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.     

Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction

A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.     

NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.

NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.     

Characterization of human pregnancy zone protein. Comparison with human alpha 2-macroglobulin

Native human pregnancy zone protein (PZP), a close homolog of alpha 2-macroglobulin (alpha 2M), can be obtained in approximately 20% yield from pooled late pregnancy plasma or serum by a combination of polyethylene glycol precipitation, euglobulin precipitation, DEAE-Sephacel chromatography, zinc-chelate affinity chromatography, and negative affinity chromatography on insolubilized antibodies against human serum proteins. Both proteins are similarly organized as disulfide-bridged dimers of 360 kDa containing 180-kDa subunits. These dimers constitute the proteinase-binding units of PZP, and in contrast to alpha 2M, they appear to be only loosely associated, indicating a subtle difference in the quaternary structure of these alpha-macroglobulins. The preparations contain functionally intact beta-cysteinyl-gamma-glutamyl thiol esters, located in the same nonapeptide sequence as found in alpha 2M, and form complexes with a variety of proteinases in which a large fraction of the proteinase is bound covalently. Proteinases bound to PZP are still active and poorly accessible to reaction with large inhibitors like alpha 1-proteinase inhibitor. The structural and functional features of PZP indicate that PZP and alpha 2M, although extremely similar, may have different yet overlapping sets of proteinases as targets. It is possible that PZP mainly controls the activity of cellular proteinases released under conditions of increased cellular turnover and that PZP could be the human equivalent to the acute phase alpha-macroglobulins known in other species.     

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.     

Trypanosoma cruzi: cruzipain and membrane-bound cysteine proteinase isoform(s) interacts with human alpha(2)-macroglobulin and pregnancy zone protein

Plasmatic levels of pregnancy zone protein (PZP) increase in children with acute Chagas disease. PZP, as well as alpha2-macroglobulin (alpha2-M), are able to interact with Trypanosoma cruzi proteinases. The interaction of alpha2-M and PZP with cruzipain, the major cysteine proteinase of T. cruzi, was investigated. Several molecular changes on both alpha-M inhibitors under reaction with cruzipain were found. PAGE analysis showed: (i) formation of complexes of intermediate mobility and tetramerization of native alpha2-M and PZP, respectively; (ii) limited proteolysis of bait region in alpha2-M and PZP, and (iii) covalent binding of cruzipain to PZP and alpha2-M. Conformational and structural changes experimented by alpha-Ms correlate with modifications of the enzyme electrophoretic mobility and activity. Cruzipain-alpha-M complexes were also detected by gelatin SDS-PAGE and immunoblotting using polyclonal anti-cruzipain antibodies. Concomitantly, alpha2-M and PZP impaired the activity of cruzipain towards Bz-Pro-Phe-Arg-pNA substrate. In addition, alpha-Ms were able to form covalent complexes with membrane isoforms of cysteine proteinases cross-reacting with cruzipain. The present study suggests that both human alpha-macroglobulin inhibitors could prevent or minimize harmful action of cruzipain on host's molecules and hypothetically regulate parasite functions controlled by cruzipain.     

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.     

