The alpha-macroglobulin bait region. Sequence diversity and localization of cleavage sites for proteinases in five mammalian alpha-macroglobulins

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

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

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

A conserved region in alpha-macroglobulins participates in binding to the mammalian alpha-macroglobulin receptor

Efforts to characterize the receptor recognition domain of alpha-macroglobulins have primarily focused on human alpha 2-macroglobulin (alpha 2M). In the present work, the structure and function of the alpha-macroglobulin receptor recognition site were investigated by amino acid sequence analysis, plasma clearance, and cell binding studies using several nonhuman alpha-macroglobulins: bovine alpha 2M, rat alpha 1-macroglobulin (alpha 1M), rat alpha 1-inhibitor 3 (alpha 1I3), and proteolytic fragments derived from these proteins. Each alpha-macroglobulin bound to the murine peritoneal macrophage alpha-macroglobulin receptor with comparable affinity (Kd approximately 1 nM). A carboxyl-terminal 20-kDa fragment was isolated from each of these proteins, and this fragment bound to alpha-macroglobulin receptors with Kd values ranging from 10 to 125 nM. The amino acid identity between the homologous carboxyl-terminal 20-kDa fragments of human and bovine alpha 2M was approximately 90%, while the overall sequence homology between all carboxyl-terminal fragments studied was 75%. The interchain disulfide bond present in the human alpha 2M carboxyl-terminal 20-kDa fragment was conserved in bovine alpha 2M and rat alpha 1I3, but not in rat alpha 1M. The clearance of each intact alpha-macroglobulin-proteinase complex was significantly retarded following treatment with cis-dichlorodiammineplatinum(II) (cis-DDP). cis-DDP treatment, however, did not affect receptor recognition of purified carboxyl-terminal 20-kDa fragments of these alpha-macroglobulins. A carboxyl-terminal 40-kDa subunit, which can be isolated from rat alpha 1M, bound to the murine alpha-macroglobulin receptor with a Kd of 5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Human fibroblast collagenase-alpha-macroglobulin interactions. Localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins

The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin, and rat alpha 1-inhibitor 3) differing in primary and quaternary structure has been investigated. Complex formation with each of these alpha-macroglobulins follows the course identified for many other proteinases, i.e. specific limited proteolysis in their bait regions inducing a set of conformational changes resulting in activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters and covalent complex formation. At collagenase: alpha-macroglobulin molar ratios of less than 1:1 3.2-3.6 mol of SH groups appear for 1 mol of collagenase bound to human and rat alpha 2-macroglobulin and to rat alpha 1-macroglobulin. For these alpha-macroglobulins it can be estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1 s-1 while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3. More than 95% of the complexed collagenase is covalently bound, and sodium dodecyl sulfate gel electrophoresis shows the typical pattern of bands corresponding to reaction products of very high apparent molecular weight. The same pattern is also seen in the covalent (greater than 98%) complex very slowly formed from Clostridium histolyticum collagenase and human alpha 2-macroglobulin. The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens. These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin, as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues. In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase. It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.     

Sequence and acute phase regulation of rat alpha 1-inhibitor III messenger RNA

cDNA clones coding for the plasma proteinase inhibitor alpha 1-inhibitor III were isolated from an acute phase rat liver library. The isolates could be divided into four groups with characteristic BamHI restriction fragment patterns. The identity of the prototype clone pRLA1I3/2J was established by comparison with the published amino acid sequence of the purified protein. It codes for a 1477-amino acid precursor polypeptide with a 24-residue signal peptide. The mature protein shares 58% overall sequence identity with rat alpha 2-macroglobulin and contains a typical internal thiolester sequence. Twenty-two of its twenty-three cysteinyl residues are conserved with alpha 2-macroglobulin implying similar tertiary structure. However, the prototype alpha 1-inhibitor III sequence differed significantly from the rat and human alpha 2-macroglobulin sequences in its bait region suggesting alpha 1-inhibitor III possesses proteinase inhibitory specificities different from those of alpha 2-macroglobulin. The variant alpha 1-inhibitor III clone pRLA1I3/2J from a second cDNA group also differed from the prototype in the bait region coding sequence, although both specify similar signal peptides and NH2 termini. The observation of variant cDNA classes suggests that acute phase rat livers produce a heterogeneous mixture of alpha 1-inhibitor III mRNA molecules. Evidence was obtained for the presence of at least four different alpha 1-inhibitor III-related genes in the rat genome. During the first 24 h of an acute phase response the abundance of hepatic alpha 1-inhibitor III mRNA was decreased 3-4-fold. This decrease was of the same order of magnitude as the reported reduction of the corresponding plasma protein concentration, suggesting that in the early phase of the acute inflammatory response the plasma concentration of this protein is mainly controlled through the abundance of its hepatic mRNA.     

