Alpha 2M Complexes Research Articles

Cerebral clearance of human amyloid‐β peptide (1–40) across the blood–brain barrier is reduced by self‐aggregation and formation of low‐density lipoprotein receptor‐related protein‐1 ligand complexes

Soluble amyloid-beta peptide (Abeta) exists in the form of monomers and oligomers, and as complexes with Abeta-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Abeta(1-40) [hAbeta(1-40)] from the rat brain across the blood-brain barrier. Incubation of [(125)I]hAbeta(1-40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [(125)I]hAbeta(1-40) dimer was significantly decreased by 92.7% compared with that of [(125)I]hAbeta(1-40) monomer. Pre-incubation with LRP-1 ligands, such as activated alpha2-macroglobulin (alpha2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [(125)I]hAbeta(1-40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [(125)I]hAbeta(1-40) monomer elimination. Purified [(125)I]hAbeta(1-40)/activated alpha2M complex and [(125)I]activated alpha2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [(125)I]activated alpha2M, and enhancement of [(125)I]hAbeta(1-40) uptake upon pre-incubation with apoE, suggesting that [(125)I]activated alpha2M and [(125)I]hAbeta(1-40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAbeta(1-40) from the brain across the blood-brain barrier.

Reaction of Factor VIIa (VIIa) with Plasma Inhibitors Ex Vivo and In Vivo.

Activated proteases are typically short-lived in the circulation where they react with plasma inhibitors and are removed as complexes by scavenger receptors. In contrast VIIa is characterized by a relatively long in vivo clearance half-life of 2–3 hr. Free VIIa exists in a latent state that is fully activated only upon binding to tissue factor (TF). Ex vivo VIIa is described to react predominantly with anti-thrombin (AT) in a slow reaction enhanced by heparin and by TF. Free VIIa is assumed to be rather inert towards plasma inhibitors also in vivo, and this may account for its slow clearance. Still it is not known whether the reaction with AT or other plasma inhibitors is an obligatory step in the in vivo clearance of VIIa. To clarify this question we studied the interaction between VIIa and plasma inhibitors in human and murine serum and plasma, and also studied this interaction during in vivo clearance of VIIa in mice. The VIIa-inhibitor complexes formed after spiking of serum with VIIa for 24 h were purified by affinity chromatography and subjected to SDS-PAGE and characterized by MALDI analysis. In the absence of heparin we identified VIIa complexes with alpha 2-macroglobulin (alpha 2M), AT, alpha 2-plasmin inhibitor and inter-alpha-trypsin inhibitor with the complex with alpha 2M as the most prominent component, whereas the AT complex was pre-dominant in the presence of heparin. The interaction between VIIa and plasma inhibitors might be differently affected by contact with the vasculature in the circulation. We therefore next studied VIIa complex formation in vivo in mice after i.v. injection of 125I-VIIa. Complex formation was followed by SDS PAGE and autoradiography, and clearance was followed by measurements of the VII antigen level. The data suggest that alpha 2M and AT complexes are formed in vivo, however, without affecting VIIa antigen clearance. This was confirmed by studies of in vivo clearance of VIIa or a preformed VIIa-AT complex. Measurements of VIIa clot activity, and of VII- and VIIa-AT antigen concentrations suggestedthat in vivo clearance of VIIa from circulation does not include complex formation with a plasma inhibitor as an obligatory intermediate,that the VIIa-AT complex is cleared with the same rate as VIIa andthat a transient VIIa-AT complex is formed subsequent to VIIa bolus injection which may account for a faster clearance of VIIa when measured by clot activity relative to VII clearance when this is measured by antigen concentration.

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.

Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes.

