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The Biochemical Journal Aug 19731. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to...
1. alpha(2)-Macroglobulin is known to bind and inhibit a number of serine proteinases. We show that it binds thiol and carboxyl proteinases, and there is now reason to believe that alpha(2)-macroglobulin can bind essentially all proteinases. 2. Radiochemically labelled trypsin, chymotrypsin, cathepsin B1 and papain are bound by alpha(2)-macroglobulin in an approximately equimolar ratio. Equimolar binding was confirmed for trypsin by activesite titration. 3. Pretreatment of alpha(2)-macroglobulin with a saturating amount of one proteinase prevented the subsequent binding of another. We conclude that each molecule of alpha(2)-macroglobulin is able to react with one molecule of proteinase only. 4. alpha(2)-Macroglobulin did not react with exopeptidases, non-proteolytic hydrolases or inactive forms of endopeptidases. 5. The literature on binding and inhibition of proteinases by alpha(2)-macroglobulin is reviewed, and from consideration of this and our own work several general characteristics of the interaction can be discerned. 6. A model is proposed for the molecular mechanism of the interaction of alpha(2)-macroglobulin with proteinases. It is suggested that the enzyme cleaves a peptide bond in a sensitive region of the macroglobulin, and that this results in a conformational change in the alpha(2)-macroglobulin molecule that traps the enzyme irreversibly. Access of substrates to the active site of the enzyme becomes sterically hindered, causing inhibition that is most pronounced with large substrate molecules. 7. The possible physiological importance of the unique binding characteristics of alpha(2)-macroglobulin is discussed.
Topics: Binding Sites; Bromelains; Cathepsins; Chromatography; Chymotrypsin; Gels; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin G; Iodine Radioisotopes; Kinetics; Macroglobulins; Models, Chemical; Molecular Conformation; Molecular Weight; Papain; Peptide Hydrolases; Tritium; Trypsin
PubMed: 4201304
DOI: 10.1042/bj1330709 -
The FEBS Journal Sep 2020Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which...
Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a k in the ≤ 10 m ·s range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.
Topics: Amino Acid Sequence; Binding Sites; Chromatography, Liquid; Humans; Kinetics; Mass Spectrometry; Molecular Docking Simulation; Myeloblastin; Peptides; Pregnancy-Associated alpha 2-Macroglobulins; Protein Binding; Protein Domains; Proteolysis; Recombinant Proteins
PubMed: 31995266
DOI: 10.1111/febs.15229 -
The Journal of Clinical Investigation Nov 1977We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay)... (Comparative Study)
Comparative Study
We studied the synthesis and secretion of alpha-2-macroglobulin by cultures of human adherent cells. Much more alpha-2-macroglobulin (measured by radioimmunoassay) accumulated in media of established strains of adherent cells derived from embryonic lung than in media of established strains derived from adult skin or rheumatoid synovium. Alpha-2-macroglobulin accumulated in media of primary cultures of adherent cells from a variety of embryonic tissues. However, the amount of alpha-2-macroglobulin accumulating in media of subsequent passage of these cells declined for all strains except those derived from lung. Immunodiffusion and double-antibody immunoprecipitation studies of cell extracts and media after incubation of cells with l-[(35)S]methionine supported the radioimmunoassay finding that adherent cells from lung synthesized and secreted more alpha-2-macroglobulin than adherent cells from skin. Intracellular alpha-2-macroglobulin could not be detected by radio-immunoassay or visualized by immunofluorescent microscopy, suggesting that synthesized alpha-2-macroglobulin is rapidly secreted. Plasminogen-rich fibrin clots were lysed in culture media of adherent cells from embryonic lung and, to a lesser extent, heart. Adherent cells from other tissues, which produced less alpha-2-macroglobulin, did not lyse fibrin clots. However, all cultures of adherent cells contained pericellular fibronectin, a large, external, transformation-sensitive glycoprotein known to be cleaved by plasmin. We speculate that production of alpha-2-macroglobulin may be a means for protease-secreting normal cells to preserve cell surface integrity and that alpha-2-macroglobulin synthesized locally in lung may protect lung tissues from a variety of proteases.
Topics: Cell Adhesion; Cells, Cultured; Chromatography, Gel; Fibroblasts; Fluorescent Antibody Technique; Humans; Lung; Myocardium; Precipitin Tests; Radioimmunoassay; Skin; alpha-Macroglobulins
PubMed: 71307
DOI: 10.1172/JCI108854 -
Journal of Clinical Laboratory Analysis 2004The lack of satisfactory methods for quantifying serum levels and a credible reference material has limited bedside use of serum alpha(2)-macroglobulin (alpha2M)... (Comparative Study)
Comparative Study Review
The lack of satisfactory methods for quantifying serum levels and a credible reference material has limited bedside use of serum alpha(2)-macroglobulin (alpha2M) measurements. Great strides have been made in the last few years. The remaining barrier to more relevant and cost effective use of serum protein data for diagnosis and prognosis is the availability of reliable reference intervals from birth to old age for both males and females. A total of 40 publications reporting reference intervals have been identified that meet the criteria used in our prior five studies, and these have been analyzed statistically. On average, previous small studies of these individual proteins agree with our life-long reference ranges over their constrained age ranges. This meta-analysis provides support for our reference ranges and places them in the perspective of previous publications.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Child; Child, Preschool; Cohort Studies; Ethnicity; Female; Humans; Infant; Infant, Newborn; Male; Middle Aged; Reference Values; alpha-Macroglobulins
PubMed: 15065216
DOI: 10.1002/jcla.20013 -
Kidney International Jan 1998In both the nephrotic syndrome (NS) and hereditary analbuminemia in the Nagase analbuminemic rat (NAR), the plasma protein concentration is nearly normal since albumin...
