Interaction of human rheumatoid synovial collagenase (matrix metalloproteinase 1) and stromelysin (matrix metalloproteinase 3) with human alpha 2-macroglobulin and chicken ovostatin. Binding kinetics and identification of matrix metalloproteinase cleavage sites

The homologous proteinase inhibitors, human alpha 2-macroglobulin (alpha 2M) and chicken ovostatin, have been compared with respect to their "bait" region sequences and interactions with two human matrix metalloproteinases, collagenase and stromelysin. A stretch of 34 amino acid residues of the ovostatin bait region sequence was determined and the matrix metalloproteinase cleavage sites identified. Collagenase cleaved a X-Leu bond where X was unidentified, whereas the major cleavage site by stromelysin was at the Gly-Phe bond, 4 residues on the COOH-terminal side of the collagenase cleavage site. Collagenase cleaved the alpha 2M bait region at the Gly679-Leu680 bond, and stromelysin at Gly679-Leu680 and Phe684-Tyr685 bonds. Sequence similarity in the bait region of members of the alpha-macroglobulin family is strikingly low. The kinetic studies indicate that alpha 2M is a 150-fold better substrate for collagenase than type I collagen. Structural predictions based on the bait region sequences suggest that a collagen-like triple helical structure is not a prerequisite for the efficient binding of tissue collagenase to a substrate. The binding of stromelysin to alpha 2M is slower than that of collagenase. Stromelysin reacts with ovostatin even more slowly. Despite the preference of chicken ovostatin for metalloproteinases, human alpha 2M, a far less selective inhibitor, reacts more rapidly with collagenase and stromelysin. These results suggest that alpha 2M may play an important role in regulating the activities of matrix metalloproteinases in the extracellular space.     

Human neutrophil elastase and cathepsin G cleavage sites in the bait region of alpha 2-macroglobulin. Proposed structural limits of the bait region

The sites of cleavage in the "bait region" of human alpha 2-macroglobulin made by both neutrophil elastase and cathepsin G, as the first step in their inactivation by this inhibitor, have been identified. These positions are at a valylhistidyl bond for elastase and a phenylalanyl-tyrosyl bond for cathepsin G. All of the proteinases tested so far, including those utilized in this study, are cleaving within a twenty-seven aminoacid peptide sequence occurring between two proline residues. It is suggested that this area represents the outer limits of the "bait region" loop.     

Inhibition of inflammatory proteinase, medullasin, by alpha 2-macroglobulin and ovomacroglobulin

The in vitro activity of inflammatory proteinase, medullasin, was stoichiometrically inhibited by a serum proteinase inhibitor, alpha 2-macroglobulin, and its homolog, chicken ovomacroglobulin. The two inhibitors were cleaved by medullasin only in the bait region. The effectiveness of alpha 2-macroglobulin to inhibit medullasin in competition with alpha -1-proteinase inhibitor was measured under a simulated in vivo condition and an estimation was made that about 60-70% medullasin is inhibited by alpha-1-inhibitor and 30-40% by alpha 2-macroglobulin.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Internalization of serratial protease into cells as an enzyme-inhibitor complex with alpha 2-macroglobulin and regeneration of protease activity and cytotoxicity

Extracellular serratial protease (56,000 Da) is known to be cytotoxic. Fluorescein isothiocyanate-labeled protease was found to form a complex with human alpha 2-macroglobulin (alpha 2M), and this enzyme-inhibitor complex was purified. The protease was found to be internalized by fibroblasts in culture as a complex with alpha 2M, which resulted in cell destruction. Regeneration of enzyme activity was confirmed in cells after 2-3 h of incubation. Chicken egg-white ovomacroglobulin, a homolog of human alpha 2M, formed a complex with this enzyme similarly and more tightly but failed to exhibit protease activity, cytotoxicity, and internalization into cells.     

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

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

Hen egg white ovomacroglobulin has a protease inhibitory activity

Hen egg white ovomacroglobulin purified by Miller and Feeney without reference to its activity was shown to have a protease inhibitory activity towards trypsin, papain, and thermolysin. It has four subunits of equal molecular weight (175,000 by SDS-PAGE) and each two of which are disulfide bonded. Upon incubation with trypsin it yields a fragment of Mr = 80,000 plus smaller ones. The subunit composition, amino acid composition and a newly found protease inhibitory activity place ovomacroglobulin as a closely related protein to human serum alpha 2-macroglobulin.     