Differential binding properties of human pregnancy zone protein- and alpha2-macroglobulin-proteinase complexes to low-density lipoprotein receptor-related protein.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Both alpha-macroglobulins (alpha-Ms) covalently bind proteinases, which is accompanied by the exposure of carboxy terminal receptor recognition domains important for the rapid clearance from the circulation and tissues. It is accepted that the molecule responsible for the clearance of alpha2-M- and PZP-proteinase complexes is the low-density lipoprotein receptor-related protein (LRP). Although both alpha-M-proteinase complexes bind to the same receptor, differences in the binding properties have been reported. In addition, although it is known that the binding of alpha2-M-proteinase complexes to LRP can be blocked by Ni2+, the effect on PZP-proteinase has never been examined. In order to investigate differences in the binding properties of both alpha-Ms to the receptor, we purified LRP from human placenta by affinity chromatography and then analyzed the specificity and affinity of binding of alpha2-M- and PZP-proteinase complexes to the receptor by enzyme immunoassay. Our results clearly established that although both alpha-M-proteinase complexes specifically bind to LRP, PZP-chymotrypsin complexes bind to the receptor with lesser apparent affinity (Kd approximately equal 320 nM) than alpha2-M-chymotrypsin complexes (Kd approximately equal 40 nM). We also demonstrated that Ni2+ blocks the binding of alpha2-M-chymotrypsin complexes, but not PZP-chymotrypsin complexes, to LRP. These data suggest that the binding to LRP involves conformational differences between both alpha-Ms in a region immediately upstream of the carboxy terminal receptor recognition domain. The possibility that PZP-proteinase complexes interact with other receptors not available to alpha2-M-proteinase complexes could be considered.     

Differences in the proteinase inhibition mechanism of human alpha 2-macroglobulin and pregnancy zone protein.

Different conformational states of human alpha 2-macroglobulin (alpha 2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i.e. the 'bait' regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and alpha 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In alpha 2M, in which the thiol ester is broken by binding of methylamine and the 'trap' is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the 'trap' and the 'bait' regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native alpha 2M. In contrast, cyanylation has no effect on proteinase-treated alpha 2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate-treated alpha 2M exposes the receptor-recognition site, but the derivative is not cleared from the circulation in mice. The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor-recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP-chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthiocyanate. The results suggest that the conformational changes in alpha 2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in alpha 2M is the thiol ester, by which the events 'trapping' and exposure of the receptor-recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the 'bait' region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.     

Binding and receptor-mediated endocytosis of pregnancy zone protein-proteinase complex in rat macrophages

125I-labelled pregnancy zone protein complex was injected intravenously in rats and after 6 min uptake into cells of the liver and spleen was determined by electron microscopic autoradiography. The liver took up 68% of the injected radioactivity; 61% was in the hepatocytes and 7% was in the liver macrophages (Kupffer cells). The spleen took up 3-4% and nearly all the radioactivity was in the macrophages of the red pulp. The uptake per cell volume was several times higher in the macrophage than in the hepatocyte. The radioactivity associated with macrophages was largely in endocytotic vacuoles and lysosomes. Binding of labelled pregnancy zone protein complex to peritoneal macrophages at 4 degrees C was 2-3 times higher than binding of the homologous alpha 2-macroglobulin complex. The two ligands competed for binding to the same receptors and the difference was due to a higher affinity of the pregnancy zone protein complex (Kd approx. 60 pM). After binding to the receptor, this ligand was internalised within 2-3 min at 37 degrees C and radioactivity inside the cells largely represented intact pregnancy zone protein complex. Radioactivity was released from the cell as iodotyrosine after a lag time of about 10 min. It is concluded that pregnancy zone protein complex is bound with a high affinity to the alpha 2-macroglobulin receptors in rat macrophages followed by receptor-mediated endocytosis and degradation of the ligand in the lysosomes.     

Demonstration of an analogue to human pregnancy zone protein in the African green monkey.

A serum protein in the grivet (Cercopithecus aethiops), immunologically cross-reacting with human pregnancy zone protein (PZP), is found in males as well as in nonpregnant females. The electrophoretic mobility, antigenic cross-reactivity, molecular weight, and carbohydrate structure appear analogous to PZP; however, contrary to the situation in man, exogenous estrogens cause a fall in serum concentration of the protein.     

