Clearance and binding of two electrophoretic "fast" forms of human alpha 2-macroglobulin

These studies explore the role of conformational change and exposed carbohydrate residues in the clearance of alpha 2-macroglobulin-trypsin (alpha 2M-T) complexes in the mouse. Human alpha 2-macroglobulin (alpha 2M) was purified and demonstrated to be homogeneous in the electrophoretic "slow" form. Two conformationally altered derivatives, alpha 2M-T and alpha 2-macroglobulin-methylamine (alpha 2M-MeNH2), were prepared and demonstrated to exist in the electrophoretic "fast" form. Radiolabeled alpha 2M-T and alpha 2M-MeNH2 were cleared rapidly with a half-life of 2-4 min following injection into mice. Radiolabeled native alpha 2M, however, remained in the circulation with a half-life of several hours. Both alpha 2M-T and alpha 2M-MeNH2 bound specifically to mouse peritoneal macrophages at 4 degrees C and occupancy of receptor sites increased with increasing time and radioligand concentration. Excess amounts of unlabeled alpha 2M-T or alpha 2M-MeNH2 cross-completed with trace amounts of the other in both clearance studies and binding assays, indicating that both derivatives were removed by the same receptor pathway. The clearance and binding of alpha 2M-T and alpha 2M-MeNH2 were not inhibited by excess amounts of unlabeled asialoorosomucoid, fucosyl-bovine serum albumin, mannosyl-BSA, or N-acetylglucosaminyl-BSA. Our results indicate that the clearance pathway removing alpha 2M-T complexes from the circulation recognizes a fundamental conformational change in alpha 2M secondary to protease binding, which can also be induced by exposure to methylamine. Therefore, other chemical or physical alterations that occur in alpha 2M upon binding trypsin, apart from the conformational change also present in alpha 2M-MeNH2, do not seem necessary for the recognition of alpha 2M-T by cells in the clearance pathway. In addition, this pathway appears distinct from several systems already described mediating clearance of glycoproteins through recognition of terminal galactose, fucose, N-acetylglucosamine, or mannose on oligosaccharide side chains.     

High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor cells in several developmental lineages and by some types of malignant cells. In vitro studies have suggested that NG2 participates in growth factor activation of the platelet-derived growth factor-alpha receptor. In this study the ability of recombinant NG2 core protein to interact with several different growth factors (epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF)165 and transforming growth factor (TGF)-beta1) was investigated using two different assay systems: enzyme-linked immunosorbent assay-type solid-phase binding and an optical biosensor (BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, KD values in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-beta1, VEGF, and EGF exhibited little or no binding to NG2 in either type of assay. These data suggest that NG2 can have an important role in organizing and presenting some types of mitogenic growth factors at the cell surface.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

Purification and characterization of a "half-molecule" alpha 2-macroglobulin from the southern grass frog: absence of binding to the mammalian alpha 2-macroglobulin receptor

An alpha-macroglobulin (alpha 2M), which is a dimer consisting of two non-disulfide-bonded subunits, was identified and purified from frog plasma by Ni2+ chelate affinity chromatography. This frog "half-molecule" alpha-macroglobulin migrated as an alpha 2-globulin in cellulose-acetate electrophoresis rather than as the previously described frog alpha 1M, which exists as a tetramer formed by the noncovalent association of disulfide-bonded pairs. A molecular weight of approximately 380 000 was obtained by gel-filtration high-pressure liquid chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the protein migrated as a single band of Mr approximately 180 000 before and after reduction. No evidence was obtained for association of this protein to a higher molecular weight species. After the preparation was heated, additional bands were obtained in SDS-PAGE with Mr approximately 60 000 and 12 000. The additional bands were not obtained after heating methylamine-treated preparations. The circular dichroic spectrum of frog alpha 2M exhibits negative ellipticity over the region 205-250 nm with a minimum at 216 nm. After reaction with proteinase, a decrease in the absolute mean residue rotation was obtained. Amino acid analysis demonstrated that frog alpha 2M and alpha 1M are similar in composition to avian and mammalian alpha-macroglobulins; however, there are sufficient differences in the composition of these two amphibian alpha-macroglobulins to support the conclusion that they are distinct proteins. Frog alpha 2M bound approximately 0.5 mol of trypsin/mol of inhibitor. This binding was abolished by pretreatment with methylamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited mutations in full-length tetrameric human alpha2-macroglobulin abrogate binding of platelet-derived growth factor-BB and transforming growth factor-beta1

