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

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

Isolation and characterization of horse alpha 2-macroglobulin protease inhibitor

Several publications have described in the past properties of partly purified horse alpha 2-macroglobulin (alpha 2M) which are strikingly different from the human alpha 2M. Horse alpha 2M was therefore isolated to purity by classical procedures, i.e. affinity chromatography, ion exchange chromatography and gel filtration, and its properties are compared with those of its human counterpart. The molecular weight of the native protein and its subunits, the isoelectrofocusing pattern and the change in electrophoretic mobility caused by interaction with protease were similar to those of human alpha 2M. Horse alpha 2M had a broad enzyme specificity and inhibited enzymatic action on macromolecules but not on small molecular weight synthetic substrates. In addition the horse and human alpha 2M were found to be immunochemically related when examined by specific antisera to human as well as to horse alpha 2-macroglobulin.     

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

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

Sertoli cell synthesizes and secretes a protease inhibitor, alpha 2-macroglobulin.

The mechanism by which the seminiferous epithelium limits the damaging effects of proteases that are released from degenerating late spermatids does not depend upon protease inhibitors in the systemic circulation since these proteins are excluded from the seminiferous tubule by the blood-testis barrier. The purpose of this study was to identify the major protease inhibitor of the testis and determine its cellular origin. Sertoli cells, the major epithelial component of the seminiferous epithelium, release a protease inhibitor, testicular alpha 2-macroglobulin, in vitro. Immunoprecipitation using [35S]methionine and a monospecific polyclonal antibody prepared against purified testicular alpha 2-macroglobulin establishes that this protein is actively synthesized and secreted by Sertoli cells. Measurements of immunoreactive protease inhibitors in tubular and rete testis fluids collected by micropuncture suggest that alpha 2-macroglobulin rather than alpha 1-antitrypsin is the major protease inhibitor in the seminiferous tubules in vivo. The ability of alpha 2-macroglobulin to inactivate proteases and growth factors such as TGF-beta by a common mechanism suggests that this protein may have a dual function in the testis.     

Inhibitory effect of alpha 2-macroglobulin on Vibrio vulnificus protease

Vibrio vulnificus, an etiologic agent of wound infections and septicemia in humans, elaborates a metalloprotease which is known to be an important virulence factor of the Vibrio. The proteolytic activity of V. vulnificus metalloprotease (VVP) toward casein and elastin was inhibited by alpha 2-macroglobulin (alpha 2 M) at the molar ratio of 1:1, although partial activity was maintained. Permeability-enhancing and hemorrhagic activities were also inhibited, but the peptidase activity toward Z-Gly-Phe-NH2 was not reduced, even by an excess amount of alpha 2 M. VVP formed a complex with alpha 2 M through cleavage of the bait regions of all four alpha 2 M subunits and elicitation of conformational change of the alpha 2 M molecule, which resulted in entrapment of VVP in the alpha 2 M molecule. The peptidase activity of alpha 2 M-VVP complex was inhibited by low-molecular-weight inhibitors such as phosphoramidon, but IgG antibody against VVP failed to neutralize its peptidase activity. Of human plasma proteins, alpha 2 M was the only inhibitor for VVP. These findings indicate that VVP produced during V. vulnificus infection is inactivated by plasma alpha 2 M that leaks from the vascular system.     

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

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

Platelet alpha2-macroglobulin and alpha1-antitrypsin.

Subcellular membrane and granule fractions derived from human platelets contain immunologically identifiable alpha2-macroglobulin and alpha1-antitrypsin. These platelet-derived inhibitors show a reaction of immunologic identity when compared to alpha2-macroglobulin and alpha1-antitrypsin purified from human plasma. Further, the platelet protease inhibitors possessed a similar subunit polypeptide chain structure to their plasma counterparts as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis. Studies of the binding of radiolabeled trypsin to the various solubilized platelet subcellular fractions suggest that the granule-associated alpha2-macroglobulin and alpha1-antitrypsin, as well as membrane-associated alpha2-macroglobulin were functionally active. Quantitatively, circulating platelets contain relatively small concentrations of these inhibitors as compared to platelet-associated fibrinogen and factor VIIIAGN. Platelet protease inhibitors may modulate the protease-mediated events involved in the formation of hemostatic plugs and thrombi.     

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.     

