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The American Journal of Pathology Sep 1999Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions....
Mice deficient in either or both mouse alpha2-macroglobulin (MAM) and murinoglobulin-1 (MUG1) were generated and proved phenotypically normal under standard conditions. Acute pancreatitis was induced with a diet deficient in choline and methionine, supplemented with ethionine. The mortality was less than 25% in wild-type mice, as opposed to at least 56% in knockout mice, and was highest (70%) in MAM-/- mice, with earliest onset at 2 days. Plasma amylase and lipase levels were increased, but pancreatic tissue appeared histologically variable in individual mice. The clinical symptoms were most severe in MAM-/- mice and, surprisingly, were not aggravated in the double knockout mice, suggesting that the lack of proteinase inhibition capacity was not the major problem. Therefore, we analyzed the expression of 21 different cytokines and polypeptide factors in the pancreas of all experimental groups of mice. Interleukin-1-receptor antagonist mRNA was consistently induced by the diet in the pancreas of MAM-/- mice, and transforming growth factor-beta, tumor necrosis factor-alpha, tumor necrosis factor-beta, beta-lymphotoxin, and interferon-gamma mRNA levels were also increased. The data demonstrate the important role of alpha2-macroglobulin (A2M) in acute pancreatitis as both a proteinase inhibitor and a cytokine carrier. Mice deficient in MAM and/or MUG thus offer new experimental models for defining in vivo the role of the macroglobulins in pancreatitis and in other normal and pathological processes.
Topics: Acute Disease; Amylases; Animals; Blood Glucose; Cytokines; Disease Models, Animal; Lipase; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Pancreatitis; Protease Inhibitors; Serum Globulins; alpha-Macroglobulins
PubMed: 10487856
DOI: 10.1016/s0002-9440(10)65198-x -
Journal of Orthopaedic Research :... Jan 2023A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically,...
A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1β (IL-1β) and subsequent upregulation of nuclear factor kappa B (NF-κB) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1β, blocking downstream NF-κB-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1β. A2M was labeled with VivoTag™ 680 to localize the protein postincubation. The degree of binding between A2M and IL-1β was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1β and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag™ 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1β. Additionally, western blot analysis revealed that A2M neutralized IL-1β and NF-κB in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-α and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1β to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.
Topics: Humans; Pregnancy; Female; NF-kappa B; Interleukin-1beta; Inflammation Mediators; Pregnancy-Associated alpha 2-Macroglobulins; alpha-Macroglobulins
PubMed: 35451533
DOI: 10.1002/jor.25348 -
Haemostasis 2001Alfimeprase is a recombinantly produced, truncated form of fibrolase, a known directly fibrinolytic zinc metalloproteinase that was first isolated from the venom of the... (Review)
Review
Alfimeprase is a recombinantly produced, truncated form of fibrolase, a known directly fibrinolytic zinc metalloproteinase that was first isolated from the venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Both fibrolase and alfimeprase have been shown to have direct proteolytic activity against the fibrinogen Aalpha chain. In vivo pharmacology studies have shown that thrombolysis with alfimeprase is up to 6 times more rapid than with plasminogen activators. Alfimeprase can be bound and neutralized by serum alpha(2)-macroglobulin, a prevalent mammalian protease inhibitor which is capable of forming a macromolecular complex with alfimeprase. As a result, systemic bleeding complications have been greatly reduced due to the inhibitory effects of alpha(2)-macroglobulin. This article reviews the biochemical in vitro and in vivo characteristics of this novel acting thrombolytic.
Topics: Animals; Fibrinolytic Agents; Humans; Metalloendopeptidases; Thrombolytic Therapy; alpha-Macroglobulins
PubMed: 11910179
DOI: 10.1159/000048057 -
The Journal of Biological Chemistry Jun 1982Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively...
Separation of free and chymotrypsin-bound alpha 2-macroglobulin by affinity chromatography. Its use to demonstrate that the two chymotrypsin-binding sites of alpha 2-macroglobulin are equivalent and independent.
Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively eluted either with acetic acid or with 40% glycerol, pH 8.0. This is the first report of a preparative separation of free and proteinase-bound alpha 2-macroglobulin. Using this affinity chromatographic system, we were able to demonstrate that the two chymotrypsin binding sites of alpha-2-macroglobulin are equivalent and independent.
