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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 Journal of Biological Chemistry Apr 2010Surfactant protein D (SP-D) is an innate immune collectin that recognizes microbes via its carbohydrate recognition domains, agglutinates bacteria, and forms immune...
Surfactant protein D (SP-D) is an innate immune collectin that recognizes microbes via its carbohydrate recognition domains, agglutinates bacteria, and forms immune complexes. During microbial infections, proteases, such as elastases, cleave the carbohydrate recognition domains and can inactivate the innate immune functions of SP-D. Host responses to counterbalance the reduction of SP-D-mediated innate immune response under these conditions are not clearly understood. We have unexpectedly identified that SP-D could interact with protein fractions containing ovomucin and ovomacroglobulin. Here, we show that SP-D interacts with human alpha(2)-macroglobulin (A2M), a protease inhibitor present in the lungs and serum. Using enzyme-linked immunosorbent assays, surface plasmon resonance, and carbohydrate competition assays, we show that SP-D interacts with A2M both in solid phase (K(D) of 7.33 nM) and in solution via lectin-carbohydrate interactions under physiological calcium conditions. Bacterial agglutination assays further show that SP-D x A2M complexes increase the ability of SP-D to agglutinate bacteria. Western blot analyses show that SP-D, but not A2M, avidly binds bacteria. Interestingly, intact and activated A2M also protect SP-D against elastase-mediated degradation, and the cleaved A2M still interacts with SP-D and is able to enhance its agglutination abilities. We also found that SP-D and A2M can interact with each other in the airway-lining fluid. Therefore, we propose that SP-D utilizes a novel mechanism in which the collectin interacts with protease inhibitor A2M to decrease its degradation and to concurrently increase its innate immune function. These interactions particularly enhance bacterial agglutination and immune complex formation.
Topics: Agglutination; Escherichia coli; Humans; Immunity, Innate; Lung; Pancreatic Elastase; Protein Binding; Pulmonary Surfactant-Associated Protein D; alpha-Macroglobulins
PubMed: 20207732
DOI: 10.1074/jbc.M110.108837 -
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 -
The development of rat alpha 2-macroglobulin. Studies in vivo and in cultured fetal rat hepatocytes.European Journal of Biochemistry Feb 1988During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase...
During inflammation and tissue injury, there is an increase in the plasma concentration of several proteins, the acute-phase proteins. The levels of some acute-phase proteins have been reported to increase in pregnant and tumour-bearing animals. Rat alpha 2-macroglobulin is classified as an acute-phase protein. In this study we report the expression of alpha 2-macroglobulin in various tissues during development of the rat embryo by analysis of mRNA. The tissues studied are liver, visceral yolk sac, placental labyrinth, decidua and trophoblast. In addition, the sites of alpha 2-macroglobulin expression are localized by in situ hybridization of cDNA for alpha 2-macroglobulin to mid-sagittal cryosections of rat embryos. The level of mRNA coding for alpha 2-macroglobulin is determined in the liver of rats aged between 12 days gestation and 2 days postnatal. alpha 2-Macroglobulin mRNA is first observed in fetal liver from 12 days of gestation and increases after day 17, reaching a maximum on day 20. At this time the level is greater than that found in the liver of an adult rat suffering from acute inflammation. alpha 2-Macroglobulin mRNA is detectable in the yolk sac, placental labyrinth, trophoblast tissue and decidua. In the decidua the alpha 2-macroglobulin message is first detected at 8 days of gestation, with high levels observed from 10 to 21 days of gestation. These observations are supported by in situ hybridization studies. Experiments using cultured hepatocytes show that cells derived from rats at 15 days and 19 days of gestation are capable of synthesizing and secreting alpha 2-macroglobulin. Both synthesis and secretion can be induced by the addition of dexamethasone to the culture medium.