alpha2-Macroglobulin (alpha2M) inhibits diverse extracellular proteases, binds growth factors such as platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-beta1 (TGF-beta1), and carries beta-amyloid peptide. alpha2M may also trigger cell signaling by binding to the low density lipoprotein receptor-related protein (LRP-1) and/or other cell surface receptors. Based on studies with recombinant alpha2M fragments expressed in bacteria and synthetic peptides, we previously localized a growth factor-binding site near the center of the alpha2M subunit. However, because intact alpha2M forms a hollow cylinder structure, an alternative model for growth factor binding involves nonspecific entrapment within the alpha2M core. To distinguish between these two models, we engineered mutations in the putative growth factor binding sequence of full-length alpha2M. These mutations did not perturb the tetrameric structure of alpha2M, reaction with proteases, the thiol ester bonds, or binding to LRP-1. A single mutation (E730R) completely blocked binding of platelet-derived growth factor-BB to intact alpha2M. E730R did not alter TGF-beta1 binding; however, this mutation in combination with mutations at Glu714 and Asp719 eliminated the increase in TGF-beta1 binding associated with alpha2M conformational change. These studies demonstrate that growth factor binding to intact alpha2M is specific, involving a defined region of the alpha2M subunit. The exact sequences required for binding different growth factors may be non-identical, mimicking the model of the bait region in which different proteases target adjacent and sometimes overlapping sequences.     

Inhibition and partial reversal of the methylamine-induced conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin by modification of the thiols.

It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J. J., & Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin (alpha 2M) normally resulting from reaction of alpha 2M with methylamine. The kinetics of reaction of DNPSCN with alpha 2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of alpha 2M with methylamine [Larsson, L. J., & Bj?rk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS is liberated per mole of free thiol in alpha 2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3(-) can also react with the methylamine-generated thiol groups of alpha 2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3(-) inhibits the conversion of alpha 2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated alpha 2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of alpha 2M.(ABSTRACT TRUNCATED AT 250 WORDS)     

The conformation-dependent interaction of alpha 2-macroglobulin with vascular endothelial growth factor. A novel mechanism of alpha 2-macroglobulin/growth factor binding

alpha(2)-Macroglobulin (alpha(2)M) is a highly conserved proteinase inhibitor present in human plasma at high concentration (2-4 mg/ml). alpha(2)M exists in two conformations, a native form and an activated, receptor-recognized form. While alpha(2)M binds to numerous cytokines and growth factors, in most cases, the nature of the alpha(2)M interaction with these factors is poorly understood. We examined in detail the interaction between alpha(2)M and vascular endothelial growth factor (VEGF) and found a novel and unexpected mechanism of interaction as demonstrated by the following observations: 1) the binding of VEGF to alpha(2)M occurs at a site distinct from the recently characterized growth factor binding site; 2) VEGF binds different forms of alpha(2)M with distinct spatial arrangement, namely to the interior of methylamine or ammonia-treated alpha(2)M and to the exterior of native and proteinase-converted alpha(2)M; and 3) VEGF (molecular mass approximately 40 kDa) can access the interior of receptor-recognized alpha(2)M in the absence of a proteinase trapped within the molecule. VEGF bound to receptor-recognized forms of alpha(2)M is internalized and degraded by macrophages via the alpha(2)M receptor, the low density lipoprotein receptor-related protein. Oxidation of both native and receptor-recognized alpha(2)M results in significant inhibition of VEGF binding. We also examined the biological significance of this interaction by studying the effect of alpha(2)M on VEGF-induced cell proliferation and VEGF-induced up-regulation of intracellular Ca(2+) levels. We demonstrate that under physiological conditions, alpha(2)M does not impact the ability of VEGF to induce cell proliferation or up-regulate Ca(2+).     