Alpha-macroglobulin from Limulus polyphemus exhibits proteinase inhibitory activity and participates in a hemolytic system.

Significant primary sequence homology between the alpha-macroglobulin family of proteinase inhibitors and the complement components C3, C4, and C5 implies that these proteins arose from a common ancestor. Hemolymph from the ancient invertebrate Limulus polyphemus contains both complement-like and proteinase inhibitory activity. In this report, we present evidence that L. polyphemus alpha-macroglobulin not only possesses proteinase inhibitory activity, but it also participates in the lytic system of the horseshoe crab. The protein is a disulfide-linked dimer of subunits of molecular mass 185 kDa. Upon reaction with proteinase or methylamine, L. polyphemus alpha-macroglobulin underwent a major conformational change and no proteinase-associated multimerization was detected. L. polyphemus alpha-macroglobulin is the only detectable inhibitor of a number of proteinases in L. polyphemus hemolymph. Proteinase inhibition follows the general "trapping" mechanism shared by most alpha-macroglobulins; however, no covalent linking of proteinases to the inhibitor was detected despite the presence of a functional thiolester. Moreover, the inhibitor demonstrated thiolester-mediated binding to sheep erythrocytes, a property also observed with complement components such as C3. Depletion of functional protein by treatment of hemolymph with methylamine destroyed the proteinase inhibitory capacity and the lytic activity of the hemolymph. Both activities were restored by adding purified protein to depleted hemolymph. Studies with purified L. polyphemus alpha-macroglobulin demonstrated that the thiolester incorporates glycerol as well as methylamine, a property shared by human C3. The data support the hypothesis that L. polyphemus alpha-macroglobulin is both a proteinase inhibitor and part of a lytic system, providing a link between the two distinct sides of the alpha-macroglobulin family. Because both properties are contained in one molecule, we propose the name "limac" to describe this Limulus alpha-macroglobulin complement-like protein.     

Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases

Human plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1-inhibitor, and alpha 2-antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 1-antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino-terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy-terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis--one near the amino-terminal end and another in the exposed reactive site loop of the inhibitor.     

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.     

Structure of a rat alpha 1-macroglobulin receptor-binding domain dimer

Alpha-macroglobulin inhibits a broad spectrum of proteinases by forming macromolecular cages inside which proteinases are cross-linked and trapped. Upon formation of a complex with proteinase, alpha-macroglobulin undergoes a large conformational change that results in the exposure of its receptor-binding domain (RBD). Engagement of this domain by alpha-macroglobulin receptor permits clearance of the alpha-macroglobulin: proteinase complex from circulation. The crystal structure of rat alpha1-macroglobulin RBD has been determined at 2.3 A resolution. The RBD is composed of a nine-stranded beta-sandwich and a single alpha-helix that has been implicated as part of the receptor binding site and that lies on the surface of the beta-sandwich. The crystallographic asymmetric unit contains a dimer of RBDs related by approximate twofold symmetry such that the putative receptor recognition sites of the two monomers are contiguous. By gel filtration and ultracentrifugation, it is shown that RBD dimers form in solution with a dissociation constant of approximately 50 microM. The structure of the RBD dimer might mimic a conformation of transformed alpha-macroglobulin in which the proposed receptor binding residues are exposed on one face of the dimer. A pair of phenylalanine residues replaces a cystine that is conserved in other members of the macroglobulin family. These residues participate in a network of aromatic side-chain interactions that appears to stabilize the dimer interface.     

