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PubMed1992

Binding of transforming growth factor-beta 1 to methylamine-modified alpha 2-macroglobulin and to binary and ternary alpha 2-macroglobulin-proteinase complexes.

The binding of 125I-labelled transforming growth factor-beta 1 (TGF-beta 1) to human alpha 2-macroglobulin (alpha 2M) was studied by native PAGE and autoradiography. TGF-beta 1 bound preferentially to alpha 2M-methylamine and minimally, if at all, to native alpha 2M. Preparations of alpha 2M-proteinase complex were generated by incubating a standard concentration of alpha 2M (0.4 microM) with different concentrations of trypsin, chymotrypsin or neutrophil elastase (0.04-2.0 microM). The 125I-TGF-beta 1-binding activity depended on the initial ratio of active proteinase to alpha 2M, or r value, used to form the alpha 2M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta 1-binding activity relative to native alpha 2M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta 1-binding activity. The results of [3H]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the 125I-TGF-beta-binding experiments. alpha 2M-trypsin and alpha 2M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta 1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, 125I-TGF-beta 1 binding to alpha 2M-methylamine was at least 80% non-covalent. Reaction of alpha 2M-methylamine with iodoacetamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of 125I-TGF-beta 1 to alpha 2M-methylamine. Cleavage of the 'bait regions' in alpha 2M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta 1 binding to alpha 2M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or amine. If both proteinase-binding sites in a single alpha 2M molecule are occupied, TGF-beta 1-binding activity is decreased or perhaps eliminated.

Animals↗

PubMed1993

Binding of platelet-derived growth factor-BB and transforming growth factor-beta 1 to alpha 2-macroglobulin in vitro and in vivo: comparison of receptor-recognized and non-recognized alpha 2-macroglobulin conformations.

alpha 2-Macroglobulin (alpha 2M) undergoes a major conformational change when reacting with proteinases or primary amines. This conformational change has been referred to as the 'slow' to 'fast' transformation based on the increase in alpha 2M mobility shown by non-denaturing PAGE. Previous studies demonstrated that many cytokines, including transforming growth factor beta 1 (TGF-beta 1) and interleukin-1 beta, bind preferentially or exclusively to alpha 2M which has undergone conformational change. In this study, we demonstrate that platelet-derived growth factor-BB (PDGF-BB) also binds preferentially to conformationally transformed alpha 2M (alpha 2M-methylamine, alpha 2M-trypsin) in vitro. Purified 125I-PDGF-BB-alpha 2M-methylamine complex cleared rapidly from the circulation of mice via the alpha 2M receptor/low-density-lipoprotein-receptor-related protein (alpha 2M-R/LRP). In order to determine whether PDGF-BB or TGF-beta 1 binds to native alpha 2M, we defined the native conformation by lack of interaction with alpha 2M-R/LRP instead of electrophoretic mobility. 125I-PDGF-BB was incubated with 4.3 microM native alpha 2M and 0.47 microM alpha 2M-methylamine. The 125I-PDGF-BB distributed evenly between slow-form and fast-form alpha 2M without shifting the electrophoretic mobility of either species. When the mixed preparation was injected intravenously in mice, 125I-PDGF-BB-fast-form-alpha 2M cleared rapidly and selectively from the circulation; 125I-PDGF-BB which was bound to slow-form alpha 2M was stable in the blood (apparently not recognized by alpha 2M-R/LRP). Therefore, while conformationally transformed alpha 2M binds PDGF-BB preferentially in vitro, non-alpha 2M-R/LRP-recognized alpha 2M binds PDGF-BB as well. Binding of 125I-PDGF-BB and 125I-TGF-beta 1 to alpha 2M was demonstrated in vivo by injecting the free growth factors intravenously into mice. Plasma samples which were subjected to non-denaturing PAGE and autoradiography demonstrated binding of both growth factors exclusively to the slow-form of alpha 2M. Therefore, under normal physiological conditions, native alpha 2M (non-alpha 2M-R/LRP-recognized) is the primary form of the proteinase inhibitor functioning as a carrier of PDGF-BB and TGF-beta 1 in the blood.

