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PubMed1996

Activated human plasma carboxypeptidase B is retained in the blood by binding to alpha2-macroglobulin and pregnancy zone protein.

A 66-kDa glycosylated carboxypeptidase, plasma pro-carboxypeptidase B (pro-plasma CPB), has recently been identified in human blood (Eaton, D. L., Malloy, B. E., Tsai, S. P., Henzel, W., and Drayna, D. (1991) J. Biol. Chem. 266, 21833-21838). The pro-enzyme binds to plasminogen and the active enzyme is specific for COOH-terminal Lys or Arg residues. These properties implicate a role in the fibrinolytic or coagulation system. However, we show that the molecular mass of the active plasma CPB is approximately 36 kDa, which is below the glomerular filtration limit. Since activated plasma CPB no longer binds plasminogen, the active enzyme may not be retained in the circulation. To investigate this, we performed plasma elimination studies in mice which showed that 125I-plasma CPB remains in the circulation despite its small size. Native polyacrylamide gel electrophoresis of blood samples removed from the mice revealed that plasma CPB migrated as a high molecular weight band. Similar bands were observed in vitro when 125I-plasma CPB was added to plasma from humans and other species. The plasma CPB-binding proteins were purified from human plasma and identified as alpha2-macroglobulin (alpha2M) and pregnancy zone protein. Only the active enzyme bound to the two alpha-macroglobulins, and the interaction was specific for alpha2M in its native conformation, but not its receptor recognized forms. The complex between human alpha2M and plasma CPB dissociated during SDS-polyacrylamide gel electrophoresis and transverse urea gel electrophoresis suggesting that the interaction was noncovalent and depended on the tertiary structure of the native alpha2M molecule. The catalytic activity of plasma CPB was not significantly affected by its binding to alpha2M. The specific binding of plasma CPB to alpha-macroglobulins suggest that these proteins may function as a "shuttle" in vivo to modulate the clearance of plasma CPB from the circulatory system.

Carboxypeptidase B↗

PubMed1998

The low density lipoprotein receptor gene family. Differential expression of two alpha2-macroglobulin receptors in the brain.

LR7/8B is a member of the low density lipoprotein receptor gene family that is specifically synthesized in the brain. Here we have functionally expressed in 293 cells the splice variant harboring eight ligand binding repeats (LR8B). As assessed by confocal microscopy, the expressed receptor is localized to the plasma membrane. Importantly, in cell binding experiments, we demonstrate that this protein is a receptor for activated alpha2-macroglobulin. Because to date low density lipoprotein receptor-related protein (LRP) has been shown to be the only alpha2-macroglobulin receptor in brain, we became interested in the expression pattern of both proteins at the cellular level in the brain. LR7/8B is expressed in large neurons and Purkinje cells of the cerebellum and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus, the arachnoidea, and the endothelium of penetrating blood vessels. Anti-LR7/8B antibody stains the plasma membrane, dendrites, and vesicular structures close to the cell membrane of neurons, especially of Purkinje cells. In contrast, LRP is present in patchy regions around large neurons and most prominently in the glomeruli of the stratum granulare of the cerebellum. This suggests that, contrary to LR7/8B, LRP is expressed in synaptic regions of the neurons; furthermore, there is a striking difference in the expression patterns of LR7/8B and LRP in the choroid plexus. Whereas LRP shows baso-lateral and apical localization in the epithelial cells, LR7/8B is restricted to the apical cell aspect facing the cerebrospinal fluid. Finally, these studies were extended to cultured primary rat neurons, where double immunofluorescence labeling with anti-LR7/8B and anti-microtubuli-associated protein 2 (MAP2) confirmed the somatodendritic expression of the receptor. Based upon these data, we propose that LR7/8B is involved in the clearance of alpha2-macroglobulin.proteinase complexes and/or of other substrates bound to alpha2-macroglobulin from the cerebrospinal fluid and from the surface of neurons.

Alternative Splicing↗

PubMed1999

Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor.

Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.

Animals↗

PubMed1963

Sequences of synthesis of gamma-1 macroglobulin and gamma-2 globulin antibodies during primary and secondary responses to proteins, salmonella antigens, and phage.