Changes in the binding of "fast"-form alpha 2-macroglobulin to 3T3-L1 cells after differentiation to adipocytes

Human alpha 2-macroglobulin (alpha 2M)-CH3NH2 specifically binds to 3T3-L1 fibroblasts and adipocytes with an apparent Kd of 0.3 nM at 4 degrees C. Binding to fibroblasts follows first-order kinetics only for the first 20-30 min of reaction, k1 = 160 microM-1 h-1, and then proceeds in a non-first-order reaction that takes 28 h to reach steady state. Receptor activity is 120 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is nondissociable. In contrast, binding to adipocytes follows first-order kinetics, k1 = 720 microM-1 h-1, and reaches steady state in 6-8 h. Receptor activity is 35 fmol of alpha 2M-CH3NH2/mg of cell protein or 60 000 molecules/cell. Binding is reversible with a k2 of 0.4 h-1. Control studies with 3T3-C2 cells, which do not differentiate after hormone treatment, indicate that these differences are not due to hormone treatment alone. Binding to both fibroblasts and adipocytes is specific for "fast"-form alpha 2M but not for native alpha 2M. Inhibition studies with neoglycoproteins demonstrate that binding does not occur via any of the known carbohydrate receptors. Some cross-reactivity with antithrombin III-trypsin complexes is demonstrated. Both fibroblasts and adipocytes take up and degrade alpha 2M-CH3NH2 at 37 degrees C. For both cell types, the concentration of alpha 2M-CH3NH2 needed for half-maximal uptake is 65 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppressive properties of electrophoretically "slow" and "fast" form alpha 2-macroglobulin. Effects on cell-mediated cytotoxicity and (allo-) antigen-induced T cell proliferation

Lymphokine activated killer cell lysis of K562 cells was inhibited by alpha 2-macroglobulin (alpha 2M), soybean trypsin inhibitor, and alpha 1-proteinase inhibitor. In serum free medium 2 mg/ml alpha 2M suppressed target cell lysis in a 4-h cytotoxic assay with about 40%. Suppression was dose and time dependent. Cytotoxicity was unaffected by alpha 2M concentrations less than 0.25 mg/ml, and by alpha 2M added later than 1.5 h from start of assay. Pre-treatment of effector (but not of target) cells with alpha 2M was even more suppressive than the presence of alpha 2M during assay. Cell-mediated cytotoxicity was not inhibited by alpha 2M treated with methylamine or by various alpha 2M-proteinase complexes. In contrast, alpha 2M-proteinase complex as well as native alpha 2M suppressed the proliferation of Ag-activated T cells. However, methylamine-treated alpha 2M did not inhibit T cell proliferation, and suppression by alpha 2M-proteinase complex was significantly reduced after inhibition of the alpha 2M-bound proteinase. On incubation at 4 degrees C with lymphokine-activated killer cells, alpha 2M reacted with cell associated proteinases and changed from electrophoretically "slow" to "fast" form. Cell associated proteinases bound by alpha 2M showed chymotrypsin- and trypsin-like specificities and their activity surpassed activity caused by cellular leakage and secretion. The present results strongly indicate that alpha 2M mediates immunosuppression in its capacity as a proteinase inhibitor and suggest inhibition of (T)cell surface-associated proteinases as a possible mode of suppression.     

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*.     

Absence of binding of pancreatic and urinary kallikreins to alpha 2-macroglobulin.

Pancreatic and urinary kallikreins failed to form the typical serine proteinase complex with alpha2M (alpha2-macroglobulin). Studies were performed to compare this with the binding of trypsin to alpha2M at various molar binding ratios, with the use of Sephadex G-200 gel filtration to separate free and alpha2M-bound enzyme fractions. The subunit conversion was totally absent with pancreatic kallikrein from lhich traces of a binding proteinase had been removed. The lack of binding is believed to be the result of the restricted specificity of the kallikreins.     