Chagas' disease patients (CDP) develop both humoral and cellular immune responses against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Here we demonstrate that complexes formed by cruzipain and alpha 2-macroglobulin (alpha 2M) are efficiently internalized by human monocytes, and that this process results in enhanced presentation of cruzipain peptides to CD4+ T cells from CDP. Purified or serum alpha 2M binds to polymorphic cruzipains, but only a fraction of the proteinases become covalently linked. Once bound to alpha 2M, fluorescein-labeled cruzipain (FITC-cruzipain) or [125I]cruzipain were more efficiently internalized by normal peripheral blood mononuclear cells (PBMC) or monocytes; this effect was abolished by (I) pre-treating the cells with receptor-associated protein (rRAP), a known antagonist the of alpha 2M receptor (alpha 2MR/LRP), and (II) inactivating [125I]cruzipain's active site prior to the reaction with alpha 2M, indicating that the exposure of receptor binding sites on alpha 2M complexes required bait region cleavage. We then sought to determine if the alpha 2MR/LRP-dependent uptake of alpha 2M:cruzipain by monocytes resulted in increased CD4+ T cell responses of PBMC-CDP (n = 13). These effects were only revealed after depletion of CD19+ B lymphocytes from PBMC-CDP; the threshold of T cell stimulation was far lower in cultures stimulated with alpha 2M:cruzipain, as compared to antigen alone. Myocardial specimens from CDP with chronic myocardiopathy (three necropsies) were analyzed by immunohistochemistry with mAb anti-cruzipain or anti-alpha 2MR/LRP (CD81+). Extracellular depots of cruzipain were localized amidst inflammatory mononuclear infiltrates, part of which contained CD91+ macrophage-like cells. Ongoing studies should clarify if T. cruzi cysteinyl proteinases play a role in the pathogenesis of Chagas' heart disease.

Quantitative immunoassay for complexes of prostate-specific antigen with alpha2-macroglobulin

We have developed two ELISAs for quantifying complexes of prostate-specific antigen (PSA) with alpha2-macroglobulin (alpha2M), using partially purified PSA:alpha2M complex as the calibrator. One ELISA was designed to evaluate )SA:alpha2M complex in fluids containing a huge excess of PSA over the amount of complex (semen-derived fluids), the other for use in fluids containing an excess of alpha2M over PSA (blood plasma). The range of the assays was 2-1000 micrograms/L for PSA complexed to alpha2M; the detection limit was 3 micrograms/: Intra- and interassay CVs were 7-13% and 11-17%, respectively, at complexed PSA concentrations of 6-500 micrograms/L. Seminal fluid from healthy men (n = 60) contained 5.2 +/- 2.6 micrograms/L PSA complexed with alpha2M. Prostatic and seminal vesicle fluids contained 6.5 +/- 2.9 ad 0.3 +/- 0.2 mg/L PSA complexed to alpha2M, respectively. When purified PSA was incubated with citrated plasma, between 45% and 65% of the added PSA was recovered as free PSA, whereas approximately 25% formed complexes with alpha2M, 10% complexed with alpha1-antichymotrypsin, and only 0.1-6% was complexed with protein C inhibitor. Of 30 patients with prostate disease, 20 showed detectable plasma PSA:alpha2M complexes; however, the potential diagnostic significance of this complex requires further investigation.

Fibronectin Degradation in Chronic Wounds Depends on the Relative Levels of Elastase, α1-Proteinase Inhibitor, and α2-Macroglobulin

The goal of our studies was to learn about the mechanism of fibronectin degradation in chronic ulcers. We found that the appearance of fibronectin fragments in chronic ulcer wound fluid correlated with elevated levels of elastase and cleavage of the proteinase inhibitors alpha2-macroglobulin (alpha2-M) and alpha 1-proteinase inhibitor (alpha1-P1). Some wound fluid samples retained the capacity to degrade fibronectin in vitro. Degradation of fibronectin by these samples was blocked by specific inhibitors of neutrophil elastase but not by inhibitors of metalloproteinases. Addition of human neutrophil elastase to mastectomy fluid, an acute wound fluid, resulted in formation of alpha1-PI and alpha2-M complexes and cleavage products resembling those observed in chronic wound fluid. Moreover, degradation of fibronectin and processing of matrix metalloproteinase MMP-9 occurred under these conditions. Taken together, our findings suggest that elevated levels of neutrophil elastase are responsible for fibronectin degradation in the chronic wound environment.