Internal structure of ovomacroglobulin studied by electron microscopy.

As a model for the molecular structure of proteins belonging to the alpha 2-macroglobulin family, ovomacroglobulin of reptilian origin was studied by electron microscopy in the original tetrameric form as well as in the dissociated forms into half- and quarter molecules. The following aspects of the molecular internal structure which had previously not been known for the homologous human alpha 2-macroglobulin or chicken ovomacroglobulin were revealed. First, the negatively stained tetrameric native protein gave an appearance of a collection of four semi-circular strings placed on the four corners of a molecule. They were connected to each other in the center of a molecule through a set of globular domains which formed a cross-figured subunit contact region. Second, two kinds of active half-molecules prepared either by the reduction of intersubunit disulfide bonds or by the disruption of noncovalent subunit interface had similarly elongated forms having semi-circular units on the two ends, indicating quasi-equivalent subunit arrangement in the two kinds of half-molecules. We thus concluded that the structure of native ovomacroglobulin can be represented by four circular strings each equipped with an extra domain to form the central intersubunit contact region. The results may also be adapted to the internal structure of human alpha 2-macroglobulin because it was sometimes possible to observe similar ring-like internal structure in the human protein.     

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.     

Analysis of site-directed mutations in human pro-alpha 2(I) collagen which block cleavage by the C-proteinase

We have used site-directed mutagenesis to obtain human pro alpha 2(I) cDNAs containing novel mutations designed to inhibit cleavage at the C-proteinase site. Deletion of six relatively conserved amino acids which surround the cleavage site did not interfere with assembly of the triple helix in transfected rat cells, but blocked cleavage of the constituent mutated chains by endogenous C-proteinase. Substitution for a conserved Asp, which forms part of the Ala-Asp bond cleaved by C-proteinase, also blocked cleavage by endogenous C-proteinase. The conserved Asp is, therefore, a necessary component of the C-proteinase cleavage site. Incubation in vitro with a purified mouse C-proteinase, confirmed both mutations to be resistant to cleavage by high concentrations of the physiologically relevant enzyme. Mutant pro alpha 2(I) chains, resistant to cleavage by C-proteinase in culture media, were processed in cell layers by a different protease which cleaved telopeptide domains. Naturally occurring mutations at the C-proteinase site have not been described in human patients. The mutations characterized here, further define the C-proteinase cleavage site and provide reagents which may be informative when introduced into transgenic mice.     

Recognition of nucleophile-treated alpha 2-macroglobulin by the alveolar macrophage alpha-macroglobulin . protease complex receptor

Rabbit alveolar macrophages exhibit high affinity surface receptors which recognize alpha 2-macroglobulin . protease complexes but not native alpha 2- macroglobulin. Binding of alpha 2-macroglobulin . protease complexes to surface receptors is independent of the protease used to form the complex. In this communication, we demonstrate that treatment of human alpha 2-macroglobulin with nucleophilic agents (methyl amine, ammonium salts) converts native alpha 2-macroglobulin into a form recognized by the surface receptor for alpha 2-macroglobulin protease complexes. Analysis of the concentration dependency of ligand binding revealed that the surface receptor did not distinguish between nucleophile-treated alpha 2-macroglobulin and alpha 2-macroglobulin . protease complexes. These results are consistent with the hypothesis that proteases or nucleophilic agents effect the hydrolysis of an internal thiol-ester bond (Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., and Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 5764-5768), leading to an alteration in alpha 2-macroglobulin conformation. The altered conformation results in recognition of the alpha 2-macroglobulin by surface receptors.     