Animals↗

PubMed1995

Synthesis of a Cys949Tyr alpha 2-macroglobulin thiol ester mutant: co-transfection with wild-type alpha 2-macroglobulin in an episomal expression system.

A full-length alpha 2-macroglobulin (alpha 2M) cDNA was cloned into the episomal expression vectors pREP7 and pMEP4. Electroporation of the cell lines WI-L2-729HF2, U-937, K-562 and an Epstein-Barr virus-transformed cell line resulted in stable transfectants only with K-562 cells. Stable expression was obtained exclusively with pMEP4-alpha 2M and was driven from the inducible human metallothionein IIA promoter. Expression of the wild-type alpha 2M cDNA resulted in a recombinant protein (r alpha 2M) that could not be distinguished from plasma alpha 2M (p-alpha 2M): the transfected K-562 cells secreted tetrameric alpha 2M with intact internal thiol esters, a functional bait domain and a latent receptor-binding domain. r alpha 2M inhibited trypsin and elastase from cleaving a high-molecular-mass substrate. When the Cys-949 involved in the formation of the internal thiol ester was mutated to tyrosine (C949Y-r alpha 2M), a tetrameric alpha 2M was secreted, with the electrophoretic mobility of methylamine-treated p-alpha 2M (p-alpha 2M/MA) and with a functional receptor-binding domain. The C949Y-r alpha 2M did not possess proteinase-inhibiting capacity. Heterozygosity was mimicked by co-transfecting the K-562 cells with wild-type and mutant expression vectors. In this case, r alpha 2M was secreted with zero, one, two, three or four internal thiol esters. A comparison of the interaction of interleukin 1 beta and basic fibroblast growth factor with native p-alpha 2M, p-alpha 2M/MA and the mutant C949Y-r alpha 2M revealed that when assayed under nondenaturing conditions, no binding occurred to 'slow' p-alpha 2M whereas quantitatively similar binding was observed to 'fast' p-alpha 2M/MA and C949Y-r alpha 2M. Covalent binding, however, was essentially limited to p-alpha 2M/MA, suggesting the involvement of Cys-949 in the process. Covalent binding of insulin, on the contrary, was only observed when it was present during hydrolysis of the internal thiol esters of p-alpha 2M by trypsin treatment, and thus involves the activated Glx residue.

Antibodies, Monoclonal↗

PubMed1995

Design of a new protease inhibitor by the manipulation of the bait region of alpha 2-macroglobulin: inhibition of the tobacco etch virus protease by mutant alpha 2-macroglobulin.

Human alpha 2-macroglobulin (alpha 2M) inhibits a broad spectrum of proteases by changing its conformation and physically confining the enzyme. The inhibitory spectrum of alpha 2M is defined by a stretch of 39 amino acids, the bait region, located near the middle of the alpha 2M monomers. To investigate whether a new inhibitory specificity can be introduced by the manipulation of the bait region, recombinant alpha 2M (r alpha 2M) was produced in which the primary cleavage site was replaced by a heptapeptide containing the cleavage specificity of tobacco etch virus (TEV) protease. This protease is not inhibited by wild-type alpha 2M. The r alpha 2M, produced in an episomal expression system, was fully functional and able to inhibit the tobacco etch virus protease according to its normal 'trap' mechanism. The manipulation of the bait region of alpha 2M thus allows the design of new, specific protease inhibitors.

Amino Acid Sequence↗

PubMed1995

Inhibition of platelet-derived growth factor-BB-induced fibroblast proliferation by plasmin-activated alpha 2-macroglobulin is mediated via an alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein-dependent mechanism.