Topics: Animals; Binding Sites; Cattle; Chromatography, Affinity; Chymotrypsin; Humans; Kinetics; Macromolecular Substances; Mathematics; Protein Binding; alpha-Macroglobulins
PubMed: 6177686
DOI: No ID Found -
Journal of Clinical Pathology Sep 1984Reference ranges for albumin bound and alpha 2-macroglobulin bound zinc concentrations have been determined in a study of sera obtained from 134 healthy adults. The...
Reference ranges for albumin bound and alpha 2-macroglobulin bound zinc concentrations have been determined in a study of sera obtained from 134 healthy adults. The concentrations of zinc bound to alpha 2-macroglobulin were remarkably constant with a mean (+/-SD) of 2.4 +/- 0.6 mumol/l; the variations in total serum zinc concentrations were almost entirely accounted for by variations in the zinc associated with albumin. There were no sex related differences in the transport of zinc in serum; neither was this sensitive to the use of oral contraceptives. These data provide a baseline for further investigations into the effects of zinc deficiency on the serum transport of the metal.
Topics: Adolescent; Adult; Carrier Proteins; Contraceptives, Oral, Combined; Female; Humans; Male; Middle Aged; Reference Values; Serum Albumin; Zinc; alpha-Macroglobulins
PubMed: 6206098
DOI: 10.1136/jcp.37.9.1050 -
Journal of Clinical Pathology Sep 1970A radial immunodiffusion technique has been used to measure levels of four serum proteins in preeclampsia with or without proteinuria and in normal pregnant and...
A radial immunodiffusion technique has been used to measure levels of four serum proteins in preeclampsia with or without proteinuria and in normal pregnant and non-pregnant controls. In preeclampsia unaccompanied by proteinuria, albumin and transferrin levels are similar to those found in the normal pregnant controls, but there are significant falls in alpha(2)-macroglobulin and IgG. When preeclampsia is accompanied by proteinuria there is a marked fall in albumin and an increase in alpha(2)-macroglobulin. Since alpha(2)-macroglobulin has antiplasmin activity it is possible that increased levels of this protein in preeclampsia accompanied by proteinuria contribute to the intravascular coagulation which has been described in this disorder.
Topics: Adolescent; Adult; Antifibrinolytic Agents; Blood Coagulation; Female; Humans; Immunodiffusion; Immunoglobulin G; Macroglobulins; Pre-Eclampsia; Pregnancy; Proteinuria; Serum Albumin; Transferrin
PubMed: 4991000
DOI: 10.1136/jcp.23.6.514 -
Pharmaceutical Biology Dec 2022α2-Macroglobulin (α2-M) is believed to be a potential anti-irradiation agent, but related mechanisms remains unclear.
CONTEXT
α2-Macroglobulin (α2-M) is believed to be a potential anti-irradiation agent, but related mechanisms remains unclear.
OBJECTIVE
We investigated the irradiation protective effect of α2-M.
MATERIALS AND METHODS
A total of 10 Gy dose of irradiation was used to damage human skin fibroblasts. The influence of α2-M (100 µg/mL) on the proliferation, migration, invasion and apoptosis of fibroblasts was observed using Cell Counting Kit-8 (CCK8), wound healing, transwell, and flow cytometry. Malondialdehyde, superoxide dismutase and catalase was measured using related ELISA kits. The levels of mitochondrial membrane potential and calcium were detected using flow cytometry. The expression of transient receptor potential melastatin 2 (TRPM2) was investigated through western blotting and immunofluorescence staining.
RESULTS
High purity of α2-M was isolated from Cohn fraction IV. α2-M significantly increased cell proliferation, migration, invasion, but suppressed cell apoptosis after irradiation. The promotion of cell proliferation, migration and invasion by α2-M exceeded over 50% compared group irradiation. The increased cell ratio in the S phase and decreased cell ratio in the G2 phase induced by irradiation were remarkably reversed by α2-M. α2-M markedly suppressed the increased oxidative stress level caused by irradiation. The mitochondrial damage induced by irradiation was improved by α2-M through inhibiting mitochondrial membrane potential loss, calcium and TRPM2 expression.
