Ontogenic emergence of a quail leukocyte/endothelium cell surface antigen

The ontogenic emergence of MB1, a quail cell surface antigen expressed by endothelial and hemopoietic cells but not erythrocytes, was followed by direct immunofluorescent staining of transverse sections of the developing blastodisc, from the stage of the cephalic fold until 22 pairs of somites. Along the developmental sequence that leads from hemangioblasts, the mesodermal precursors of both endothelium and hemopoietic cells, to vessels containing blood cells, MB1 is first expressed by arising endothelial cells. These first emerge as flattened cells at the periphery of hemangioblastic clusters in the area opaca from the stage of one pair of somites and slightly later as unicellular angioblasts in the area pellucida and in the embryo. MB1 expression is then maintained on endothelium as vessels develop, in contrast with extraembryonic blood islands in which primitive erythroblasts remain MB1-negative. A small proportion of blood island cells and budding of endothelium contribute a population of MB1-positive hemopoietic cells appearing soon after the onset of angiogenesis.     

Preparation and initial characterization of an intermediate, half-cleaved form of human alpha 2-macroglobulin

A form of human alpha 2-macroglobulin (alpha 2M) has been prepared that has properties intermediate to those of native alpha 2-macroglobulin and 2:1 protease-alpha 2 M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of alpha 2M and migrates as a diffuse band. Two bait regions and two thiol esters per alpha 2M tetramer are cleaved, although no chymotrypsin is detectable in the modified alpha 2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form alpha 2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form alpha 2M at rates not distinguishable within experimental error from those of their reactions with native alpha 2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native alpha 2 M, indicating a nonequivalence of the two reactive sites on alpha 2M. This nonequivalence may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted alpha 2M with those from spin-labeling of the free SH groups in intermediate-form alpha 2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form alpha 2M.     

alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Membrane D2 T antigen: characterization and comparison with its nuclear counterpart

Up to 40% of membrane T antigen (D2 T antigen) produced by the adenovirus 2--simian virus 40 hybrid, Ad2+D2, remained tightly associated with membranes under alkaline conditions up to pH 11.5. The antigen could not be totally solubilized by treatment with ionic detergents, nonionic detergents, or both. These properties are characteristic of integral membrane proteins. Sephacryl S-300 chromatography in high salt in the presence of Brij-99 showed that the nuclear form of D2 T antigen was dissociated to low molecular weight species, while the membrane form eluted as a complex of high molecular weight. The membrane form, therefore, is able to bind more detergent than the nuclear form, indicating a difference in supramolecular structure.     

Immunocytochemical identification of the human alpha 2-macroglobulin receptor in monocytes and fibroblasts: monoclonal antibodies define the receptor as a monocyte differentiation antigen

The alpha 2-macroglobulin receptor was recently purified from rat liver and human placenta. Three different monoclonal antibodies have now been raised against the human receptor and expression of the 440-kDa receptor protein is demonstrated in human placenta, fibroblasts, liver, and monocytes by immunoblot analysis. Flow cytometric studies showed that anti-alpha 2-macroglobulin receptor monoclonal antibodies bind to 90-100% of the blood monocyte population and not to other blood cells. This defines the alpha 2-macroglobulin receptor as a monocyte differentiation antigen, different from any of the classified leucocyte cluster determinants. Electron microscopic gold immunocytochemistry revealed the subcellular distribution of the receptor in human cultured monocytes and fibroblasts. In these cells, 18-33% of the gold particles were found on the outside of the plasma membrane, and in fibroblasts, especially, in coated invaginations. The intracellular receptors were mainly distributed in vesicles and tubular structures.     

Studies on the interaction between leukocyte elastase, antileukoproteinase and the plasma proteinase inhibitors alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The dominating inhibitor of leukocyte elastase in human respiratory tract secretions is a low molecular mass inhibitor, designated antileukoproteinase. An equimolar antileukoproteinase-elastase complex was produced and subjected to gel filtration after differing time intervals and was found to be stable. On addition to human serum, however, elastase dissociated from antileukoproteinase and formed a complex with alpha 1-proteinase inhibitor. A small amount of elastase was also found bound to alpha 2-macroglobulin. Antileukoproteinase was capable of inhibiting elastase bound to alpha 2-macroglobulin. This inhibition was more complete and more rapid when the alpha 2-macroglobulin-elastase complex was in a molar ratio of 1:1 than in a ratio of 1:2.     