Chymotrypsin inhibitory activity of normal C1-inhibitor and a P1 Arg to His mutant: evidence for the presence of overlapping reactive centers.

C1-inhibitor is a serine proteinase inhibitor that is active against C1s, C1r, kallikrein, and factor XII. Recently, it has been shown that it also has inhibitory activity against chymotrypsin. We have investigated this activity of normal human C1-inhibitor, normal rabbit C1-inhibitor, and P1 Arg to His mutant human C1-inhibitors and find that all are able to inhibit chymotrypsin and form stable sodium dodecyl sulfate-resistant complexes. The Kass values show that the P1 His mutant is a slightly better inhibitor of chymotrypsin than normal human C1-inhibitor (3.4 x 10(4) compared with 7.3 x 10(3)). The carboxy-terminal peptide of normal human C1-inhibitor, derived from the dissociated protease-inhibitor complex, shows cleavage between the P2 and P1 residues. Therefore, as with alpha 2-antiplasmin, C1-inhibitor possesses two overlapping P1 residues, one for chymotrypsin and the other for Arg-specific proteinases. In contrast, with the P1 His mutant, the peptide generated from the dissociation of its complex with chymotrypsin demonstrated cleavage between the P1 and P'1 residues. Therefore, unlike alpha 2-antiplasmin, chymotrypsin utilizes the P2 residue as its reactive site in normal C1-inhibitor but utilizes the P1 residue as its reactive site in the P1 His mutant protein. This suggests that the reactive center loop allows a degree of induced fit and therefore must be relatively flexible.     

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.     

Engineered rat insulin I analogue having a B16 Tyr/Asp replacement exhibits unchanged susceptibility to cleavage by insulin proteinase

An analogue of rat insulin I was produced by oligonucleotide-directed mutagenesis of a cloned rat preproinsulin I cDNA, followed by expression of a resulting mutant gene in Escherichia coli K-12 and proteolytic cleavage of mutant proinsulin isolated from this bacterium. The Tyr-to-Asp replacement at residue B16 in the insulin analogue had been expected to diminish the rate of cleavage of the molecule by the enzyme insulin proteinase, since the bond TyrB16-LeuB17, invariant in all mammalian species, had been proposed by other authors as one of the early, major sites of proteolytic attack. In the event the substitution had no measurable effect on the rate of degradation by insulin proteinase. Thus we find no support in these experiments for the hypothesis that the site in question is of primary importance in the degradation of rat insulin I by the enzyme.     

alpha-Macroglobulins are present in some gram-negative bacteria: characterization of the alpha2-macroglobulin from Escherichia coli

alpha-Macroglobulins (alphaMs) are large glycoproteins that have been identified in a wide range of vertebrate and invertebrate species and are mostly thiol ester containing proteinase inhibitors. A recent analysis of bacterial genomes ( Budd, A., Blandin, S., Levashina, E. A., and Gibson, T. J. (2004) Genome Biol. 5, R38 ) identified many alpha-macroglobulin-like sequences that appear to have been acquired by Gram-negative bacteria from their metazoan hosts. We report the first expression and characterization of such a bacterial alpha-macroglobulin, that from Escherichia coli. This is also the first alpha-macroglobulin to be characterized that is predicted to be membrane-anchored. We found that the 183-kDa protein contains an intact thiol ester, is monomeric, and is localized to the periplasmic space. Reaction with proteinase results in limited cleavage within a bait region, rapid activation of the thiol ester, cross-linking to the attacking proteinase or other available nucleophiles, and partial protection of the proteinase against macromolecular substrates. Given these properties and the co-occurrence of the alphaM gene with one for a repair transglycosylase, this suggests a possible role for bacterial alphaMs in cell defense following host attack. Such a role would make bacterial alphaMs appropriate novel targets for antibiotic drugs.     