The nature of the antibodies produced by the rabbit during the primary and secondary responses to T(2) phage, proteins, and the O and H antigens of Salmonella typhosa has been determined. Immune sera have been fractionated by zone electrophoresis, sucrose density ultracentrifugation, and anion exchange chromatography. The resulting fractions have been assayed by phage neutralization or hemagglutination (antisera to proteins) or bacterial agglutination. In confirmation and extension of earlier work from this laboratory, the primary response to these antigens, with the exception of the O antigen of the Salmonella, included the early synthesis of 19S, gamma-1 globulin antibody, and the later synthesis of 7S, gamma-2 globulin antibody. The primary response to the O antigen consisted of the synthesis of only a macroglobulin agglutinin. The secondary response to the proteins, including the H antigen of the Salmonella, comprised the early synthesis of large amounts of the 7S gamma-2 globulin antibody to the same level attained during the primary response. The secondary response to the phage consisted in the synthesis of 7S, gamma-2 globulin antibody alone. Treatment of the macroglobulin phage-neutralizing antibody with mercaptoethanol resulted in complete loss of its neutralizing activity. A working hypothesis to explain these observations was presented. A salient feature of this hypothesis was the suggestion that different cells synthesized the two distinct molecular forms of antibody. The significance of the sequential synthesis of the two forms of antibody is not known. It was proposed that the system for synthesis of macroglobulin antibody is an auxiliary system for antibody synthesis, perhaps the first to develop phylogenetically and ontogenetically. It is felt that the present observations indicate a clear-cut qualitative distinction between the primary and secondary responses to immunization whereby these responses might be identified in various experimental situations. It is also felt that these findings with the primary and secondary responses to various antigens in the rabbit may be of widespread occurrence in nature among a variety of species.

Animals↗

PubMed1976

Synthesis and secretion of alpha2-macroglobulin by cultured human fibroblasts.

The following observations indicate that cultured human WI-38 fibroblasts synthesize and secrete alpha2-macroglobulin into serum-free medium: (a) after incubation of cultures with [35S]L-methionine, a labeled protein appeared in the medium which was precipitated by antiserum directed against alpha2-macroglobulin; (b) after incubation of cultures with [35S]L-methionine, a major band of radioactivity detected by polyacrylamide gel electrophoresis of the proteins in medium co-migrated with alpha2-macroglobulin; and (c) the amount of alpha2-macroblobulin in the medium, estimated both functionally and immunologically, increased with time in normal but not not puromycin-treated cultures.

Cells, Cultured↗

PubMed1977

Plasmin inhibitor interactions. The effectiveness of alpha2-plasmin inhibitor in the presence of alpha2-macroglobulin.

alpha2-Plasmin inhibitor and alpha2-macroglobulin were allowed to compete for the protease plasmin. The binding of the enzyme to these inhibitors was assessed by two different but comparable methods. The interactions were completed in 10 s of incubation, and transfer of plasmin from one inhibitor to the other did not occur. Almost as much plasmin was bound to alpha2-plasmin inhibitor in mixtures containing a large molar excess of alpha2-macroglobulin relative to plasmin or alpha2-plasmin inhibitor, as was bound in mixtures not containing alpha2-macroblobulin. These studies demonstrate directly the effectiveness of alpha2-plasmin inhibitor in binding and inhibiting plasmin in the presence of alpha2-macroglobulin, and suggest that the alpha2-plasmin inhibitor may be the major circulating plasmin inhibitor.

Binding, Competitive↗

PubMed1976

A plasma glycoprotein depressed in vitamin A deficiency in the rat: alpha 1-macroglobulin.

Plasma glycoprotein synthesis in normal and vitamin A-deficient rats was investigated by injecting the rats with labeled carbohydrate precursors and then fractionating their plasmas on DEAE-Sephadex. Plasma from deficient rats showed a consistent depression of 30% in the uptake of label into a peak eluting with 0.23 M NaCl. The major component of this peak was identified as the rat alpha1-macroglobulin, based on its molecular weight (800,000), its mobility on cellulose acetate electrophoresis and its ability to bind trypsin. Although the alpha1-macroglobulin synthesis appeared to be depressed by 30%, its fractional turnover rate was not affected by vitamin A deficiency (t 1/2 = 18 hours). The trypsin-binding ability of this glycoprotein was used as a comparative measure of its concentration, and the results confirmed that serum levels of this glycoprotein were lower in deficient rats. In severe deficiency, alpha1-macroglobulin levels dropped to between 10% and 20% of normal levels.

Animals↗

PubMed1983

A versatile method for the coupling of protein to DNA: synthesis of alpha 2-macroglobulin-DNA conjugates.