DISCUSSION AND CONCLUSIONS
α2-M significantly promoted the decreased fibroblast viability and improved the mitochondria dysfunction caused by irradiation. α2-M might present anti-radiation effect through alleviating mitochondrial dysfunction caused by irradiation. This study could provide a novel understanding about the improvement of α2-M on irradiation-induced injury.
Topics: Apoptosis; Calcium; Female; Fibroblasts; Humans; Membrane Potential, Mitochondrial; Mitochondria; Pregnancy; Pregnancy-Associated alpha 2-Macroglobulins; TRPM Cation Channels
PubMed: 35881053
DOI: 10.1080/13880209.2022.2096077 -
The Journal of Biological Chemistry Dec 2020Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use...
Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines ( on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.
Topics: Acetylation; Amino Acid Sequence; Chromatography, High Pressure Liquid; Esters; Histidine; Humans; Hydrogen-Ion Concentration; Hydroxides; Mutagenesis, Site-Directed; Peptides; Protease Inhibitors; Recombinant Proteins; Sequence Alignment; Sulfhydryl Compounds; Tandem Mass Spectrometry; Thermolysin; alpha-Macroglobulins
PubMed: 32978260
DOI: 10.1074/jbc.RA120.015694 -
Scientific Reports Sep 2017Recently we have reported that asthma is associated with enhanced plasma thrombin formation, impaired fibrinolysis and platelet activation. In the present study we...
Recently we have reported that asthma is associated with enhanced plasma thrombin formation, impaired fibrinolysis and platelet activation. In the present study we investigated whether described prothrombotic blood alterations might predispose to thromboembolic events or asthma exacerbations. In 164 adult asthmatics we assessed clinical events during 3-year follow-up and analyzed their associations with measured at baseline prothrombotic blood parameters. Data were obtained from 157 (95.7%) of the asthma patients. We documented 198 severe asthma exacerbations (64/year), which occurred in 53 subjects (34%). These patients were older (p = 0.004), had worse asthma control (p = 0.02) and lower spirometry values (p = 0.01), at baseline. Interestingly, this subgroup had longer clot lysis time (CLT), as well as lower α-macroglobulin (p = 0.038 and p = 0.04, respectively, after adjustment for potential confounders). Increased CLT and lower α-macroglobulin were demonstrated as independent predictors of asthma exacerbation in multiple regression model. Moreover, we documented two episodes of deep vein thrombosis (1.3%), and eight acute coronary syndromes (5.1%). Patients who experienced thromboembolic events (n = 10, 6.4%, 2.1%/year) had lower α-macroglobulin (p = 0.04), without differences in efficiency of fibrinolysis and thrombin generation. Impaired fibrinolysis and lower levels of α-macroglobulin might predispose to a higher rate of asthma exacerbations, suggesting new links between disturbed hemostasis and asthma.
Topics: Asthma; Female; Fibrinolysis; Humans; Male; Middle Aged; Plasma; Pregnancy-Associated alpha 2-Macroglobulins; Risk Assessment
PubMed: 28887505
DOI: 10.1038/s41598-017-11467-8 -
FEBS Letters Jan 1983The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell... (Comparative Study)
Comparative Study
The biosynthesis and secretion of alpha 2-macroglobulin was studied in rat hepatocyte primary cultures. After immunoprecipitation of alpha 2-macroglobulin from a cell homogenate and the hepatocyte medium, two forms of alpha 2-macroglobulin with app. Mr of 176000 and 182000, respectively, were identified. A precursor-product relationship for the two alpha 2-macroglobulin forms was demonstrated by a pulse-chase experiment. The cellular form of alpha 2-macroglobulin could be deglycosylated by endoglucosaminidase H, whereas the medium form of alpha 2-macroglobulin remained unaffected. On the other hand, only the medium form of alpha 2-macroglobulin was found to be susceptible to neuraminidase. In vitro translation of rat liver poly(A)+ RNA resulted in a translation product of an app. Mr of 162000.
Topics: Animals; Cell-Free System; Cells, Cultured; Hexosaminidases; Immunosorbent Techniques; Liver; Male; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Neuraminidase; Poly A; Protein Biosynthesis; Protein Precursors; RNA; RNA, Messenger; Rats; Rats, Inbred Strains; alpha-Macroglobulins
PubMed: 6186524
DOI: 10.1016/0014-5793(83)80331-7