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.     

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.     

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)     

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.     

Cloning and characterization of a secreted form of angiotensin-converting enzyme 2

Angiotensin-converting enzyme 2 (ACE2) is a newly discovered, membrane-bound aminopeptidase responsible for the production of vasodilatory peptides such as angiotensin 1-7 (Ang 1-7). Thus, ACE2 is important in counteracting the adverse, vasoconstrictor effects of angiotensin II (Ang II). The objective of the present study was to clone and characterize a constitutively secreted form of ACE2 as a prelude to an investigation into its therapeutic potential in hypertension. A truncated form of ACE2 was cloned into a lentiviral vector behind the human elongation factor 1 alpha promoter (lenti-shACE2). Transfection experiments demonstrated that secreted human ACE2 (shACE2) was secreted constitutively into the medium. The kinetic properties of shACE2 were comparable to the human recombinant enzyme (rACE2). Transduction of human coronary artery endothelial cells and rat cardiomyocytes with lenti-shACE2 showed a significant secretion of the enzyme into the medium compared to its native, membrane-bound homolog (human ACE2 [hACE2]). In addition, systemic administration of lenti-shACE2 into neonatal rats resulted in a eightfold increase in ACE2 activity in the serum above control values. These observations establish that lenti-shACE2 can be used to transduce cardiovascularly relevant cells for the secretion of functional ACE2 enzyme both in vitro and in vivo. Collectively, these results set the stage for the use of these vectors to investigate the consequences of ACE2 over-expression in the pathogenesis of hypertension.     

Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Levels of alpha 1-antitrypsin and alpha 2-macroglobulin in blood serum and its antitryptic capacity

Immunochemical analysis in combination with gel filtration and isoelectric focusing made it possible to state that in blood serum of healthy people 81.3 +/- 0.5% of administered trypsin is bound with alpha 1-antitrypsin and 18.7 +/- 0.6%--with alpha 2-macroglobulin. The latter is functionally heterogeneous, only 40% of it is bound with trypsin and in the formed complex the antigenic properties of trypsin and alpha 2-macroglobulin are lost. A great number of blood serum alpha 1-antitrypsin cannot fix trypsin. The content of such alpha 1-antitrypsin rises sharply with pathology available. In the immunochemical estimation of the organism inhibitory potential relative to proteolytic enzymes not only the amount of the inhibitor but also its functional activity should be taken into account. The data of immunochemical research of the blood serum isoelectrophoregrams show that the most considerable changes under conditions of pathology occur in alpha 2-macroglobulin.     

Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.     

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.     

Inactivation of alpha- and beta-thrombin by antithrombin-III, alpha 2-macroglobulin and alpha 1-proteinase inhibitor.

Inactivation of alpha- and beta-thrombin by alpha 2-macroglobulin, by alpha 1-proteinase inhibitor and by antithrombin-III and heparin was studied. The amount of alpha- and beta-thrombin inactivated by antithrombin-III was proportional to the concentration of the inhibitor, but the inactivation rates of the two forms of thrombin were different. Heparin facilitated complex-formation between alpha-thrombin and antithrombin-III, whereas inactivation of beta-thrombin by antithrombin was only slightly influenced, even at a heparin concentration two orders of magnitude higher. alpha 2-Macroglobulin inhibited both alpha- and beta-thrombin activity similarly, i.e. the amount of alpha- and beta-thrombin inactivated as well as the rates of their inhibition were the same. alpha 1-Proteinase inhibitor also formed a complex with alpha- and beta-thrombin, similarly to antithrombin-III, although the inactivation of the enzyme needed high inhibitor concentration and long incubation time. These results suggest that the inactivation of beta-thrombin, if it occurs in the plasma, is also controlled by plasma inhibitors.     

Isolation of rat serum alpha 1-microglobulin. Identification of a complex with alpha 1-inhibitor-3, a rat alpha 2-macroglobulin homologue.