Classification of alpha 2-macroglobulin-cytokine interactions based on affinity of noncovalent association in solution under apparent equilibrium conditions.

alpha 2-Macroglobulin (alpha 2M) binds numerous cytokines; however, since binding affinities have not been determined, it is difficult to compare various alpha 2M-cytokine interactions or predict whether alpha 2M-cytokine complexes will form in the presence of other cytokine-binding macromolecules. In this investigation, we used a novel method to demonstrate that transforming growth factor-beta 1 (TGF-beta 1), TGF-beta 2, nerve growth factor-beta (NGF-beta), platelet derived growth factor-BB (PDGF-BB), tumor necrosis factor-alpha (TNF-alpha), and basic fibroblast growth factor (bFGF) reversibly associate with alpha 2M-methylamine to form noncovalent complexes. Apparent equilibrium was achieved in less than 15 min. Noncovalent alpha 2M-cytokine complexes were converted into covalent complexes; however, this occurred slowly. Therefore, a rapid equilibrium assumption was applied and equilibrium dissociation constants were determined using a single binding site model. KD values for the binding of cytokines to alpha 2M-methylamine varied by 2 orders of magnitude. The rank order of affinity was TGF-beta 2 (13 +/- 2 nM) > TGF-beta 1, NGF-beta > PDGF-BB > or = bFGF > TNF-alpha. Native alpha 2M bound TGF-beta 1, TGF-beta 2, NGF-beta, PDGF-BB, and TNF-alpha. Interferon-gamma did not bind to native alpha 2M or alpha 2M-methylamine. Each cytokine bound native alpha 2M with lower affinity than alpha 2M-methylamine except for TGF-beta 2 which bound both forms with equal affinity. In non-equilibrium systems, alpha 2M-methylamine appeared to bind more TGF-beta 2 due to the more rapid dissociation of TGF-beta 2-native alpha 2M complex. The classification of alpha 2M-cytokine complexes according to binding affinity should predict which complexes are most likely to form in cell culture and under various conditions in vivo.

Vascular endothelial growth factor is inactivated by binding to alpha 2-macroglobulin and the binding is inhibited by heparin.

Vascular endothelial growth factor (VEGF) is a mitogen for cultured endothelial cells, and a potent angiogenic factor in vivo. Incubation of 125I-VEGF with human or bovine serum led to the formation of 125I-VEGF containing complexes that had a molecular mass greater than 300 kDa. These complexes were specifically immunoprecipitated with anti-human alpha 2-macroglobulin (alpha 2M) antibodies. Similar high molecular weight complexes were formed when 125I-VEGF was incubated with commercially available alpha 2M. The 125I-VEGF.alpha 2M complexes were resistant to boiling in the presence of SDS. The formation of 125I-VEGF.alpha 2M complexes was inhibited by iodoacetic acid, indicating that free sulfhydryl groups are required for complex assembly. Tryptic digestion of alpha 2M did not affect its VEGF binding ability. Tryptic digestion of 125I-VEGF.alpha 2M complexes on the other hand, resulted in the degradation of bound 125I-VEGF, indicating that alpha 2M does not protect bound 125I-VEGF from proteolytic digestion. The binding of 125I-VEGF to alpha 2M was partially inhibited by an excess of basic fibroblast growth factor. Other growth factors which bind to alpha 2M, such as platelet-derived growth factor and insulin, did not inhibit the binding of 125I-VEGF. The binding of VEGF to alpha 2M inhibited its receptor binding ability, indicating that alpha 2M may function as a VEGF removal and inactivation factor. Heparin and heparan sulfate, but not other glycosaminoglycans such as chondroitin sulfate, efficiently inhibited the binding of 125I-VEGF to alpha 2M. It is possible that heparin-like molecules released from extracellular matrixes could prevent the inactivation of VEGF by alpha 2M resulting in the potentiation of processes such as tumor angiogenesis.

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.

Differential inhibition of transforming growth factor beta 1 and beta 2 activity by alpha 2-macroglobulin.

Affinity labeling and immunoprecipitation studies demonstrate that alpha 2-macroglobulin (alpha 2M) is the major serum-binding protein for transforming growth factors beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2). Purified alpha 2M inhibits the binding of both 125I-TGF-beta 1 and 125I-TGF-beta 2 to cell surface receptors at I50 values of 200 and 10 micrograms/ml, respectively. alpha 2M (200 micrograms/ml) does not block TGF-beta 1 inhibition of CCL-64 mink lung cell growth but reduces this activity of TGF-beta 2 10-fold. The electrophoretic migration of 125I-TGF-beta.alpha 2M complexes on polyacrylamide gels under nondenaturing conditions demonstrates that alpha 2M has 10-fold greater affinity for TGF-beta 2 than for TGF-beta 1. Each of these complexes comigrates as a single band with the fast form of alpha 2M. We suggest that alpha 2M is an important differential regulator of the biological activities of TGF-beta 1 and TGF-beta 2 in vivo.

