lysenin protein  (Biosynth Carbosynth)

Journal: The Journal of Biological Chemistry

Article Title: Acid sphingomyelinase recruits palmitoylated CD36 to membrane rafts and enhances lipid uptake

doi: 10.1016/j.jbc.2025.110213

Figure Lengend Snippet: OxLDL increases ASMase expression and promotes sphingomyelin (SM) catabolism. A , schematic illustration of SM catabolism. SM is hydrolyzed by ASMase to generate ceramide and phosphatidylcholine. Ceramidases convert ceramides into sphingosine and fatty acids. B , Western blot detection of ASMase expression is performed in cells treated with varying concentrations of oxLDL (50–120 μg/ml; one-way ANOVA followed by Tukey’s post hoc test, n = 3). C , RT-qPCR analysis measures ASMase mRNA levels in oxLDL (50 μg/ml) treated macrophages. D , ASMase enzymatic activity is assessed in cells treated with oxLDL at concentration of 50 μg/ml. E , OxLDL-treated cells are seeded on coverslip and incubated with SM-binding protein, lysenin-His (1 μg/ml). Membrane SM is detected using immunofluorescence staining and quantified by flow cytometry. F – H , total cellular levels of SM and ceramides, and SM/ceramide ratio are measured by LC-MS/MS in oxLDL-treated cells (50 μg/ml). I and J , lipidomics analysis of specific SM and ceramide species in cells treated with oxLDL (50 μg/ml). All comparisons were conducted with Student's t test except in ( B ) (mean ± SD, n = 3). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. ns, not significant.

Article Snippet: After blocking with 2% BSA/PBS, 1 μg/ml lysenin-His (TargetMol) dissolved in the blocking solution was added and incubated at 4 °C overnight.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Activity Assay, Concentration Assay, Incubation, Binding Assay, Membrane, Immunofluorescence, Staining, Flow Cytometry, Liquid Chromatography with Mass Spectroscopy

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis

doi: 10.1161/ATVBAHA.122.318443

Figure Lengend Snippet: Alterations in sphingomyelin levels by genetic manipulation of SMS1 (sphingomyelin synthase 1) or SMS2 modulate TF (tissue factor) activity. A , Increased SM levels in human monocyte-derived macrophage (MDMs) infected with adenovirus encoding SMS1 or SMS2. MDMs were treated with a control vehicle (CV) or infected with a control adenovirus (AdV) or AdV encoding SMS1, SMS2, or both (10 pfu/cell). After culturing MDMs for 48 hours, the cells were fixed and stained with an sphingomyelin (SM)-binding protein, lysenin, as described in Methods. Cells were imaged by confocal microscopy, and the fluorescence intensity of SM (red color) in the plasma membrane (30 cells) was quantified using Image J (FIJI). DAPI was used to label nuclei (blue color). The left panel shows representative images, and the right panel shows quantified data by 1-way ANOVA followed by Dunnett post hoc test. The fluorescence intensity measured in MDMs treated with a CV was taken as 100%. B , Increased synthesis of SM by overexpression of SMS1 or SMS2 does not alter TF antigen levels on the cell surface. MDMs, infected with a control, SMS1, SMS2, or both AdV as described in A , were immunostained with TF mAb. As controls, MDMs treated with a control vehicle were stained with control IgG (Con IgG) or TF mAb (CV). Immunostained cells were subjected to FACS analysis. Please note that histograms of TF immunostained cells of various treatments were completely overlapped. C and D , SMS1 or SMS2 overexpression attenuates ATP-induced increased TF activity on the cell surface ( C ) and EVs ( D ). MDMs were treated with a control vehicle (CV) or infected with control, SMS1, SMS2, SMS1+SMS2 AdVs as described in A . After culturing cells for 48 hours, they were treated with a control vehicle (control) or ATP (200 µM) for 15 minutes. At the end of 15-minute treatment, cell supernatants were collected, and EVs from cell supernatants were isolated by centrifugation. TF activity associated with cells and EVs were determined in a factor X activation assay by adding FVIIa (10 nM) and FX (175 nM) and measuring the rate of factor Xa generation in a chromogenic assay. The data were analyzed by 1-way ANOVA followed by Dunnett post hoc test (n=12 for C and n=9 for D ). P were calculated relative to CV|ATP. E , Reduced expression of SM (red) in MDMs transfected with SMS1 or SMS2 siRNA. MDMs were transfected with a mock transfection reagent (Mock), scrambled RNA (scRNA), or siRNA specific for SMS1, SMS2, or both. After culturing cells for 48 hours, SM was stained and quantified as described in A . Nuclei were stained with DAPI (blue). The left panel shows representative images of SM staining, and the right panel shows quantification of SM staining in the plasma membrane by 1-way ANOVA followed by Dunnett post hoc test (n=30). F , Silencing of SMS1, SMS2, or both does not affect cell surface expression of TF in MDMs. Non-permeabilized MDMs, transfected with Mock, scRNA, or siRNA specific for SMS1, SMS2, or both, were immunostained for TF and subjected to FACS analysis. As controls, Mock transfected cells were stained with control IgG (Con IgG). Histograms of TF immunostained cells of various treatments were completely overlapped. G and H , Silencing of SMS1 or SMS2 led to increased cell surface TF activity ( G ) and EV-associated TF activity ( H ). MDMs were transfected with scRNA, or siRNA specific for SMS1, SMS2, or both. MDMs were treated with a control vehicle or ATP, and TF activity associated with the cell surface and EVs were measured as described in C and D . The data were analyzed by 1-way ANOVA followed by Dunnett post hoc test (n=7 for G and n=8 for H ). P were calculated relative to Mock Control.