Alpha 1-Microglobulin (alpha 1-m), or protein HC, a low molecular weight plasma protein with immunoregulatory properties, was isolated from rat serum by affinity chromatography using Sepharose-coupled monoclonal anti-alpha 1-m antibodies. High molecular weight forms of alpha 1-m were then separated from the low molecular weight alpha 1-m by gel chromatography of the eluted proteins. The apparent Mr (28,000), the charge heterogeneity, the N-linked carbohydrate, and yellow-brown chromophore suggest that the low molecular weight alpha 1-m is the serum counterpart to urinary alpha 1-m, which was purified previously. A high molecular weight complex of alpha 1-m was also isolated by the gel chromatography. It was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. The molecule was bound by antibodies against human alpha 2-macroglobulin, and experiments with antisera against the three alpha-macroglobulin variants in rat serum, alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor-3 (alpha 1I3) suggested that alpha 1I3 was the complex-partner of alpha 1-m. An antiserum raised against high molecular weight alpha 1-m was then used to isolate the complex-partner of alpha 1-m from rat serum with affinity chromatography, and this molecule was positively identified as alpha 1I3 by its physicochemical properties. Gel chromatography of the alpha 1I3.alpha 1-m complex suggested a molecule with an Mr of 266,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, it migrated as three major molecular species with apparent molecular weights of 224,000, 205,000, and 194,000 and several minor species of both higher and lower molecular weights, suggesting a complex subunit structure. alpha 1-m and alpha 1I3 could be detected in all three major species by Western blotting, and NH2-terminal amino acid sequencing suggested a molar ratio of 1:1 of alpha 1-m and alpha 1I3 in all three species. alpha 1I3.alpha 1-m was colorless, did not show light absorbance beyond 300 nm which is typical of low molecular weight alpha 1-m and was electrophoretically homogeneous, suggesting that it lacks the chromophore. Finally, the serum concentrations of the alpha 1I3.alpha 1-m complex and free alpha 1-m were determined as 0.16 and 0.010 g/liter, respectively. Thus, alpha 1I3.alpha 1-m constitutes 1-3% of the total alpha 1I3 in rat serum (w/w) and approximately 60% of the total alpha 1-m.     

Evolution of alpha 2-macroglobulin. The purification and characterization of a protein homologous with human alpha 2-macroglobulin from plaice (Pleuronectes platessa L.) plasma.

A papain-binding protein (PB-protein) was purified to homogeneity from the plasma of plaice (Pleuronectes platessa L.). PB-protein inhibited the activity of trypsin and pancreatic elastase (serine proteinases), thermolysin (a metalloproteinase) and papain (a cysteine proteinase). Presaturation of PB-protein with trypsin prevented the subsequent inhibition of thermolysin, and vice versa. Only catalytically active endopeptidases were bound by PB-protein. The catalytic activity of trypsin bound by PB-protein was inhibited by 95% against an insoluble protein substrate, but only by 38% against a low-molecular-weight synthetic substrate. The remaining activity of the bound trypsin was partially protected against further inhibition by soya-bean trypsin inhibitor. Trypsin bound by PB-protein showed a decrease of 67% in its reactivity with antibodies. The inhibitory activity of PB-protein was inactivated at pH 8.0 by methylamine (0.2M) or dithiothreitol (1 mM). The inhibition of proteinases by plaice PB-protein shows the distinctive characteristics of inhibition by human alpha 2-macroglobulin, and it is concluded that the plaice protein is a homologue of the human macroglobulin.     

Sequence similarity between alpha 2-macroglobulin from the horseshoe crab, Limulus polyphemus, and proteins of the alpha 2-macroglobulin family from mammals.

1. Purified alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus was cleaved with trypsin and 20 of the tryptic peptides were sequenced and compared with the sequences of human alpha 2M, rat alpha 1M, alpha 2M, and alpha 1-inhibitor 3, and human complement proteins C3 and C4. 2. Ten of the peptides (233 residues), including that containing the thiol ester site, could be aligned unambiguously with stretches in mammalian alpha 2M, with a degree of identity greater than 30%. 3. The 12-residue thiol ester-containing peptide of Limulus alpha 2M showed 67% identity with the same stretch of human alpha 2M.