alpha 2-Macroglobulin (alpha 2M) is a potentially important regulator of platelet-derived growth factor-BB (PDGF-BB)-stimulated cell growth due to our previous observation that PDGF-BB binds to alpha 2M noncovalently (Bonner, J. C., Goodell, A. L., Lasky, J. A., and Hoffman, M. R. (1992) J. Biol. Chem. 267, 12837-12844). We examined the in vitro effect of native and plasmin-activated (receptor-recognized) alpha 2M on the PDGF-BB-induced proliferation of mouse Swiss 3T3 and rat lung fibroblasts. Nondenaturing polyacrylamide gel electrophoresis showed that plasmin converted alpha 2M to its electrophoretically "fast" form at a 2:1 molar ratio and that 125I-PDGF-BB bound both alpha 2M and alpha 2M-plasmin. PDGF-BB-induced growth was not affected by native alpha 2M (0.3 microM) or plasmin (0.6 microM). The combination of plasmin and alpha 2M (2:1 molar ratio) inhibited PDGF-BB-induced cell proliferation 80-90%. Complexes of PDGF-BB.alpha 2M purified by gel filtration chromatography retained growth promoting activity, but the PDGF-BB.alpha 2M-plasmin complex did not. Preincubation of fibroblasts (37 degrees C for 24 h) with alpha 2M-plasmin did not change 125I-PDGF-BB binding or affect gene expression of the 6.5-kilobase PDGF-alpha receptor or 5.2-kilobase PDGF-beta receptor mRNA. However, preincubation with alpha 2M-plasmin (0-4 degrees C for 4 h) increased 125I-PDGF-BB binding 2-fold, and this increase was blocked by a receptor-associated protein antagonist of the alpha 2M-receptor/low density lipoprotein receptor-related protein. The receptor-associated protein antagonist blocked 125I-alpha 2M-methylamine binding, inhibited PDGF-BB-alpha 2M-plasmin uptake from fibroblast-cultured supernatants, and abolished the inhibitory effect of alpha 2M-plasmin on PDGF-stimulated growth. These data suggest that inhibition of PDGF-stimulated proliferation by alpha 2M-plasmin is mediated in part by clearance of PDGF-BB-alpha 2M-plasmin through the lipoprotein receptor-related protein.

Animals↗

PubMed1996

Selective mutations in cloned and expressed alpha-macroglobulin receptor binding fragment alter binding to either the alpha2-macroglobulin signaling receptor or the low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor.

alpha2-Macroglobulin (alpha2M) activated with methylamine binds to two distinct cell-surface receptors: low density-lipoprotein receptor-related protein/alpha2M receptors and alpha2M signaling receptors. Binding to lipoprotein receptor-related protein/alpha2M receptor but not alpha2M signal receptor is inhibitable by another ligand, receptor-associated protein. Direct binding studies with a recombinant receptor binding fragment (RBF) from rat alpha1M and murine macrophages demonstrate two classes of binding sites of apparent Kd = 90 pM (1500 sites/cell) and 40 nM (60,400 sites/cell). Receptor-associated protein competes with RBF for binding to the lower but not the higher affinity site. Site-directed mutation of Lys-1374 (human numbering) in RBF to Arg or Ile residues almost completely abolishes signal transduction as compared to wild-type RBF. Direct binding studies with K1374R demonstrated no significant alteration in binding to the lower affinity site; however, binding to the high affinity site is reduced by 83%. Mutation of Lys-1370 to Ala resulted in a 4-5-fold increase in the Kd for binding to the lower affinity site with no significant alteration in binding to the high affinity site or signal transduction properties. Studies demonstrate comparable internalization and degradation of wild-type RBF and K1374R; however, internalization and degradation of K1370A is negligible. These studies suggest that regions around Lys-1370 and Lys-1374 are involved in lipoprotein receptor-related protein/alpha2M receptor and alpha2M signaling receptor binding, respectively.

Amino Acid Sequence↗

PubMed1998

Binding of receptor-recognized forms of alpha2-macroglobulin to the alpha2-macroglobulin signaling receptor activates phosphatidylinositol 3-kinase.

Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation.

Androstadienes↗

PubMed1999

Coordinate regulation of the alpha(2)-macroglobulin signaling receptor and the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor by insulin.