ATP-activated, high-molecular-mass proteinase-I from rat skeletal muscle is a cysteine proteinase-alpha 1-macroglobulin complex

From rat skeletal muscle tissue we have isolated and purified a proteolytic activity of molecular mass 750 kDa. The enzyme, designated 'proteinase I', which has been found to be located in capillaries of skeletal muscle tissue, catalyzes the hydrolysis of Z-Phe-Arg-MCA and [14C]methylcasein and this process is activated about 2-fold by ATP. As judged by SDS-polyacrylamide gel electrophoresis the subunit pattern of 'proteinase I' is similar to alpha-macroglobulin. Immunoelectrophoretic analyses of 'proteinase I' with antisera to rat alpha 1-macroglobulin, alpha 2-macroglobulin, and rat liver cathepsins reveal that this high-molecular-mass proteinase is a complex of alpha 1-macroglobulin and the cysteine proteinases cathepsin B, H and L. A similar 'proteinase' has been isolated from rat serum. Two ATP-activated high molecular-mass proteinases that have been previously identified in liver and heart muscle by other investigators equally show a positive immunological reaction with the antiserum raised against 'proteinase I'. From these data, together with results presented in an accompanying paper (Kuehn, L., Dahlmann, B., Gauthier, F. and Neubauer, H.-P. (1989) Biochim. Biophys. Acta 991, 263), we conclude that the ATP-stimulated high-molecular-mass proteolytic activity is partly due to the presence of a complex of alpha-macroglobulin and cysteine proteinases.     

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.     

Proteinase inhibitory activity in the plasma of a mollusc: evidence for the presence of alpha-macroglobulin in Biomphalaria glabrata.

1. A methylamine-sensitive inhibitor was present in the plasma of B. glabrata. 2. This inhibitor decreased trypsin activity against a protein substrate, however trypsin retained activity against a low molecular weight substrate in the presence of the inhibitor. 3. Snail plasma protected trypsin from inhibition by soybean trypsin inhibitor. 4. The results give evidence for an alpha-macroglobulin proteinase inhibitor in the plasma of this gastropod mollusc.     

Isolation of rat serum alpha 1-microglobulin. Identification of a complex with alpha 1-inhibitor-3, a rat alpha 2-macroglobulin homologue.

Alpha 1-Microglobulin (alpha 1-m), or protein HC, a low molecular weight plasma protein with immunoregulatory properties, was isolated from rat serum by affinity chromatography using Sepharose-coupled monoclonal anti-alpha 1-m antibodies. High molecular weight forms of alpha 1-m were then separated from the low molecular weight alpha 1-m by gel chromatography of the eluted proteins. The apparent Mr (28,000), the charge heterogeneity, the N-linked carbohydrate, and yellow-brown chromophore suggest that the low molecular weight alpha 1-m is the serum counterpart to urinary alpha 1-m, which was purified previously. A high molecular weight complex of alpha 1-m was also isolated by the gel chromatography. It was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. The molecule was bound by antibodies against human alpha 2-macroglobulin, and experiments with antisera against the three alpha-macroglobulin variants in rat serum, alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor-3 (alpha 1I3) suggested that alpha 1I3 was the complex-partner of alpha 1-m. An antiserum raised against high molecular weight alpha 1-m was then used to isolate the complex-partner of alpha 1-m from rat serum with affinity chromatography, and this molecule was positively identified as alpha 1I3 by its physicochemical properties. Gel chromatography of the alpha 1I3.alpha 1-m complex suggested a molecule with an Mr of 266,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, it migrated as three major molecular species with apparent molecular weights of 224,000, 205,000, and 194,000 and several minor species of both higher and lower molecular weights, suggesting a complex subunit structure. alpha 1-m and alpha 1I3 could be detected in all three major species by Western blotting, and NH2-terminal amino acid sequencing suggested a molar ratio of 1:1 of alpha 1-m and alpha 1I3 in all three species. alpha 1I3.alpha 1-m was colorless, did not show light absorbance beyond 300 nm which is typical of low molecular weight alpha 1-m and was electrophoretically homogeneous, suggesting that it lacks the chromophore. Finally, the serum concentrations of the alpha 1I3.alpha 1-m complex and free alpha 1-m were determined as 0.16 and 0.010 g/liter, respectively. Thus, alpha 1I3.alpha 1-m constitutes 1-3% of the total alpha 1I3 in rat serum (w/w) and approximately 60% of the total alpha 1-m.