We describe a simple, general method to link proteins covalently to DNA. The method uses two reagents, N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine and 2-iminothiolane. The former reacts specifically with nonpaired quanine residues and upon reduction generates a free sulfhydryl group. The latter reacts with a protein to provide another sulfhydryl group which is subsequently conjugated to DNA by an intermolecular disulfide interchange reaction. Using this method alpha 2-macroglobulin was conjugated to plasmid DNA encoding the Herpes simplex virus-1 thymidine kinase gene or a DNA fragment containing the E. coli chloramphenicol acetyltransferase gene. Up to 20% of the total DNA was conjugated to alpha 2-macroglobulin and the alpha 2-macroglobulin-DNA conjugate had a protein/DNA molar ratio of approximately two. The whole reaction takes place under very mild conditions in aqueous solution. The structure of DNA appears not to be significantly affected by the chemical modification. This method may prove useful in ligand directed gene transfer studies.

Cross-Linking Reagents↗

PubMed1991

A tissue inhibitor of metalloproteinases and alpha-macroglobulins in the ovulating rat ovary: possible regulators of collagen matrix breakdown.

Immature female Sprague-Dawley rats were primed with 20 IU eCG at 28 days of age and treated with 10 IU hCG 48 h later. Ovulation followed at 12-14 h. Ovaries were extracted at various times after hCG by use of Triton X-100 and 10 mM CaCl2 (Triton extract) followed by heating to 60 degrees C for 6 min with 0.1 M CaCl2 in 50 mM Tris/0.15 M NaCl, pH 7.5 (heat extract). These extracts were tested for their ability to inhibit tissue metalloproteinases by use of the small uterine metalloproteinase (UMP) of the rat. The ovary contains three plasma-derived inhibitors (alpha 1-macroglobulin [alpha 1 M], alpha 2-macroglobulin [alpha 2 M], and alpha 1 inhibitor3 [alpha 1I3]) and one tissue-derived inhibitor of the tissue inhibitor of metalloproteinases (TIMP) family. alpha 1 I3 was shown to inhibit UMP and rat collagenase. The concentration of the tissue inhibitor rose 5-fold from 0.6 micrograms UMP blocked per gram wet tissue in ovaries not primed with eCG to 3.2 micrograms UMP blocked at 8 h after hCG. Over this same time interval, the sum of alpha 1M + alpha 2M per gram of ovary rose 7-fold from 3.2 to 22.4 micrograms UMP inhibited and alpha 1I3 rose 2-fold from 4.4 to 10.7 micrograms UMP inhibited. The increases in the tissue inhibitor are interpreted to be due to increased synthesis by the tissue, whereas the changes in alpha-macroglobulins are postulated to be due to increased vascularity and increased permeability of the vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

Levels of alpha1-proteinase inhibitor and alpha2-macroglobulin in the tear film of patients with keratoconus.

PURPOSE: Levels of alpha1-proteinase inhibitor and alpha2-macroglobulin in the tear film of patients with keratoconus were measured to elucidate their possible roles in the pathogenesis of keratoconus. METHODS: Tear samples were collected from 15 keratoconus patients and 14 age-similar human control subjects. Levels of alpha1-proteinase inhibitor and alpha2-macroglobulin in each tear sample were quantified and compared between the two groups. RESULTS: Mean values for alpha1-proteinase inhibitor were 101.0+/-35.5 and 106.1+/-41.7 ng/microg protein for the keratoconus and control groups, respectively. The corresponding mean values for alpha2-macroglobulin were 13.5+/-6.8 and 14.8+/-7.5 ng/microg protein. Neither inhibitor showed a statistically significant difference between the keratoconus and control specimens. Subset analysis to evaluate the effects of contact lens wear and the presence of a graft in the fellow eye did not reveal a statistically significant difference. CONCLUSION: The tear film of patients with keratoconus contains normal levels of protease inhibitors. Therefore, the tear film may not be a source of the reduced inhibitor levels shown in the corneas of patients with keratoconus.

Adolescent↗

PubMed1994

Serum concentrations of albumin, C-reactive protein, alpha 2-macroglobulin, prealbumin, fibronectin, fibrinogen, transferrin, and retinol binding protein in 55 patients with hepatic glycogen storage diseases.

