α2m human plasma  (Millipore)

Journal: Journal of Medical Biochemistry

Article Title: Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration

doi: 10.5937/jomb0-39557

Figure Lengend Snippet: α2M level decreased in degenerated NP tissue. Representative images of (A) MRI, the yellow arrows indicated the operation section, (B) HE staining (magnification: 200×) (C) IHC targeting α2M of both mildly and severely degenerated NP tissue. (magnification: 200×) (D) Quantification analysis of IHC. NP tissue from the 16 patients was lysed to measure (E) α2M with ELISA methods and (F) total ROS level. The values are mean ± SD of three independent experiments. (***P<0.001)

Article Snippet: Additionally, α2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) was used to reverse HOCl.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medical Biochemistry

Article Title: Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration

doi: 10.5937/jomb0-39557

Figure Lengend Snippet: α2M stimulation decreased ROS, MMP13, and ADAMTS4 of severely degenerated NP tissue. We cultured severely degenerated NP tissue in α2M (50, 150, 200 nmol/L) growth medium for 3 days. The protein expression level of α2M was determined by (A) IHC (magnification: 200×) and (B) quantification analysis. (C) Total ROS level of NP tissue. (D) The content of MMP13 and ADAMTS4 was assayed by ELISA. The values are mean ± SD of three independent experiments. (*P<0.05, **P<0.01)

Article Snippet: Additionally, α2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) was used to reverse HOCl.

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medical Biochemistry

Article Title: Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration

doi: 10.5937/jomb0-39557

Figure Lengend Snippet: α2M stimulation reversed HOCl-induced oxidative stress of NP cells. NP cells of mildly degenerated NP tissue were treated with HOCl (from 10 to 50 μmol/L) for 6 h, or treated with 30 μmol/L from 3 h to 24 h; Besides, NP cells were pretreated with 30 μmol/L HOCH for 6 h and then cultured with α2M (from 50 to 200 nmol/L) for another 24 h, or cultured with 200 nmol/L α2M from 3 h to 24 h. (A, B) The protein expression level of α2M was determined by ELISA. (C, D) Total ROS level of NP cells. The values are mean ± SD of three independent experiments.

Article Snippet: Additionally, α2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) was used to reverse HOCl.

Techniques: Cell Culture, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Medical Biochemistry

Article Title: Antioxidative behavior of a2-macroglobulin in intervertebral disc degeneration

doi: 10.5937/jomb0-39557

Figure Lengend Snippet: α2M level decreased in degenerated NP tissue. Representative images of (A) MRI, the yellow arrows indicated the operation section, (B) HE staining (magnification: 200×) (C) IHC targeting α2M of both mildly and severely degenerated NP tissue. (magnification: 200×) (D) Quantification analysis of IHC. NP tissue from the 16 patients was lysed to measure (E) α2M with ELISA methods and (F) total ROS level. The values are mean ± SD of three independent experiments. (***P<0.001)

Article Snippet: Additionally, α2M from human plasma (Sigma-Aldrich, St. Louis, MO, USA) was used to reverse HOCl.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: α2M is efficiently cleaved by active MMP-9. a Illustration of the α2M inhibition mechanism. The tetrameric structure of α2M (± 720 kDa) is formed by a non-covalent interaction between two covalently linked dimers (± 360 kDa). Each individual monomer (± 180 kDa) contains a protease bait region (red lines) and a buried receptor binding domain (gray triangles). Upon cleavage of the bait region, α2M becomes ‘activated’ (α2M*) and undergoes a conformational change. This results in physical trapping of the protease, the exposure of a reactive thioester bond which hooks to protease lysine residues (red hooks) and the surface exposure of the receptor binding domains for optimal clearance of α2M*/protease complexes from the circulation. b (Top panel) Structural model of an activated human MMP-9 monomer [29]. The active site (yellow), fibronectin repeats (blue), Zn2+-binding domain (orange), O-glycosylated domain (black) and hemopexin domain (red) are shown. The gray arrowheads and lines symbolize the flexibility and relative distances between the active site and the hemopexin domains. (center panel) Structural model for activated trimeric MMP-9 [36]. The three gray symbols indicate the relative “swelling” of the MMP-9 trimer. (Bottom panel) Structure of activated human α2M* (PDB ID: 4AXQ) [6]. Each α2M monomer is shown in a different shade of green. c, Incubation of α2M with active MMP-9 (17 nM MMP-9/67 nM α2M) results in a single cut of α2M (α2M*) and the generation of ± 90 kDa fragments. This process is reduced in the presence of an MMP-inhibitor (SB-3CT). d Cleavage of α2M by MMP-9 is fast (25% instant activation) and increases time-dependently (20 nM MMP-9/140 nM α2M). Relative quantification of three experiments (bottom panel). Y-axis indicates the percentage (%) of cleaved α2M. e Cleavage of α2M by MMP-9 (α2M at 140 nM with decreasing concentrations of MMP-9) requires a high α2M/MMP-9 ratio, likely due to the protease-inhibitory effect of the α2M/MMP-9 interaction. Relative quantification of three experiments (bottom panel). All images were obtained by Western-blot analysis under denaturing/reducing condition and detected with anti-α2M. Y axis indicates the percentage (%) of cleaved α2M. Quantification data were fitted with a four-parameter dose–response fit and are representative for three independent experiments (see Supplementary figure S2)