We have studied insulin-dependent regulation of macrophage alpha(2)-macroglobulin signaling receptors (alpha(2)MSR) and low density lipoprotein receptor-related protein/alpha(2)M receptors (LRP/alpha(2)MR) employing cell binding of (125)I-alpha(2)M*, inhibition of binding by receptor-associated protein (RAP) or Ni(2+), LRP/alpha(2)MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of alpha(2)M* high (alpha(2)MSR) and low (LRP/alpha(2)MR) affinity binding sites from 1, 600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni(2+) blocked the binding of (125)I-alpha(2)M* to alpha(2)MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/alpha(2)MR. Insulin significantly increased LRP/alpha(2)MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented (125)I-alpha(2)M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in alpha(2)MSR receptor synthesis was reflected by augmented generation of IP(3) and increased [Ca(2+)](i) levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and alpha(2)M* stimulation significantly reduced the insulin-augmented increase in IP(3) and [Ca(2+)](i) levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new alpha(2)MSR. In conclusion, we show here that insulin coordinately regulates macrophage alpha(2)MSR and LRP/alpha(2)MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.

Animals↗

PubMed2001

Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha 2-macroglobulin. Implications regarding the molecular mechanisms of complex formation between alpha 2-macroglobulin and growth factors, cytokines, and hormones.

The biological activities of transforming growth factor-beta isoforms (TGF-beta(1,2)) are known to be modulated by alpha(2)-macroglobulin (alpha(2)M). alpha(2)M forms complexes with numerous growth factors, cytokines, and hormones, including TGF-beta. Identification of the binding sites in TGF-beta isoforms responsible for high affinity interaction with alpha(2)M many unravel the molecular basis of the complex formation. Here we demonstrate that among nine synthetic pentacosapeptides with overlapping amino acid sequences spanning the entire TGF-beta(1) molecule, the peptide (residues 41-65) containing Trp-52 exhibited the most potent activity in inhibiting the formation of complexes between (125)I-TGF-beta(1) and activated alpha(2)M (alpha(2)M*) as determined by nondenaturing polyacrylamide gel electrophoresis and by plasma clearance in mice. TGF-beta(2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta(1) peptide, whereas the corresponding TGF-beta(3) peptide lacking Trp-52, was inactive. The replacement of the Trp-52 with alanine abolished the inhibitory activities of these peptides. (125)I-TGF-beta(3), which lacks Trp-52, bound to alpha(2)M* with an affinity lower than that of (125)I-TGF-beta(1). Furthermore, unlabeled TGF-beta(3) and the mutant TGF-beta(1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-beta(1) in blocking I(125)-TGF-beta(1) binding to alpha(2)M*. TGF-beta(1) and TGF-beta(2) peptides containing Trp-52 were also effective in inhibiting I(125)-nerve growth factor binding to alpha(2)M*. Tauhese results suggest that Trp-52 is involved in high affinity binding of TGF-beta to alpha(2)M*. They also imply that TGF-beta and other growth factors/cytokines/hormones may form complexes with alpha(2)M* via a common mechanism involving the interactions between topologically exposed Trp and/or other hydrophobic residues and a hydrophobic region in alpha(2)M*.

Amino Acid Sequence↗

PubMed2002

The three-dimensional structure of the human alpha 2-macroglobulin dimer reveals its structural organization in the tetrameric native and chymotrypsin alpha 2-macroglobulin complexes.

Three-dimensional electron microscopy reconstructions of the human alpha(2)-macroglobulin (alpha(2)M) dimer and chymotrypsin-transformed alpha(2)M reveal the structural arrangement of the two dimers that comprise native and proteinase-transformed molecules. They consist of two side-by-side extended strands that have a clockwise and counterclockwise twist about their major axes in the native and transformed structures, respectively. This and other studies show that there are major contacts between the two strands at both ends of the molecule that evidently sequester the receptor binding domains. Upon proteinase cleavage of the bait domains and subsequent thiol ester cleavages, which occur near the central region of the molecule, the two strands separate by 40 A at both ends of the structure to expose the receptor binding domains and form the arm-like extensions of the transformed alpha(2)M. During the transformation of the structure, the strands untwist to expose the alpha(2)M central cavity to the proteinase. This extraordinary change in the architecture of alpha(2)M functions to completely engulf two molecules of chymotrypsin within its central cavity and to irreversibly encapsulate them.