Pregnancy zone protein, a proteinase-binding macroglobulin. Interactions with proteinases and methylamine

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). Its properties and its reactions with a number of enzymes, particularly chymotrypsin, and with methylamine have been investigated. It is concluded that native PZP molecules are dimers of disulfide-bridged 180-kDa subunits and that proteinase binding results in covalent 1:1 (tetrameric)PZP-enzyme complexes. Native PZP is unstable, and storage should be avoided, but when kept unfrozen at 0 degree C most PZP preparations stay native 1-3 months. The reaction of PZP with chymotrypsin involves (i) proteolysis of bait regions, (ii) cleavage of beta-cysteinyl-gamma-glutamyl thiol ester groups, (iii) some change of the conformation and quaternary structure of PZP, and (iv) the formation of covalent 1:1 chymotrypsin-PZP(tetramer) complexes in which chymotrypsin is active but shows less activity than free chymotrypsin. The emission spectra of intrinsic fluorescence show significant differences between the PZP-chymotrypsin complex and its native components, whereas no differences are observed between methylamine-reacted PZP and native PZP. Methylamine reacts with the beta-cysteinyl-gamma-glutamyl thiol ester groups of PZP in a second-order process with k = (13.6 +/- 0.5) M-1 s-1, pH 7.6, 25 degrees C. The reaction product is PZP(dimers); no PZP(tetramers) are formed. The proteinase-binding specificity of PZP is far more restricted than that of alpha 2M. Certain chymotrypsin-like and trypsin-like enzymes are bound much less efficiently than is chymotrypsin itself.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of homogeneous preparations of pregnancy zone protein lyophilized in the presence of saccharose. Structural and functional studies

Human pregnancy zone protein (PZP) is a macromolecule of 360 kDa, organized as a disulfide-linked homodimer of two 180 kDa subunits, with an amino acid sequence and structure remarkably similar to that of human alpha2-Macroglobulin. Homogeneous PZP samples undergo fast aging forming oligomeric aggregates of high molecular weight. This aged PZP loses its ability to interact with proteinases and consequently, non-recognition of receptors occurs. In the present work, we assessed the effect of saccharose on the stability of native PZP on lyophilized samples kept for a long period of time. Herein, we demonstrate that the addition of 0.25 M saccharose to homogeneous PZP and further lyophilization is enough to prevent aging and preserve functional activity for more than 1 year. Hence, high quality samples, in terms of purity, stability and functional activity will allow to develop biochemical studies in order to know the PZP role in physiological and pathological states where the protein levels are increased, such as pregnancy and tumoral disorders.     

A two-hybrid dual bait system to discriminate specificity of protein interactions.

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.     

Human pregnancy zone protein and alpha 2-macroglobulin. High-affinity binding of complexes to the same receptor on fibroblasts and characterization by monoclonal antibodies

Pregnancy zone protein (PZP) was isolated from late pregnancy serum and examined for binding to normal skin fibroblasts in culture. A high-affinity binding site on these cells is demonstrated for PZP reacted with methylamine. Experiments with alpha 2-macroglobulin (alpha 2M) and PZP, both modified by methylamine, showed this receptor to be identical to the previously characterized receptor for alpha 2M-proteinase complexes (Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1979) J. Biol. Chem. 254, 5155-5160). With available monoclonal antibodies directed toward alpha 2M and prepared toward PZP, only a limited cross-reaction was observed. We obtained a monoclonal antibody which defines a neo-antigenic site on PZP-methylamine, completely analogous to the monoclonal antibody F2B2, which was previously shown to define a neo-antigenic site on alpha 2M complexes (Marynen, P., Van Leuven, F., Cassiman, J.J., and Van den Berghe, H. (1981) J. Immunol. 127, 1782-1786). These results provide evidence for the homologous function of alpha 2M and PZP as proteinase scavengers. The need for an extra proteinase inhibitor of the alpha 2M-type in pregnancy is discussed. The monoclonal antibodies now available will prove helpful in quantitation and eventually isolation of proteinase complexes of alpha 2M and PZP.     