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Inhibition, Binding Assay, Incubation, Activation Assay, Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: Active MMP-9 is covalently trapped by α2M and presents as high-molecular weight complexes on gelatin zymograms. a MMP-9 converts native α2M into its electrophoretically ‘fast’ form (α2M*). Native electrophoresis with a protein ladder in the left lane for size standardization. Protein masses are indicated in kilodaltons (kDa). b Active MMP-9 forms four covalent complexes with α2M under denaturing conditions (Western blot analysis of MMP-9/α2M (17 nM/67 nM)) The presence of the MMP-inhibitor SB-3CT prevents the formation of these complexes. Proteins were separated under non-reducing conditions and visualized with an anti-MMP-9 antibody. c Covalent α2M*/MMP-9 (17 nM/67 nM) complexes are detected by modified gelatin zymography analysis. d The complex formation between α2M and MMP-9 (20 nM MMP-9/140 nM α2M) is time-dependent and occurs fast (± 20% instant activation). Relative quantification of three experiments (bottom panel). e Formation α2M*/MMP-9 complexes is dose-dependent (α2M at 140 nM with decreasing concentrations of MMP-9) and requires a high α2M/MMP-9 ratio. Relative quantification of three experiments (bottom panel). d, e Western-blot analysis under non-reducing conditions and detected with an anti-α2M antibody. Quantification data were fitted with a four-parameter dose–response fit and are representative for three independent experiments (see Supplementary figure S2)

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Molecular Weight, Electrophoresis, Western Blot, Modification, Zymography, Activation Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: Active MMP-9 efficiently cleaves and complexes α2M in human plasma. a In human plasma, activated MMP-9 (A) cleaves α2M (0.5 pmol MMP-9/µl plasma) with a single cut, splitting the molecule in halves with comparable sizes (α2M*). This process is reduces in the presence of the MMP inhibitor SB-3CT and does not occur with an inactive MMP-9 mutant (E). Images represent an SDS-PAGE analysis (reducing conditions) followed by Western blot with an anti-α2M antibody. b Activated MMP-9 (A) forms four covalent complexes with α2M in human plasma. This process is reduced in the presence of the MMP inhibitor SB-3CT and does not occur with an inactive MMP-9 mutant (E). Images represent an SDS-PAGE analysis (non-reducing conditions) followed by Western blot analysis (anti-MMP-9 antibody). c The formation of α2M*/MMP-9 complexes in human plasma is detected by modified gelatin zymography analysis. Images in panels A-C are representative of 3 experiments with plasma from three different plasma donors (see Supplementary Figure S4). d, e Gradual activation of proMMP-9 was induced by addition of the catalytic domain of MMP-3 (cdMMP-3). In the presence of human plasma, fully activated (± 80 kDa) MMP-9 was efficiently converted into high-molecular weight complexes, undetectable by standard gelatin zymography analysis. d Standard gelatin zymography analysis. e Western-blot analysis with anti-MMP-9 (AB6008)

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Mutagenesis, SDS Page, Western Blot, Modification, Zymography, Activation Assay, Molecular Weight