Chymotrypsin↗

PubMed2000

Fluorescence monitoring of the conformational change in alpha 2-macroglobulin induced by trypsin under second-order conditions: the macroglobulin acts both as a substrate and a competitive inhibitor of the protease.

The reaction of bovine pancreatic trypsin with human plasma alpha(2)-macroglobulin (alpha(2)M) was studied at 25 degrees C, using equimolar mixtures of E and I in 50 mM potassium phosphate buffer, pH 7. The conformational change in alpha(2)M was monitored through the increase in protein fluorescence at 320 nm (exc lambda, 280 nm). At [alpha(2)M](0) =[E](0) =11.5-200 nM, the fluorescence change data fit the integrated second-order rate equation, (F(infinity) -F(0) )/(F(infinity) -F(t) )=1+k(i,obsd) [alpha(2)M](0) t, indicating that cleavage of the bait region in alpha(2)M was the rate-determining step. The apparent rate constant (k(i,obsd)) was found to be inversely related to reactant concentration. The kinetic behavior of the system was compatible with a model involving reversible, nonbait region binding of E to alpha(2)M, competitively limiting the concentration of E available for bait region cleavage. The intrinsic value of k(i) was (1.7+/-0.24) x 10(7) M(-1) s(-1).K(p), the inhibitory constant associated with peripheral binding, was estimated to be in the submicromolar range. The results of the present study point to a potential problem in interpreting kinetic data relating to protease-induced structural changes in macromolecular substrates. If there is nonproductive binding, as in the case of trypsin and alpha(2)M, and the reactions are monitored under pseudo first-order conditions ([S](0) >>[E](0) ), an intrinsically second-order process (such as the rate-limiting bait region cleavage in alpha(2)M) may become kinetically indistinguishable from an intrinsically first-order process (e.g. rate-limiting conformational change). Hence an excess of one component over the other should be avoided in kinetic studies addressing such systems.

Animals↗

PubMed1990

The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin.

Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.

Animals↗

PubMed1982

Comparison of the structure and aspects of the proteinase-binding properties of cystic fibrotic alpha-macroglobulin with normal alpha 2-macroglobulin.

Considerable attention has been focused recently on alpha 2-macroglobulin (alpha 2M), a major endopeptidase inhibitor in blood plasma, as a possible source of the primary defect in cystic fibrosis (CF). We report here studies designed to compare the structure of CF alpha 2M with normal alpha 2M to determine if there is a difference. The physicochemical properties of purified alpha 2M as revealed by various electrophoretic techniques, covalent proteinase binding properties, and primary structural studies on a variety of partial hydrolyzates of CF alpha 2M and normal alpha 2M are compared. These studies were carried out on eight different individual isolates of CF alpha 2M and three age-matched normal alpha 2M preparations and alpha 2M isolated from fetal cord blood. Three properties of CF alpha 2M were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): (1) the existence of four identically-sized subunits in the native molecule (10), (2) the cleavage of this subunit into fragments of approximately 100,000 daltons upon interaction with proteinases (10), and (3) the cleavage of an alkaline/heat sensitive bond to produce 120,000 and 60,000 dalton fragments (11). Both CF and normal alpha 2M were cleaved to the extent of 79-87%, CF alpha 2M behaves identically with normal alpha 2M with regard to all these properties. Salvesen and Barrett (24) have demonstrated that varying proportions of several [125I]-labeled proteinases form SDS-stable, non-reducible links to normal alpha 2M. Two of the CF alpha 2M preparations were studied to determine if similar covalent binding of proteinases occurred. The positions of the labeled and % of proteinase bound bands in SDS/reduced PAGE system were identical for normal alpha 2M and CF alpha 2M. These results indicate that CF alpha 2M behaves normally with regard to covalent binding of proteinases. Qualitative comparison of the peptide fragments separated by SDS-PAGE or isoelectric focusing of CF and normal alpha 2M produced by partial proteolysis with trypsin, chymotrypsin or Staphylococcus aureus V-8 proteinase did not reveal any difference unique to CF alpha 2M. The cyanogen bromide fragmentation studies and the cysteine cleavage studies also indicated that no major change in the positions of methionyl residues or cysteinyl/cystinyl residues has occurred in CF alpha 2M. The failure of all these different studies and those reported by others to demonstrate any differences between CF and normal alpha 2M makes it highly unlikely that there is a primary defect in alpha 2M in CF.