Kinetic evidence that Glut4 follows different endocytic pathways than the receptors for transferrin and alpha2-macroglobulin

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and α(2)-macroglobulin (α-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k(ex)) for both Glut4 and Tf. However, the k(ex) of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k(en)) of Glut4 was also five times slower than Tf. Insulin did not affect the k(en) of either protein. In basal cells, the k(en) for α-2-M/LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k(en) for α-2-M/LRP-1 by 30%. In contrast, the k(ex) for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k(en) of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k(en) can be entirely accounted for by the well established increase in Glut4 k(ex).     

The temperature-dependent reaction between alpha 2-macroglobulin and streptokinase-plasmin(ogen) complex

The reactions of alpha 2-macroglobulin (alpha 2M) with plasmin or streptokinase-plasmin(ogen) (SkPl) were studied as a function of temperature. alpha 2M and plasmin reacted relatively rapidly at all temperatures. The initial rates of reaction were identical at 24 and 37 degrees C. At 4 degrees C, the initial reaction rate was somewhat slower; however, the reaction was 90% complete in 2 min. The reaction of alpha 2M with SkPl was markedly temperature-dependent. Initial rates of reaction at 0 and 24 degrees C were only 3 and 40% of the rate of 37 degrees C, respectively. When these reactions occur, only the plasmin moiety is incorporated in the alpha 2M molecule. These data explain the inconsistencies in previous reports of SkPl interactions with alpha 2M, since experiments have been performed at different temperatures in various studies. In the present work, we present a model which interprets the data in terms of a possible high-energy transition state in the alpha 2M-SkPl reaction.     

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

Evolution of alpha 2-macroglobulin. The demonstration in a variety of vertebrate species of a protein resembling human alpha 2-macroglobulin.

The amino acid sequence of the Pronase-released heads of neuraminidase subtype N2 from the A/Tokyo/3/67 strain of influenza virus was determined by a combination of peptide and nucleic acid sequence analysis. The results show that the Pronase-released heads contain 396 amino acid residues and extend from residue 74 in the original protein to the C-terminus at residue 469. The heads contain five potential glycosylation sites at asparagine residues 86, 146, 200, 234 and 402, but only the first four are glycosylated. The sequence homology with the corresponding region of the previously published sequence of the neuraminidase subtype N1 [Fields, Winter & Brownlee (1981) Nature (London) 290, 213-217] is 45%. Detailed evidence for the sequence data has been deposited as Supplementary Publication SUP 50116 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1981) 193, 5.     

Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.

The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase     

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.     

Differential scanning calorimetry of alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes.

Differential scanning calorimetry is shown to detect substantial structural alterations occurring on the association of proteinases with the serum glycoprotein alpha 2-macroglobulin. At pH 7.5, the thermally induced unfolding of the macroglobulin occurs at approx. 60 degrees C with a transition enthalpy of 17 J/g. Association of active thermolysin, trypsin and papain shifts the transition temperature to 77 degrees C (transition enthalpy 5 J/g), indicating that a substantial conformational change accompanies the binding event. The stoicheiometry of the thermolysin--alpha 2-macroglobulin association producing this change appears to be unity, implying the presence of co-operative subunit interactions in the mechanism of association. The calorimetric method provides a novel approach for the evaluation of conformational variants induced on protein-protein association or pre-existing in the purified macroglobulin.     

Structural analysis of acute-phase alpha 2-macroglobulin.

High resolution images of rat acute-phase alpha 2-macroglobulin (AP alpha 2M) have been obtained by using dark-field electron microscopy. No staining or artifact-inducing procedures were used. Analysis of unfiltered electron microscope plates, exposed to minimal electron beam radiation, revealed highly contrasted particles of variable morphology with dimensions of approx. 19 nm X 14 nm. An electron-dense core with four to six projections could be seen. Two-fold symmetry was evident in selected images, supporting the four-subunit composition of the protein. Image processing and filtering confirmed the presence and configuration of the projections by demonstrating exact molecular dimensions of 16 nm X 9.5 nm and a shape with six projections like that of the Russian letter zh. SDS/polyacrylamide-gel electrophoresis revealed that this molecule was in the proteinase-bound form. C.d. data revealed a surprisingly low content of alpha-helical secondary structure (12%) and an atypically large content of beta-form structure (33%). Comparison of the amino acid compositions of AP alpha 2M and human alpha 2-macroglobulin indicated a high degree of homology between the two molecules. It is concluded that the conformation of rat AP alpha 2M, both at the molecular and secondary structural levels, is strikingly similar to that of human alpha 2-macroglobulin.     

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.     

Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex

The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.