New member of the trefoil factor family of proteins is an alpha-macroglobulin protease inhibitor

The amino acid sequence of the monomeric alpha-macroglobulin (alphaM) from the American bullfrog, Rana catesbiana, was determined. The mature protein consisted of 1469 amino acid residues and shared sequence identity with other members of the alphaM family of protein. The central portion of the frog monomeric alphaM contained Cys residues positioned analogously to the Cys residues in human alpha(2)-macroglobulin (alpha(2)M), known to be involved in disulfide bridges. Additionally, the frog monomeric alphaM contained six Cys residues in a approximately 60 residue COOH-terminal extension not present in previously characterized alphaMs. The spacing of the Cys residues and the overall sequence identity of this COOH-terminal extension were consistent with a trefoil motif. This is the first time a member of the trefoil factor family has been identified in the circulatory system. The "bait region" was located between Arg(675)-Lys(685) and contained mainly basic amino acid residues. The COOH-terminal receptor-binding domain was not exposed prior to proteolysis of this highly susceptible region. The proximity of the receptor-binding and trefoil domains implied that the trefoil domain is similarly concealed before bait region cleavage.     

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin

Human α₂-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M’s unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible “bait region.” As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the “tabula rasa” bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.     

Interaction of hemorrhagic metalloproteinases with human alpha 2-macroglobulin.

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

IgA1 proteases from Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitidis, and Streptococcus sanguis: comparative immunochemical studies

IgA1 proteases from H. influenzae, N. meningitidis, S. pneumoniae, and S. sanguis were compared with respect to site of cleavage in the IgA1 molecule and EDTA sensitivity. Proteases from S. sanguis and S. pneumoniae cleaved the Pro (227)-Thr (228) bond within the hinge region of the alpha 1 chain and were inhibited by EDTA. H. influenzae IgA1 protease cleaved the Pro (231)-Ser (232) peptide bond. The activity of IgA1 proteases from H. influenzae and N. meningitidis was unaffected by EDTA. Purified and denatured alpha 1 chain was cleaved only in the hinge region. Other component chains of secretory IgA (secretory component, light and J chains) were not susceptible. In addition to IgA1 protease, S. pneumoniae released exo- and endoglycosidases that removed a considerable portion of carbohydrate side chains of IgA1; this activity was absent from crude IgA1 protease preparations of the other three bacterial species. Association in vitro of polymeric IgA1 with SC did not inhibit the degradation of IgA1 proteases. The considerable resistance of secretory IgA to cleavage by IgA1 proteases may be explained in part by the presence of IgA1 protease-neutralizing antibodies in secretory IgA.     

Subunits of human alpha 2-macroglobulin produced by specific reduction of interchain disulfide bonds with thioredoxin

Disulfide bonds in alpha 2-macroglobulin (alpha 2M) were reduced with the thioredoxin system from Escherichia coli. Under the conditions selected, 3.5-4.1 disulfide bonds were cleaved in each alpha 2M molecule, as determined by the consumption of NADPH during the reaction and by the incorporation of iodo[3H]acetate into the reaction product. This extent of disulfide bond reduction, approximately corresponding to that expected from specific cleavage of all four interchain disulfide bonds of the protein, coincided with the nearly complete dissociation of the intact alpha 2M molecule to a species migrating as an alpha 2M subunit in gel electrophoresis, under both denaturing and nondenaturing conditions. The dissociation was accompanied by only small changes of the spectroscopic properties of the subunits, which thus retain a near-native conformation. Reaction of isolated subunits with methylamine or trypsin led to the appearance of approximately 0.55 mol of thiol group/mol of subunits, indicating that the thio ester bonds are largely intact. Moreover, the rate of cleavage of these bonds by methylamine was similar to that in the whole alpha 2M molecule. Although the bait region was specifically cleaved by nonstoichiometric amounts of trypsin, the isolated subunits had minimal proteinase binding ability. Reaction of subunits with methylamine or trypsin produced changes of farultraviolet circular dichroism and near-ultraviolet absorption similar to those induced in the whole alpha 2M molecule, although in contrast with whole alpha 2M no fluorescence change was observed. The methylamine- or trypsin-treated subunits reassociated to a tetrameric species, migrating as the "fast" form of whole alpha 2M in gradient gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of alpha 2-macroglobulin-plasmin complexes: complete subunit cleavage alters receptor recognition in vivo and in vitro

