary structure of the human 39-kDa polypeptide, termed alpha 2M receptor-associated protein, by cDNA cloning. The deduced amino acid sequence contains a putative signal sequence that precedes the 323-residue mature protein. Comparative sequence analysis revealed that alpha 2M receptor-associated protein has 73% identity with a rat protein reported to be a pathogenic domain of Heymann nephritis antigen gp 330 and 77% identity to a mouse heparin-binding protein termed HBP-44. The high overall identity suggests that these molecules are interspecies homologues and indicates that the pathogenic domain, previously thought to be a portion of gp 330, is in fact a distinct protein. Further, the 120-residue carboxyl-terminal region of alpha 2M receptor-associated protein has 26% identity with a region of apolipoprotein E containing the low density lipoprotein receptor binding domain. Pulse-chase experiments revealed that the newly formed alpha 2M receptor-associated protein remains cell-associated, while surface labeling experiments followed by immunoprecipitation suggest that this protein is present on the cell surface forming a complex with the alpha 2M receptor heavy and light chains.