Hepatic glycogen storage diseases are hereditary metabolic disorders involving the metabolism of glycogen. This study was designed to investigate the serum protein status in such diseases. Fifty-five patients with glycogen storage disease types I, III, VI, and IX, whose ages ranged from 1 month to 27 years, were included in this work. C-reactive protein, fibrinogen, alpha 2-macroglobulin, albumin, transferrin, fibronectin, retinol binding protein, and prealbumin serum concentrations were measured in each patient. In patients affected with type I glycogen storage disease, serum concentrations of alpha 2-macroglobulin, fibrinogen, C-reactive protein, and transferrin were significantly increased. In patients with types III, VI, and IX glycogen storage diseases, the concentration of alpha 2-macroglobulin was the only one that was significantly increased. Thus, even though this study raises more questions than it answers, it seems likely that the hepatic synthesis of some proteins may be increased in patients affected by hepatic glycogen storage diseases. This may indicate some degree of mild hepatic dysfunction in such metabolic disorders. However, further investigations are required to elucidate the discrepancies observed among the different types of diseases.

Adolescent↗

PubMed1989

Rabbit serum alpha-2-macroglobulin binds to liver ferritin: association causes a heterogeneity of ferritin molecules.

Rabbit liver ferritin is unusual since it forms two discrete electrophoretic bands at the beta position of molecular dimers (Santambrogio & Massover, 1987). The present studies have sought to identify the nature of a 170 kDa non-ferritin polypeptide that is uniquely present in the larger beta band. Ultrastructural, immunological and biochemical results all indicate that this polypeptide is a subunit of the plasma protein. alpha-2-macroglobulin. Experimental results show that rabbit serum alpha-2-macroglobulin will bind liver ferritin, and this association induces the de novo formation of the larger beta band. These results thus demonstrate that molecular heterogeneity of ferritin can be caused by its association with a non-ferritin protein. We conclude that alpha-2-macroglobulin is a binder of rabbit tissue ferritin in the circulation; this binding could provide additional means for the receptor-mediated uptake of circulating ferritin.

Animals↗

PubMed1976

Distribution of serum zinc between albumin and alpha2-macroglobulin in patients with decompensated hepatic cirrhosis.

Lower concentrations of total serum zinc (540 +/- 111 mug/1, mean +/- SEM), and of albumin-bound serum zinc (295 +/- 113 mug/1) and a higher concentration of alpha2-macroglobulin-bound zinc (245 +/- 69 mug/1) were found in 25 patients with decompensated hepatic cirrhosis, compared to 28 healthy subjects (835 +/- 91; 679 +/- 83; 156 +/- 27 mug/1 respectively). Levels of total and albumin-bound zinc were significantly and positively correlated with serum albumin levels. Higher levels of alpha2-macroglobulin-bound zinc were associated with higher levels of alpha2-macroglobulin in these patients (2.8 +/- 0.8 g/1) compared to normals (2.3 +/- 0.6). Hence, not only do decompensated cirrhotics exhibit a lower serum zinc level but a greater proportion of this zinc is associated with the tightly bound, and presumably metabolically more inert, serum fraction. This situation exaggerates the zinc deficiency state of the severe cirrhotic.

Humans↗

PubMed1981

Polarization fluorescence studies on proteolytic activity of alpha 2-macroglobulin-trypsin complexes.

When digestive enzymes are released into the blood, they may be completely inactivated by a variety of inhibitor present (alpha-1-protease inhibitor, antithrombin III, alpha 2-plasmin inhibitor, etc.) or only partially neutralized by alpha 2-macroglobulin. In this study, polarization fluorescence is used to demonstrate that complexes of alpha 2-macroglobulin with trypsin fluorescence is used to demonstrate that complexes of alpha 2-macroglobulin with trypsin can digest beta-endorphin, adrenocorticotropin, and beta-lipotropin. Furthermore, it has been shown that a small trypsin inhibitor (trasylol, mol. wt. 6500) can prevent this digestion, but that larger inhibitory proteins (i.e. soybean trypsin inhibitor, mol. wt. 21 500; alpha 1-protease inhibitor, mol. wt. 50 000) cannot.

Adrenocorticotropic Hormone↗

PubMed1982

Small-angle X-ray scattering study of the interaction between human alpha 2-macroglobulin and trypsin.

The interaction between human alpha 2-macroglobulin and trypsin yields a complex of two trypsin molecules and one alpha 2-macroglobulin molecule as the main product. This is indicated from small-angle X-ray titration measurements in 0.02 M Tris/HCl buffer of pH 7.40 containing 0.2 M NaCl and 5 mM EDTA. The radius of gyration for the complex was determined to be 7.1 nm. The complex formation coincides with a decrease in the first side maximum of the scattering curve for alpha 2-macroglobulin. A comparison of the experimental data with those calculated from various models indicates that the data are consistent with the scattering from a hollow cylinder containing two sites, each of which is partly filled with one trypsin molecule.