Adolescent↗

PubMed1995

Alpha 2-macroglobulin conformation determines binding affinity for activin A and plasma clearance of activin A/alpha 2-macroglobulin complex.

Activin A is a member of the transforming growth factor-beta family of growth factors and a potent regulator of cellular activity. A number of binding proteins for activin A have been identified, including alpha 2-macroglobulin (alpha 2M). Alpha 2M has several conformational states that are known to have different growth factor-binding properties. The effect of alpha 2M conformation on activin A binding has not been characterized. The aims of this study were to determine 1) whether activin A binds preferentially to the native (alpha 2M-N) or "activated" (alpha 2M*) conformation of alpha 2M, 2) the affinity of different alpha 2M conformations for activin A, and 3) the fate of activin A complexed with alpha 2M-N or alpha 2M* in vivo. [125I]Activin A associated with alpha 2M in plasma and follicular fluid and with purified alpha 2Ms. In this qualitative assay, more activin A was associated with alpha 2M* than with alpha 2M-N. The affinity of the activin A-alpha 2M interaction was determined. The Kd values for activin A-alpha 2M* and activin A-alpha 2M-N were 190 +/- 30 and 510 +/- 60 nM, respectively. The plasma clearance profiles and tissue distribution of uncomplexed activin A and purified alpha 2M*-activin A complex were determined. Radiolabeled activin A cleared in a biphasic manner, with rapid clearance over the initial 10 min and substantially slower clearance over the subsequent 20 min. During the slow phase of clearance, activin A formed a complex with circulating alpha 2M-N. In contrast, radiolabeled activin A-alpha 2M* complexes were rapidly cleared from plasma with a half-life of approximately 5 min and were specifically targeted to alpha 2M receptors in vivo. These studies reveal that alpha 2M can maintain activin A in the circulation or rapidly target the hormone for plasma clearance depending on the conformational state of the carrier protein in vivo.

Activins↗

PubMed1998

Human leptin forms complexes with alpha 2-macroglobulin which are recognized by the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein.

OBJECTIVE: To identify binding proteins of leptin in human plasma. METHODS: Binding was evaluated by electrophoresis, size exclusion chromatography (SEC), Western blotting, and radioisotope labeling. Quantification of leptin and the different forms of alpha2-macroglobulin (alpha2-M) was performed by ELISA. RESULTS: Leptin interacts with the proteinase inhibitor, alpha2-M. 125I-labeled leptin specifically binds to the transformed inhibitor, which arises by reaction with proteinases or with reactive primary amines. No leptin binding was observed to the native alpha2-M, which abundantly occurs in plasma. The complex formation between leptin and alpha2-M was found to proceed within minutes and was stable, as it resisted separation by SEC and electrophoresis. The Kd of the complex was 2.14 +/- 0.78 micromol/l. Complex formation with transformed alpha2-M did not interfere with the immunological determination of leptin in plasma. The leptin-alpha2-M complex was found to be recognized by the alpha2-M receptor/low density lipoprotein receptor-related protein. By computer analysis, a simple model is presented showing that the degree of transformation of alpha2-M may significantly influence the leptin concentration in blood. CONCLUSIONS: The proteinase inhibitor, alpha2-M, may act as a leptin-binding protein in human plasma. Binding of leptin to transformed alpha2-M and its rapid clearance by the alpha2-M receptor may significantly influence the bioavailability of leptin in human plasma.

Adult↗

PubMed1999

The trypsin-inhibitory efficiency of human alpha 2-macroglobulin in the presence of alpha 1-proteinase inhibitor: evidence for the formation of an alpha 2-macroglobulin--alpha 1-proteinase inhibitor complex.