Identical or overlapping sequences in the primary structure of human alpha(2)-macroglobulin are responsible for the binding of nerve growth factor-beta, platelet-derived growth factor-BB, and transforming growth factor-beta

alpha(2)-Macroglobulin (alpha(2)M) functions as a proteinase inhibitor and as a carrier of diverse growth factors. In this study, we localized binding sites for platelet-derived growth factor-BB (PDGF-BB) and nerve growth factor-beta (NGF-beta) to a linear sequence in the 180-kDa human alpha(2)M subunit which includes amino acids 591-774. A glutathione S-transferase fusion protein containing amino acids 591-774 (FP3) bound PDGF-BB and NGF-beta in ligand blotting assays whereas five other fusion proteins, which collectively include amino acids 99-590 and 775-1451 did not. The K(D) values for PDGF-BB and NGF-beta binding to immobilized FP3 were 300 +/- 40 and 180 +/- 30 nM, respectively; these values were comparable with those determined using methylamine-modified alpha(2)M, suggesting that higher-order alpha(2)M structure is not necessary for PDGF-BB and NGF-beta binding. PDGF-BB and NGF-beta blocked the binding of transforming growth factor-beta1 (TGF-beta1) to FP3. Furthermore, murinoglobulin, which is the only known member of the alpha-macroglobulin family that does not bind TGF-beta, also failed to bind PDGF-BB and NGF-beta. These results support the hypothesis that either a single linear sequence in human alpha(2)M or overlapping sequences are responsible for the binding of TGF-beta, PDGF-BB, and NGF-beta, even though there is minimal sequence identity between these three growth factors. FP3 blocked the binding of PDGF-BB to a purified chimeric protein, in which the extracellular domain of the PDGF beta receptor was fused to the IgG(1) Fc domain, and to PDGF receptors on NIH 3T3 cells. Thus, FP3 may inhibit the activity of PDGF-BB.     

Reaction of human alpha 2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of alpha 2-macroglobulin with plasmin increases binding of transforming growth factors-beta 1 and beta 2.

The binding of 125I-transforming growth factors-beta 1 and beta 2 (TGF-beta 1 and TGF-beta 2) to alpha 2-macroglobulin (alpha 2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-beta and native or reacted forms of alpha 2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native alpha 2M with plasmin or methylamine markedly increased the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2 to alpha 2M. The alpha 2M-plasmin/TGF-beta complexes were minimally dissociated by heparin. Reaction of alpha 2M with thrombin or trypsin reduced the binding of 125I-TGF-beta 1 and 125I-TGF-beta 2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-beta 2 and native or reacted forms of alpha 2M were less dissociable by heparin than the equivalent complexes with TGF-beta 1. These studies demonstrate that the TGF-beta-binding activity of alpha 2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that alpha 2M-plasmin preferentially binds TGFs-beta suggest a mechanism by which alpha 2M may regulate availability of TGFs-beta to target cells in vivo.     

Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.     

Binding of transforming growth factor-beta 1 to immobilized human alpha 2-macroglobulin.

Native alpha 2-macroglobulin (alpha 2M) and alpha 2M-methylamine were immobilized in 96-well microtiter plates. 125I-labeled transforming growth factor-beta 1 (TGF-beta 1) bound to both alpha 2M variants; however, greater binding was observed with alpha 2M-methylamine. Binding of 125I-TGF-beta 1 (0.2 nM) to immobilized alpha 2M-methylamine was inhibited by nonradiolabeled TGF-beta 1 (up to 74% with 0.4 microM TGF-beta 1). Approximately 10% of the TGF-beta 1-alpha 2M-methylamine complex was covalent. Treatment of alpha 2M-methylamine with iodoacetamide prior to immobilization completely eliminated covalent TGF-beta 1 binding; the total amount of 125I-TGF-beta 1-alpha 2M-methylamine complex detected was unchanged. The binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was not significantly inhibited by increasing the ionic strength to 1.0 M. Binding and complex dissociation were also unaffected by changes in pH within the range 6.9-8.9. Acidic pH dramatically decreased binding and promoted complex dissociation; no binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine was detected at pH 3.5. The interaction of TGF-beta 1 with immobilized alpha 2M-methylamine was not significantly changed by 1.0 mM EDTA or 1.0 mM CaCl2. ZnCl2 (1.0 mM) completely eliminated binding. This result was not due to TGF-beta 1 precipitation or aggregation. Inhibition of 125I-TGF-beta 1 binding to alpha 2M-methylamine was 50% complete (IC50) with 30 microM ZnCl2. Native alpha 2M, thrombospondin, and alpha 2M-methylamine (in solution) decreased binding of 125I-TGF-beta 1 to immobilized alpha 2M-methylamine. The IC50 values for these three proteins were 520, 160, and 79 nM, respectively. The TGF-beta 1-binding activity of native alpha 2M may have reflected, at least in part, trace-contamination with alpha 2M-proteinase complex.     