Humans↗

PubMed1990

The H(+)-induced dissociation of human plasma alpha 2-macroglobulin. An investigation using small-angle neutron scattering and test of trypsin binding activity.

The dissociation of the tetrameric alpha 2-macroglobulin molecule into two half-molecular fragments, which occurs at pH less than 4.5, has been investigated using the small-angle neutron scattering method, and test of trypsin binding activity. Best fit with the relative forward scattering of neutrons is obtained for a model where the dissociation of the protein is driven by the uptake of H+ on altogether four acid-base groups, one per monomeric subunit of alpha 2-macroglobulin. These groups are not (or only slightly) accessible in the native tetramer, but become exposed to the solvent after dissociation of the protein. The H(+)-binding constant obtained for these groups, after dissociation of the protein, log K1 in the range 4.2-4.5, suggests that they are most probably carboxylate groups. From the about 10% increase in the radius of gyration, which occurs when lowering the pH from 4.5 to 2.0, we can conclude that the dissociation is associated with a change in structure of the protein. Tests of trypsin binding show that there is also an irreversible loss in trypsin binding activity, which is directly related to the fraction of dissociated protein. Thus, at pH less than 4.5, there is a transition of alpha 2-macroglobulin which results simultaneously in dissociation, disorganisation of the conformation of the subunits and loss in activity.

Binding Sites↗

PubMed1992

The primary sequence and the subunit structure of mouse alpha-2-macroglobulin, deduced from protein sequencing of the isolated subunits and from molecular cloning of the cDNA.

Mouse plasma alpha-2-macroglobulin (m alpha 2M) was isolated and the N-terminal amino-acid sequences determined after separation of the 165-kDa and 35-kDa subunits. These sequences were compared to the protein sequence predicted by the cDNA, which was cloned from a mouse liver library and sequenced. From these data it is evident that both subunits are encoded by one mRNA of approximately 5 kb expressed predominantly in liver. The smaller subunit, with the N-terminal sequence DLSSSDLT, comprises the C-terminal 257 residues of m alpha 2M and is derived from a single-chain precursor probably by proteolytic processing at an arginine residue in the sequence PTRDLSS. Analysis of the predicted protein further showed all the salient features of a proteinase inhibitor of the macroglobulin family: a bait region that deviates from all known sequences in this family, a very conserved internal thiolester site and conserved cysteine residues and putative N-glycosylation sites. The synthesis of m alpha 2M in adult liver was demonstrated by Northern blotting and in fetal liver by in-situ hybridization. Transient transfection of COS cells with the cDNA under control of a viral promoter demonstrated the secretion and partial processing of m alpha 2M in the culture medium. In plasma the level of m alpha 2M was found to be stable as expected for the murine counterpart of human plasma alpha-2-macroglobulin. The possibilities of using the mouse as a genetic model to study this proteinase inhibitor in vivo are discussed.

Amino Acid Sequence↗

PubMed1993

Hydrocortisone induces the synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells.

The effects of lactogenic hormones on protein secretion by 25.5-G4.2.3 cells, a rat mammary myoepithelial cell line immortalised with a temperature-sensitive T-antigen, were investigated. Insulin, prolactin, estradiol and progesterone had no effect but hydrocortisone induced the secretion of two proteins with molecular masses of 175 kDa (p175) and 146 kDa (p146), 10-30-fold and 5-fold respectively. The induction of p175 and p146 synthesis by hydrocortisone was greater at 39.5 degrees C than at 33 degrees C reflecting the increased differentiation of 25.5-G4.2.3 cells at the higher temperature. Rat mammary epithelial cells did not synthesise p175. After addition of hydrocortisone to 25.5-G4.2.3 cells, there was a lag phase of 10 h before the synthesis of p175 was induced. Half-maximal induction of p175 synthesis required a hydrocortisone concentration of 0.5 microM. p175 was identified as alpha 2-macroglobulin by N-terminal amino-acid sequence determination and immunoprecipitation with a specific antibody. Hydrocortisone induced a 5-kb alpha 2-macroglobulin-specific mRNA transcript in 25.5-G4.2.3 cells. Myoepithelial cells are responsible for synthesising the basement membrane around the rapidly expanding mammary alveoli during pregnancy. Myoepithelial cells also secrete metalloproteinases which are probably involved in turnover of the basement membrane. We suggest that increased levels of hydrocortisone during pregnancy induce the synthesis of alpha 2-macroglobulin, which is believed to be a potent inhibitor of metalloproteinases, by rat mammary myoepithelial cells to reduce proteolytic degradation of the basement membrane.

Amino Acid Sequence↗