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: Inhibition and interaction of α2M with monomeric and trimeric MMP-9. a The degradation of gelatin by active MMP-9 monomers (blue) was more efficiently inhibited by purified α2M in comparisons with MMP-9 trimers (red) and their mixtures (yellow). Data fitted with a standard dose–response fit, n = 3 (each from an independent experiment). b The degradation of gelatin by active MMP-9 monomers (blue) was more efficiently inhibited by human plasma in comparisons with MMP-9 trimers (red) and their mixtures (yellow). Data fitted with a standard dose–response fit, n = 7 (7 plasma samples from different donors, analyzed in three independent experiments). c both MMP-9 monomers and trimers cleave α2M (purified α2M and human plasma) with a single cut, splitting the molecule in halves with comparable sizes (α2M*). Images represent SDS-PAGE analysis (reducing conditions) followed by Western blot analysis (anti-α2M antibody). d MMP-9 monomers and trimers form complexes with α2M representing, respectively, complexes 2 and 4 and complexes 1 and 3. This effect is seen with purified α2M and in human plasma. Images on the left represent SDS-PAGE analysis (non-reducing conditions) followed by Western blot analysis (anti-MMP-9 antibody). Images on the right represent modified gelatin zymography analysis (33 nM MMP-9/333 nM α2M or 0.5 µl plasma per pmol MMP-9). e MMP-9 monomers and trimers (20 nM MMP-9/140 nM α2M) cleave α2M equally well in a time-dependent manner. f MMP-9 monomers and trimers equally well digest α2M in a dose-dependent manner (α2M at 140 nM with decreasing concentrations of MMP-9). g The profile of complex formation between α2M and MMP-9 monomers and trimers (20 nM MMP-9/140 nM α2M) is similar over time. h The complex formation between α2M and MMP-9 monomers/trimers is comparable across different doses. Panels E–H represent relative quantifications of Western-blot images of three independent experiments. Data were not significantly different as confirmed by extra sum-of-squares F test and fitted by one joint four-parameter dose-response fit (black). See Supplementary Figure S5 for associated Western-blot images

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Inhibition, Purification, SDS Page, Western Blot, Modification, Zymography

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: α2M*/MMP-9 trimer complexes remain partially active and escape LRP-1-mediated endocytosis. a Proposed model for the interaction between active MMP-9 monomers/trimers and α2M. Given the size and flexibility of MMP-9 trimers, protease active sites remain exposed in the environment and travel as proteolytically active proteases linked to α2M. b Native PAGE analysis of α2M*/MMP-9 complexes reveals the existence of a slower form in samples containing MMP-9 trimers (α2M*tri) (666 nM α2M with a ½ dilution of MMP-9 monomers, trimers or their mixture). Top, Coomassie blue stained native PAGE gel. Bottom, Western-blot analysis with an anti-α2M antibody. See Supplementary Figure S6 for repeat experiments. c α2M*/MMP-9 trimer complexes immobilized on anti-α2M beads retain a significant amount of their proteolytic activity against gelatins. Each data point represents an independent experiment (n = 3). Relative activities represent the fold change in fluorescence compared to α2M alone (background signal). d α2M*/MMP-9 trimer complexes from human plasma, immobilized on anti-α2M beads, retain a significant amount of their proteolytic activity against gelatins. Relative activities represent the fold change in fluorescence compared to α2M alone. Each data point represents an independent experiment (n = 5). *p < 0.05, **p < 0.01, as determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Bars represent median values with indication of the interquartile range as error bars. e, The relative amounts of α2M*/MMP-9 monomers found in endocytic vesicles are significantly increased compared to α2M*/MMP-9 trimers. Data represent four independent experiments, with each experimental replicates shown in the same color. Bars represent mean values. Data were normalized to the highest value in each experiment. *p < 0.05, as determined by Mann–Whitney test on the mean values of each experiment. f Representative images of α2M endocytosis. Bright green fluorescence represents the uptake of pH-rhodo-labeled α2M complexes and presence in acidic vesicles. Size bars indicate 200 µm. g Flow cytometry analysis of U-87 cells for the presence of cell surface LRP-1 when treated with combinations of α2M and MMP-9 proteoforms. α2M, MMP-9 monomers and MMP-9 trimers reduce the amount of LRP-1 present on cell surfaces. Complexation of α2M to MMP-9 monomers results in an additional reduction in comparison with MMP-9 trimers indicating more LRP-1 internalization and endocytosis. Data are representative for two independent experiments (See Supplementary Figure S7 for repeat experiments)

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Clear Native PAGE, Staining, Western Blot, Activity Assay, Fluorescence, MANN-WHITNEY, Labeling, Flow Cytometry, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Homotrimeric MMP-9 is an active hitchhiker on alpha-2-macroglobulin partially escaping protease inhibition and internalization through LRP-1

doi: 10.1007/s00018-019-03338-4

Figure Lengend Snippet: Inhibitory capacity of purified α2M and human plasma on gelatinolysis by MMP-9 monomers and trimers

Article Snippet: Activation of proMMP-9 was confirmed by a band shift of approximately 10 kDa, corresponding to the removal of the propeptide domain and by detection of gelatinolytic activity with a previously described gelatin degradation assay [ 51 ]. α2M purified from human plasma was purchased from Sigma Aldrich (Cat. No. M6159).

Techniques: Purification