Immunolocalization of collagenase in neoplastic epithelial cells

The distribution of interstitial collagenase in a rat mammary carcinoma model system has been studied by immunocytochemistry. Rabbit antibodies were raised against collagenase from neoplastic epithelial cells which were derived from an anaplastic, invasive, rat mammary carcinoma (BC1). Specificity of the antibodies was determined by Western blot analysis which showed reactivity with the inactive procollagenase from conditioned culture medium of BC1 cells as well as with purified, active BC1 collagenase. Anti-BC1 collagenase antibodies did not recognize BC1 collagenase entrapped by the inhibitor, rat alpha-2-macroglobulin (alpha 2M), or collagenase derived from TPA-stimulated human fibroblasts. Anti-human fibroblast collagenase antibodies did not recognize BC1 collagenase, suggesting that the human-mesenchymal and rat-epithelial enzymes are immunologically distinct molecules. Collagenase was immunolocalized intracellularly in BC1 cells cultured in the presence of monensin. Neither BC1 collagenase, alpha 2M nor enzyme-inhibitor complexes were demonstrated in or around invading tumours by immunostaining of tissue sections of rat mammary carcinomas.

α2-macroglobulin is a binding protein for basic fibroblast growth factor

Abstract After incubation with human serum or plasma, 125I-basic fibroblast growth factor (bFGF) (molecular mass 18.5 kDa) exhibits molecular mass forms greater than 200 kDa as determined by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. These high molecular mass forms of bFGF are immunoprecipitable with antiserum raised against alpha 2-macroglobulin (alpha 2M). Purified alpha 2M and 125I-bFGF form a covalent complex in a specific, saturable manner. Excess unlabeled bFGF competes with 125I-bFGF for complex formation. Complex formation is complete after 4 h and is inhibited by pretreating alpha 2M with dithiothreitol, iodoacetamide, iodoacetic acid, and N-ethylmaleimide. The complex is resistant to acidic conditions and denaturants such as urea. Heparin, which binds bFGF, has no effect on complex formation. Methylamine, which blocks protease binding to alpha 2M, increases the amount of 125I-bFGF that can be bound 2-fold. Plasmin and trypsin treatment of alpha 2M has no effect on 125I-bFGF binding. The ability of growth factors to compete for binding is specific, as aFGF and TGF-beta compete for binding to alpha 2M, whereas platelet-derived growth factor does not. 125I-bFGF.alpha 2M complexes do not bind to low affinity bFGF binding sites and bind poorly to high affinity bFGF binding sites on BHK-21 cells. In addition, 125I-bFGF bound to alpha 2M has decreased ability to stimulate plasminogen activator production in bovine capillary epithelial cells.

Receptors for α2-Macroglobulin- and pregnancy zone protein-proteinase complexes in the human placental syncytiotrophoblast

Receptors for complexes between alpha 2-macroglobulin (alpha 2M) and proteinases (e.g. trypsin) were identified and characterized in the human placenta. The receptors also bound the complex formed between pregnancy zone protein (PZP) and chymotrypsin, although with slightly lower affinity, whereas binding of alpha 2M or PZP in their native forms was negligible. Treatment with methylamine to cleave the internal thiol esters caused an increase in binding affinity of alpha 2M to the level of alpha 2M-trypsin but only a minor increase in the affinity of PZP. Chorionic villi prepared from normal full-term placentae were approximately half occupied by endogenous alpha 2M or PZP complexes. These ligands, as well as prebound 125I-labelled alpha 2M-trypsin, were rapidly removed by the addition of 5 mM EDTA. Binding was similar in villi from eight-week and full-term placentae. Autoradiography showed that labelled alpha 2M-trypsin was associated with the syncytiotrophoblast. The kinetics of 125I-labelled alpha 2M-trypsin binding at 4 degrees C was similar in isolated villi and microvillous membranes. Association was slow, with apparent equilibrium by about 16 h. Dissociation of prebound tracer was slow but markedly accelerated in the presence of unlabelled ligand at a saturating concentration. The concentration-dependence of binding at equilibrium yielded a non-linear Scatchard plot. Most of the binding of ligand at tracer concentration was accounted for by high-affinity receptors with a dissociation constant (Kd) of about 50 pM. The content of high-affinity receptors in one placenta was estimated as approximately 125 pmol, i.e., a significant fraction of the total receptor population in the pregnant woman.

Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta.

125I-Labeled human platelet-derived transforming growth factor beta (125I-TGF-beta) and human alpha 2-macroglobulin (alpha 2M) formed a complex as demonstrated by 5% native polyacrylamide gel electrophoresis. The 125I-TGF-beta.alpha 2M complex migrated at a position identical to that of the fast migrating form of alpha 2M. Most of the 125I-TGF-beta.alpha 2M complex could be dissociated by acid or urea treatment. When 125I-TGF-beta was incubated with serum, the high molecular weight form of 125I-TGF-beta could be immunoprecipitated by anti-human alpha 2M anti-sera as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha 2M purified from platelet-rich plasma also showed the latent transforming growth factor activity and immunoreactivity of TGF-beta. These results suggest that TGF-beta.alpha 2M complex is a latent form of TGF-beta.

Binding of alpha-2-macroglobulin trypsin complex to human monocytes in culture

125I-labelled alpha 2-macroglobulin-trypsin (alpha 2MT) complex bound specifically to freshly isolated human blood monocytes at 4 degrees C but not to B or T lymphocytes, polymorphonuclear leucocytes, erythrocytes or thrombocytes. Binding of 12 pmol/l labelled alpha 2MT to freshly isolated monocytes was low. However, when monocytes were cultured in vitro, binding increased, reaching 10- to 20-fold higher specific binding by 2-4 weeks. Non-specific binding was absent. Considerable variations were observed in binding to monocytes from different cultures (individuals). The half-time of 125I-labelled alpha 2MT complex association to 14-days-old monocyte cultures was about 6 h at 4 degrees C. Dissociation of labelled complex after the addition of a saturing concentration of unlabelled complex was biphasic. About half of the labelled complex dissociated with a half-time of about 1 h, whereas the other half dissociated extremely slowly. At near steady state, half of the receptors were occupied at a complex concentration of about 200 pmol/l and Scatchard analysis showed that the data were adequately described by assuming one class of receptors. It is concluded that human monocytes express a marked increase in binding of alpha 2M complex when developing to macrophage-like cells in tissue cultures.

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.

The receptor-binding domain of human alpha 2-macroglobulin. Isolation after limited proteolysis with a bacterial proteinase.

Limited proteolysis of human alpha 2-macroglobulin (alpha 2M) by a novel bacterial proteinase resulted in the isolation of a soluble 20-kDa domain. The isolated fragment contained the receptor recognition site, expressed on alpha 2M complexes, as it competed effectively with alpha 2M-trypsin for binding to the receptor on skin fibroblasts. The fragment also reacted with two monoclonal antibodies which define epitopes that are part of the receptor recognition site. Characterization of the 20-kDa domain showed it to contain an intact disulfide bridge, while its susceptibility to N-glycanase and reaction with concanavalin A indicated the presence of N-linked carbohydrate. The NH2-terminal sequence (Glu-Glu-Phe-Pro-Phe-Ala-Leu-Gly-Val-Glu-Thr-Leu-Pro-Glu-Thr-Cys-Asp-Glu -Pro) proved this fragment to constitute the COOH terminus of human alpha 2M. Proteolysis occurred at Lys1313-Glu which together with the observation that tosyllysine chloromethyl ketone was an effective inhibitor of the bacterial proteinase, would indicate the latter to hydrolyze preferentially peptide bonds carboxyl-terminal to lysine residues.

Solubilization and affinity purification of the alpha 2-macroglobulin receptor from human fibroblasts.

Plasma membranes showing specific binding of alpha 2-macroglobulin (alpha 2M) complexes were isolated from normal human fibroblasts. The membrane preparation was solubilized with Triton X-100, and specific binding to solubilized receptor was demonstrated by precipitation of the alpha 2M X protease receptor complexes with polyethylene glycol. The solubilized receptor could be quantitatively adsorbed on immobilized alpha 2M complexes. Adsorbed receptor was then dissociated from the alpha 2M complexes with either EDTA, bacitracin, the monoclonal anti-receptor-recognition site antibody F2B2 , or low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity-purified receptor preparation revealed polypeptides of 360, 130, and 83 kDa.

Relation of internal thioesters to conformational change and receptor-recognition site in alpha 2-macroglobulin complexes.

The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M. The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated. Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups. Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M. Other thiol-specific reagents were less effective. When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active. Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes. The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.