In both the nephrotic syndrome (NS) and hereditary analbuminemia in the Nagase analbuminemic rat (NAR), the plasma protein concentration is nearly normal since albumin is replaced by several high molecular weight proteins. In rats these include the protease inhibitors alpha 2-macroglobulin (alpha 2M), a 720 kDa positive acute phase protein (APP) and alpha 1-inhibitor 3 (alpha 1-I3), a 180 kDa negative APP. There is no known stimulus to increase alpha 1-I3 synthesis, but like albumin and other negative APPs its synthesis decreases during inflammation by transcriptional down-regulation. In hypoalbuminemic states gene transcription of other positive and negative APPs is increased. We report that alpha 2M was increased significantly (12-fold) in NAR and by approximately 50-fold in rats with NS compared to control. The alpha 1-I3 concentration was twice normal in NAR or NS compared to controls, providing approximately half of the total plasma protein. Infusion of human albumin into NAR to raise albumin levels > 20 mg/ml for 24 hours caused a significant decrease in alpha 1-I3 (24.8 +/- 0.6 to 18.7 +/- 0.6 mg/ml, P < 0.0001), equal in magnitude to that caused by 250 micrograms/100 g of endotoxin (23.0 +/- 1.1 to 18.6 +/- 0.6, P < 0.01). The effect of albumin was not an acute phase response since it also suppressed alpha 2M (239 +/- 10 to 205 +/- 11 micrograms/ml, P < 0.005). Turnover of 125I labeled alpha 2M and alpha 1-I3 was then measured in controls, NAR and in two models of the nephrotic syndrome in rats (Heymann nephritis, HN; adriamycin-induced, ADR), yielding fractional catabolic rates (FCR), which at steady state equals synthesis. The serum alpha 2M concentration was increased approximately equal to 50-fold and was proportional to synthesis (r = 0.91 P < 0.001). alpha 2-Macroglobulin synthesis increased by 12-fold in NAR and 50-fold in NS. In contrast, hepatic alpha 2M mRNA increased only 30% in NAR and twofold in NS, suggesting post-transcriptional regulation. Fractional catabolic rates were not decreased and played no role in increasing serum alpha 2M in NS or NAR. The alpha 1-I3 concentration and synthesis increased twofold from controls in both NAR and NS. However, hepatic alpha 1-I3 mRNA was not increased in NAR and increased only 50% in NS. Unlike alpha 2M, serum alpha 1-I3 correlated negatively with FCR (-r = 0.66, P < 0.01). In conclusion, both alpha 1-13 and alpha 2M concentration are increased in hypooncotic states by increased synthesis regulated post-transcriptionally, supporting plasma protein concentration when albumin is lost in urine or not synthesized.
Topics: Acute-Phase Proteins; Animals; Humans; Lipopolysaccharides; Male; Nephrotic Syndrome; Protease Inhibitors; RNA, Messenger; Rats; Rats, Sprague-Dawley; Serum Albumin; Transcription, Genetic; alpha-Macroglobulins
PubMed: 9453001
DOI: 10.1046/j.1523-1755.1998.00734.x -
Nagoya Journal of Medical Science Feb 2019The major hallmarks of Alzheimer's disease (AD) are the extracellular accumulation of pathological amyloid beta (Aβ) in the brain parenchyma and Aβ deposition in...
The major hallmarks of Alzheimer's disease (AD) are the extracellular accumulation of pathological amyloid beta (Aβ) in the brain parenchyma and Aβ deposition in cerebral blood walls (cerebral amyloid angiopathy; CAA). Although CAA occurs in more than 80% of AD patients, the mechanisms of Aβ deposition and clearance around the vessel walls are unknown. We found Aβ-degrading activity in human serum during analysis of the regulatory mechanism of Aβ production in human endothelial cells. To elucidate the metabolic dynamics of Aβ surrounding the brain microvessels, we identified Aβ-degrading activity in human serum (blood Aβ-degrading activity: BADA) by column chromatography and LC/MS. BADA exhibited characteristics of an acidic protein, pI 4.3, which had two different protein surface charges (low and high affinity cations). Both BADA fractions had a relative molecular mass of greater than 400 kDa. Furthermore, BADA in the low affinity cation fraction was inhibited by the serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). We clarified alpha-2-macroglobulin (a2M) and several serine proteases from this BADA by LC-MS. Moreover, we demonstrated that BADA is increased by approximately 5000-fold in human serum by column chromatography. Therefore, BADA may play an important role in the circulation and metabolism of Aβ in human brain microvessels.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Brain; Cerebral Amyloid Angiopathy; Chromatography, Liquid; Human Umbilical Vein Endothelial Cells; Humans; Macroglobulins; Mass Spectrometry; Microvessels; Serine Proteases
PubMed: 30962655
DOI: 10.18999/nagjms.81.1.55 -
The Journal of Experimental Medicine Jun 1977alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a...