Evolution of human alpha 2-macroglobulin

A papain-binding protein (PBP) resembling human alpha 2-macroglobulin (alpha 2M) but of Mr half that of alpha 2M was purified from plaice (Pleuronectes platessa L.) plasma. The plaice protein displayed most of the distinctive inhibitory properties of the human macroglobulin, and was therefore considered, despite its smaller molecular size, to be homologous with alpha 2M. Plaice PBP was shown to consist of four dissimilar subunits; two I chains (Mr 105 000) and two II chains (Mr 90 000). Each of the larger I chains contained a "bait region" sensitive to proteolytic attack by a variety of proteinases, and an autolytic site analogous to the autolytic site of alpha 2M. Subunit I, almost certainly at the autolytic site, formed SDS-stable, covalent links with methylamine or a proportion of the trapped proteinase molecules. A scheme is proposed for the evolution of human alpha 2M from the smaller fish protein, and the possibility of a shared evolutionary origin for alpha 2M and the complement components C3 and C4 is discussed.     

Pregnancy zone protein, a proteinase binding alpha-macroglobulin. Stopped-flow kinetic studies of its interaction with chymotrypsin

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein, strongly related to alpha 2-macroglobulin (alpha 2M). The proteinase binding reaction of PZP is investigated using chymotrypsin as a model enzyme. The time-course of the interaction is studied by measuring the change in intrinsic protein fluorescence of PZP-chymotrypsin reaction mixtures as a function of time after rapid mixing in a stopped-flow apparatus. Titrations show the changes of fluorescence at equilibrium to correspond with the formation of a chymotrypsin-PZP(tetramer) species. The kinetic results show the formation of the species to take place in an overall second-order process dependent on the concentrations of chymotrypsin and of PZP(dimers), k = 5 x 10(5) M-1 x s-1. Reactions of PZP-thiol groups do not give rise to fluorescence changes. The fluorescence changes most likely reflect the formation of an intermediate with intact thiol esters. Further analysis of the kinetic results suggests that the chymotrypsin-PZP(tetramer) intermediate is formed in two reaction steps: (1) initially native PZP(dimers) are cleaved at bait regions by enzyme molecules, and that is the rate determining reaction of the fluorescence changes; (2) association with another PZP(dimer) or PZP(dimer)-chymotrypsin complex in a very fast reaction that leads to the formation of 1:1 -chymotrypsin-PZP(tetramer) intermediate, probably with intact thiol esters. The interactions studied apparently are established early in the path of the reaction and the fluorescence changes probably reflect noncovalent enzyme-PZP contacts, which are not changed when covalent binding occurs. Further, fluorescence changes are seen only in reactions of PZP with enzymes, not with methylamine.     

Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine

The 1H NMR spectrum of human alpha 2-macroglobulin, Mr 716,000, consists of predominantly extremely broad unresolved resonances but also has nine relatively sharp (delta nu 1/2 less than 25 Hz) resonances from aromatic residues. By treatment of alpha 2-macroglobulin with methylamine, chymotrypsin, and subtilisin, it has been shown that eight of these resonances arise from bait region residues. More specifically, assignment has been made of resonances at 6.80 and 7.11 ppm to the ortho and meta protons, respectively, of tyrosine-685 and tentative assignment of a resonance at 7.29 ppm to the aromatic protons of phenylalanine-684. C2 proton resonances from five histidine residues are also visible. Four of these are attributed to residues in the bait region or immediately adjacent to this, at positions 675, 694, 699, and 704. The sharpness of resonances from bait region residues demonstrates the great flexibility of this region of the polypeptide. It is proposed that the flexible region extends from residue 675 to residue 710. These resonances are all affected by proteolytic cleavage in the bait region but are not influenced by the subsequent conformational rearrangement of the whole protein tetramer. The significance of these findings is discussed in relation to the current structural models of alpha 2-macroglobulin.     

Human alpha2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

Human alpha2M (alpha2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human alpha2M to be made. We describe here the expression and characterization of three alpha(2)M domains predicted to be involved in the stabilization of the thiol ester in native alpha2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the alpha2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of alpha2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1-MG8 of C3. TED is, as predicted, an alpha-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these alpha2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of alpha2M, and the consequent thiol ester-stabilizing domain-domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.