Topics: Animals; Cells, Cultured; DNA; Female; Fetus; Gene Expression Regulation; Gestational Age; Liver; Nucleic Acid Hybridization; Placenta; Pregnancy; RNA, Messenger; Rats; Rats, Inbred Strains; Trophoblasts; Yolk Sac; alpha-Macroglobulins
PubMed: 2450021
DOI: 10.1111/j.1432-1033.1988.tb13842.x -
The Journal of Biological Chemistry Sep 1989The amino acid sequence of a 90-residue segment of human pregnancy zone protein containing its bait region has been determined. Human alpha 2-macroglobulin, human... (Comparative Study)
Comparative Study
The amino acid sequence of a 90-residue segment of human pregnancy zone protein containing its bait region has been determined. Human alpha 2-macroglobulin, human pregnancy zone protein, and rat alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor 3 variants 1 and 2 constitute a group of homologous proteins; but the sequences of their bait regions are not related, and they differ in length (32-53 residues). The alpha-macroglobulin bait region is located equivalently with residues 666-706 in human alpha 2-macroglobulin. In view of the extreme sequence variation of the bait regions, the evolutionary constraints for these regions are likely to differ from those of the remainder of the alpha-macroglobulin structure. The sites of specific limited proteolysis in the bait regions of human pregnancy zone protein and rat alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor 3 variants 1 and 2 by a variety of proteinases differing in specificity have been determined and compared with those identified earlier in human alpha 2-macroglobulin. The sites of cleavage generally conform to the substrate specificity of the proteinase in question, but the positions and nature of the P4-P4' sites differ. Most cleavages occur in two relatively small segments spaced by 6-10 residues; and in each case, bait region cleavage leads to alpha-macroglobulin-proteinase complex formation. The rate at which a given proteinase cleaves alpha-macroglobulin bait regions is likely to show great variation. Possible structural features of the widely different bait regions and their role in the mechanism of activation are discussed.
Topics: Amino Acid Sequence; Animals; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Genetic Variation; Humans; Macromolecular Substances; Molecular Sequence Data; Peptide Fragments; Peptide Hydrolases; Protein Conformation; Rats; alpha-Macroglobulins
PubMed: 2476433
DOI: No ID Found -
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 -
Journal of Controlled Release :... Nov 2015Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based...
Synthetic microstructures can be engineered to deliver bioactive compounds impacting on their pharmacokinetics and pharmacodynamics. Herein, we applied dextran-based layer-by-layer (LbL) microcapsules to deliver alpha-2-macroglobulin (α2MG), a protein with modulatory properties in inflammation. Extending recent observations made with dextran-microcapsules loaded with α2MG in experimental sepsis, we focused on the physical and chemical characteristics of these microstructures and determined their biology on rodent and human cells. We report an efficient encapsulation of α2MG into microcapsules, which enhanced i) human leukocyte recruitment to inflamed endothelium and ii) human macrophage phagocytosis: in both settings microcapsules were more effective than soluble α2MG or empty microcapsules (devoid of active protein). Translation of these findings revealed that intravenous administration of α2MG-microcapsules (but not empty microcapsules) promoted neutrophil migration into peritoneal exudates and augmented macrophage phagocytic functions, the latter response being associated with alteration of bioactive lipid mediators as assessed by mass spectrometry. The present study indicates that microencapsulation can be an effective strategy to harness the complex biology of α2MG with enhancing outcomes on fundamental processes of the innate immune response paving the way to potential future development in the control of sepsis.
Topics: Animals; Capsules; Cells, Cultured; Dextrans; Drug Compounding; Human Umbilical Vein Endothelial Cells; Humans; Immunity, Innate; Leukocytes; Macrophages; Male; Mice, Inbred C57BL; alpha-Macroglobulins
PubMed: 26385167
DOI: 10.1016/j.jconrel.2015.09.021 -
Scientific Reports Mar 2023Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many...
Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (αM), an extracellular chaperone. We also established a lysosomal internalization assay for αM, which revealed that αM mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of αM and clusterin, another extracellular chaperone, indicated that αM preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.
Topics: Female; Pregnancy; Humans; Pregnancy-Associated alpha 2-Macroglobulins; Protein Folding; Proteostasis; Proteolysis; Transcription Factors; Lysosomes
PubMed: 36977730
DOI: 10.1038/s41598-023-31104-x -
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 -
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