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.     

Mechanism of insulin incorporation into alpha 2-macroglobulin: implications for the study of peptide and growth factor binding.

In recent years, many studies have suggested a direct role for alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor, in growth factor regulation. When coincubated in the presence of either trypsin, pancreatic elastase, human neutrophil elastase, or plasmin, 125I-insulin rapidly formed a complex with alpha 2M which was greater than 80% covalent. The covalent binding was stable to reduction but abolished by competition with beta-aminopropionitrile. Neither native alpha 2M nor alpha 2M pretreated with proteinase or methylamine incorporated 125I-insulin. Experiments utilizing alpha 2M cross-linked with cis-dichlorodiammineplatinum(II) indicated that 125I-insulin must be present during alpha 2M conformational change to covalently bind. A maximum stoichiometry of 4 mol of insulin bound per mole of alpha 2M and the short half-life of the alpha 2M intermediate capable of covalent incorporation were consistent with thiol ester involvement. Protein sequence analysis of unlabeled insulin-alpha 2M complexes, together with results of beta-aminopropionitrile competition, confirmed that insulin incorporation occurs via the same gamma-glutamyl amide linkage responsible for covalent proteinase and methylamine binding to alpha 2M. Although intact insulin apparently incorporated through its sole lysine residue on the B chain, we found that isolated A chain also bound covalently to alpha 2M. Phenyl isothiocyanate derivatization of the N-terminus had no effect on A-chain binding, supporting the possibility of heretofore unreported gamma-glutamyl ester linkages to alpha 2M.     

Cellular response to platelet-derived growth factor (PDGF)-AB after down-regulation of PDGF alpha-receptors. Evidence that functional binding does not require alpha-receptors.

Platelet-derived growth factor (PDGF) and its receptor exist in multiple forms. PDGF exists in three dimeric combinations of A and B subunit chains, which are the products of separate genes. The PDGF receptor is similarly encoded by genes for two distinct receptor proteins, alpha and beta. A recent model proposed PDGF binding involves the association of the two receptor proteins into three possible dimeric forms. An essential prediction of that model is that PDGF alpha-receptors are required for cells to bind and respond to the heterodimeric AB isoform of PDGF. In contrast, we found both binding and functional response to PDGF-AB was retained in Balb/c-3T3 cells after PDGF alpha-receptors had been down-regulated by PDGF-AA pretreatment. The observation that PDGF-AB could still elicit these responses suggests that at 37 degrees C, PDGF-AB may bind directly to beta-receptors in either monomeric or dimeric forms and that initial receptor activation may occur independently of the formation of alpha beta-receptor heterodimers.     

Comparison of the binding of chicken alpha-macroglobulin and ovomacroglobulin to the mammalian alpha 2-macroglobulin receptor

Chicken alpha-macroglobulin (alpha M) and ovomacroglobulin were purified by Ni+2 chelate chromatography. These proteins had similar subunit structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Chicken alpha M bound 1.0 mol and ovomacroglobulin bound 0.8 mol 125I-trypsin per mol inhibitor, respectively. Ovomacroglobulin cleared rapidly from the circulation of mice, and the clearance was inhibited by asialoorosomucoid, but native chicken alpha M cleared slowly (t 1/2 greater than 1 h). After reaction with trypsin, this alpha-macroglobulin cleared rapidly (t 1/2 = 3 min), and this clearance was inhibited by a 1000-fold molar excess of human alpha 2M-methylamine. Ovomacroglobulin-trypsin did not inhibit the binding of 0.2 nM 125I-labeled human alpha 2M-methylamine to mouse peritoneal macrophages in vitro, but chicken alpha M reacted with trypsin inhibited the binding by 50% at 1.9 nM. A kappa I of 1.1 nM was calculated for the binding of chicken alpha M-trypsin to the mammalian alpha-macroglobulin receptor. This affinity is comparable to that obtained with human and bovine alpha 2M.