The inhibition of bovine pancreatic trypsin was studied at pH 7, 25 degrees C, using mixtures of purified human alpha 2-macroglobulin (alpha 2M) and alpha 1-proteinase inhibitor (alpha 1 PI). The partitioning of the enzyme between the two inhibitors was determined by comparing control esterase activity, assayed with N-benzoyl-L-arginine ethyl ester as substrate, with that remaining after incubation with inhibitory mixtures. (At [I]0 > [E]0, remaining esteratic activity reflects the concentration of alpha 2M-associated enzyme (alpha 2M-E*) and the concentration of alpha 1PI-associated, inactive enzyme (alpha 1PI-E*) is given by the difference, [E]0-[alpha 2M-E*].) The pattern of product distribution was found to be incompatible with an inhibitory model involving parallel, second-order reactions of E with alpha 2M and alpha 1PI. The data pointed to complex formation between the two inhibitors, limiting the level of alpha 2M readily available for reaction with E. Analysis based on the binding equilibrium, alpha 2M (dimeric unit) + alpha 1PI reversible alpha 2M-alpha 1PI, yielded Kd = 2.1 +/- 0.3 microM. Complex formation between alpha 2M and alpha 1PI was verified by gel permeation experiments. alpha 2M was found to restrict the volume of distribution of alpha 1PI in Sephadex G200 beds. Kd, deduced from gel permeation behaviour, was 0.8 +/- 0.32 microM. Preliminary kinetic experiments with dialyzed plasma suggested that the alpha 2M-alpha 1PI interaction is effective also in vivo. Given Kd and the mean plasma levels of the two inhibitors ([alpha 2M] = 2 microM; [alpha 1PI] = 36 microM), it was estimated that > 90% of alpha 2M in human circulation must be complexed to alpha 1PI and lack immediate antiproteinase activity.

Animals↗

PubMed2002

Binding of alpha2-macroglobulin to collagen type I: modification of collagen matrix by alpha2-macroglobulin induces the enhancement of macrophage migration.

Alpha2-macroglobulin (a2M) secreted by tissue macrophages and fibroblasts functions in the environment of extracellular matrix macromolecules. We supposed that it may interact with these molecules and change the properties of extracellular matrix. Modified variant of ELISA was used to prove the direct binding of human a2M to collagen. Native and transformed by plasmin a2M, as well as plasmin, used as the control, were labeled by biotin. It has been found that the transformed, but not the native a2M form binds to type I collagen molecules: K(d)=(1.168 +/- 1.14) x 10(-11) M. The data obtained give a strong evidence of high power of the interaction between a2M and type I collagen: practically no reverse dissociation may be seen for such a binding. The modification of three-dimensional collagen matrix by binding to the transformed a2M resulted in the enhancement of migration of macrophages, carrying the receptors for a2M, but not splenocytes that lack for such receptors. Our results allow to suggest that a2M may be one of the components of extracellular matrix, and may change the properties of microenvironment for immunocompetent cells during the processes of inflammation, reparation and tumor invasion.

Cell Movement↗

PubMed1992

Purified alpha 2-macroglobulin receptor/LDL receptor-related protein binds urokinase.plasminogen activator inhibitor type-1 complex. Evidence that the alpha 2-macroglobulin receptor mediates cellular degradation of urokinase receptor-bound complexes.

Complexes between 125I-labeled urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) bound to purified alpha 2-macroglobulin (alpha 2M) receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP). No binding was observed when using uPA. The magnitude of uPA.PAI-1 binding was comparable with that of the alpha 2MR-associated protein (alpha 2MRAP). Binding of uPA.PAI-1 was blocked by natural and recombinant alpha 2MRAP, and about 80% inhibited by complexes between tissue-type plasminogen activator (tPA) and PAI-1, and by a monoclonal anti-PAI-1 antibody. In human monocytes, uPA.PAI-1, like uPA and its amino-terminal fragment, bound to the urokinase receptor (uPAR). Degradation of uPAR-bound 125I-uPA.PAI-1 was 3-4-fold enhanced as compared with uncomplexed uPAR-bound uPA. The inhibitor-enhanced uPA degradation was blocked by r alpha 2MRAP and inhibited by polyclonal anti-alpha 2MR/LRP antibodies. This is taken as evidence for mediation of internalization and degradation of uPAR-bound uPA.PAI-1 by alpha 2MR/LRP.

Amino Acid Sequence↗