alpha2-Macroglobulin levels in the supernates of cultures of different subpopulations of human peripheral blood mononuclear leukocytes were assayed by a radioimmunoassay. Unfractionated mononuclear leukocytes produced greater amounts of the macroglobulin (4.0 vs. 0.8 ng/10(6) cells) than did subpopulations enriched in T or B+T lymphocytes, by passage through nylon wool or cotton wool columns, respectively. Still higher concentrations of alpha2-macroglobulin (40 ng/10(6) cells) were measured in the supernates of glass-adherent mononuclear leukocyte cultures. These results suggest that cells of monocyte-macrophage lineage are mainly, if not exclusively, responsible for the appearance of alpha2- macroglobulin in the supernate of human peripheral blood leukocyte cultures. The de novo synthesis and release of alpha2-macroglobulin by cultured monocytes was demonstrated by immunoprecipitation of radioactivity from supernates of 32S-methionine-labeled glass-adherent cells. Antiserum against purified alpha2-macroglobulin was used in both Ouchterlony double diffusion and double antibody precipitation tests. SDS-polyacrylamide gel electrophoresis of immunoprecipitates showed that most of the radioactivity comigrated with authentic alpha2-macroglobulin subunit at about 160,000 daltons.
Topics: B-Lymphocytes; Cells, Cultured; Chemical Precipitation; Electrophoresis, Polyacrylamide Gel; Fluorescent Antibody Technique; Humans; Leukocytes; Macrophages; Monocytes; Peptides; alpha-Macroglobulins
PubMed: 68095
DOI: 10.1084/jem.145.6.1580 -
Journal of Biochemistry Feb 1988Green turtle plasma alpha-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their...
Green turtle plasma alpha-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their conformational changes were studied by HPLC gel chromatography, circular dichroism, and electron microscopy. The Stokes radii of native green turtle alpha-macroglobulin and ovomacroglobulin were estimated to be 84.3 +/- 0.5 A, and 93.0 +/- 0.5 A, respectively, by means of an HPLC experiment. After reaction with methylamine or proteinases, the Stokes radius of alpha-macroglobulin changed to 83.0 +/- 0.5 A or 85.4 +/- 0.5 A, respectively, and that of ovomacroglobulin to 93.0 +/- 0.5 A or 87.1 +/- 0.5 A. The circular dichroic spectra of native alpha-macroglobulin and ovomacroglobulin exhibited a negative band at around 215 nm, indicating the presence of beta-structure. Reaction of the two macroglobulins with methylamine resulted in a slight decrease in the ellipticity and reaction with proteinases led to a slight increase. The electron micrographic images of native alpha-macroglobulin and ovomacroglobulin can be described as deformed rings for the former and rugby balls for the latter. A common characteristic feature of the two molecules was that the central parts of the molecules were only thinly occupied by subunit. After reaction of macroglobulins with proteinases, the void spaces became partially filled and their overall shape more rectangular. Methylamine treatment caused a structural change only in alpha-macroglobulin but not in ovomacroglobulin. The difference in the susceptibility of the macroglobulins to methylamine was taken as an indication of evolutional divergence of the two homologous proteins within the last 300 million years.
Topics: Animals; Chromatography, High Pressure Liquid; Circular Dichroism; Egg Proteins; Macroglobulins; Microscopy, Electron; Protein Conformation; Turtles; alpha-Macroglobulins
PubMed: 2453504
DOI: 10.1093/oxfordjournals.jbchem.a122251 -
Scientific Reports Jan 2018Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such...
Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.
Topics: Bacterial Proteins; Operon; Pregnancy-Associated alpha 2-Macroglobulins; Protein Multimerization; Pseudomonas aeruginosa
PubMed: 29323132
DOI: 10.1038/s41598-017-18083-6 -
The Biochemical Journal Feb 1993The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class.... (Comparative Study)
Comparative Study
Identification of monomeric alpha-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric alpha-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbeiana.
The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.
Topics: Amino Acid Sequence; Animals; Binding Sites; Biological Evolution; Chickens; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Humans; Macromolecular Substances; Mice; Molecular Sequence Data; Protease Inhibitors; Rana catesbeiana; Rats; Turkeys; alpha-Macroglobulins
PubMed: 7679897
DOI: 10.1042/bj2900085