When human alpha 2-macroglobulin (alpha 2M) binds proteinases, it undergoes subunit cleavage. Binding of small proteinases such as trypsin results in proteolysis of each of the four subunits of the inhibitor. By contrast, previous studies suggest that reaction of plasmin with alpha 2M results in cleavage of only two or three of the inhibitor subunits. In this paper, we demonstrate that the extent of subunit cleavage of alpha 2M is a function of plasmin concentration. When alpha 2M was incubated with a 2.5-fold excess of plasmin, half of the subunits were cleaved; however, at a 20-fold enzyme to inhibitor ratio, greater than 90% of the subunits were cleaved with no additional plasmin binding. This increased cleavage was catalyzed by free rather than bound plasmin. It is concluded that this "nonproductive" subunit cleavage is dependent upon the molar ratio of proteinase to inhibitor. The consequence of complete subunit cleavage on receptor recognition of alpha 2M-plasmin (alpha 2M-Pm) complexes was studied. Preparations of alpha 2M-Pm with only two cleaved subunits bound to the murine macrophage receptor with a Kd of 0.4 nM and 60 fmol of bound complex/mg of cell protein. When preparations of alpha 2-M-Pm with four cleaved subunits were studied, the Kd was unaltered but ligand binding increased to 140 fmol/mg of cell protein. The receptor binding behavior of the latter preparation is equivalent to that observed when alpha 2M is treated with small proteinases such as trypsin. This study suggests that receptor recognition site exposure is not complete in the alpha 2M-Pm complex with half of the subunits cleaved. Proteolytic cleavage of the remaining subunits of the inhibitor results in a further conformational change exposing the remaining receptor recognition sites.     

alpha2-Macroglobulin from hemolymph of the freshwater crayfish Astacus astacus.

1. A high mol. wt proteinase inhibitor has been purified from the haemolymph of the freshwater crayfish Astacus astacus. 2. The protein is a disulphide-bonded dimer (Mr 390,000) of two identical polypeptide chains (Mr 185,000). 3. The inhibitor displays a broad specificity and protects trypsin from inhibition by soybean trypsin inhibitor and thus is similar to vertebrate alpha 2-macroglobulin. 4. The alpha 2-macroglobulin-like inhibitor from Astacus interacts with bovine trypsin in an equimolar stoichiometry thereby decreasing tryptic hydrolysis of N-benzoyl-L-arginine-ethylester to 50% residual activity. In contrast, the activity of Astacus protease, a digestive zinc proteinase from crayfish toward succinyl-alanyl-alanyl-alanyl-4-nitroanilide is inhibited almost completely. 5. Sensitivity of the inhibitor to methylamine and autolytic cleavage suggests the presence of an internal thioester bond. 6. The N-terminal amino acid sequence of Astacus alpha 2-macroglobulin is strongly related to the alpha 2-macroglobulins from Pacifastacus leniusculus (91% identity) and from the lobster Homarus americanus (72% identity). In contrast, only 25% of the residues are identical with the alpha 2-macroglobulin from the horseshoe crab Limulus polyphemus. There is also a faint similarity to human complement protein C3 and human alpha 2-macroglobulin.     

A functional, thioester-containing alpha 2-macroglobulin homologue isolated from the hemolymph of the American lobster (Homarus americanus)

An alpha 2-macroglobulin-like protease inhibitor was isolated from the cell-free hemolymph of the american lobster (Homarus americanus) by ion-exchange chromatography and gel filtration. Whereas the undissociated molecule has a molecular weight of 342,000 as determined by ultracentrifugation studies, under reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a subunit molecular weight of 180,000. On the basis of this and other evidence, we conclude that the lobster protein is a dimer consisting of two disulfide-bonded monomers. The purified protein inhibits proteolytic enzymes but protects the esterolytic activity of trypsin toward low molecular weight substrates from inactivation by soybean trypsin inhibitor. The methylamine sensitivity of this activity suggests the presence of an internal thioester bond. This was confirmed by the covalent incorporation of [14C]methylamine, by the formation of Mr 55,000 and 125,000 autolytic cleavage fragments in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and, more directly, by the amino acid sequence of a tryptic peptide containing the putative thioester region. Whereas the N-terminal amino acid sequence (22 residues) of the protein revealed an overall identity of only 18% when compared with the human protein, the sequence of the thioester-containing peptide was highly conserved, both with respect to human alpha 2-macroglobulin and to other proteins having a thioester bond. The protein showed the "slow to fast" conformational change typical in alpha 2-macroglobulins in nondenaturing gel electrophoresis after treatment with trypsin, but not after incubation with methylamine.