Article Snippet: To label sphingomyelin, non-permeabilized cells were incubated with sphingomyelin-specific binding protein, lysenin (0.5 µg/mL; Peptide Institute, Inc, Osaka, Japan) in 2% bovine serum albumin in PBS for 4 hours.

Techniques: Activity Assay, Derivative Assay, Infection, Staining, Binding Assay, Confocal Microscopy, Fluorescence, Over Expression, Isolation, Centrifugation, Activation Assay, Chromogenic Assay, Expressing, Transfection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis

doi: 10.1161/ATVBAHA.122.318443

Figure Lengend Snippet: Silencing of ASMase (acid sphingomyelinase), nSMase (neutral sphingomyelinase) 2, or nSMase 3, and not nSMase 1, increases sphingomyelin (SM) levels in the plasma membrane and reduces cell surface TF (tissue factor) activity and TF activity associated with extracellular vesicles (EVs). A , Silencing of ASMase, nSMase 2, or nSMase 3, and not nSMase 1, increases SM levels in the plasma membrane of monocyte-derived macrophages (MDMs). MDMs were transfected with scrambled RNA (scRNA) or siRNA specific for ASMase, nSMase 1, nSMase 2, or nSMase 3 (100 nM). After culturing cells for 48 hours, cells were fixed and stained for SM using lysenin as described in Figure A. The cells were analyzed by confocal microscopy, and the fluorescence intensity of SM staining in the plasma membrane was quantified from 30 cells. The data were analyzed by 1-way ANOVA followed by Dunnett post hoc test. P were calculated relative to Mock. B , Silencing of SMases had no effect on TF expression. MDMs were transfected with scrambled RNA (scRNA) or siRNA specific for ASMase, nSMase 1, nSMase 2, or nSMase 3. TF expression on the cell surface was analyzed by flow cytometry by immunostaining non-permeabilized cells with TF mAb. As controls, mock-transfected cells were stained with control IgG (Con IgG) or TF mAb (Mock). Histograms of all TF immunostained cells of various treatments were overlapped, therefore individual histograms are not discernible. C and D , Silencing of ASMase, nSMase 2, or nSMase 3, and not nSMase 1, reduces TF activity at the cell surface and curtails TF + EVs release. MDMs, transfected with scRNA or siRNA specific for ASMase, nSMase 1, nSMase 2, or nSMase 3, were stimulated with a control vehicle or ATP for 15 minutes. At the end of 15 minutes, cell supernatants were collected and EVs were isolated from the cell supernatants. Cell surface TF activity and TF activity associated with EVs were measured in a factor X activation assay. Cellular TF activity in C (n=5) and EV associated TF activity in D (n=4) were analyzed by 1-way ANOVA followed by Dunnett post hoc test. P were calculated relative to Mock ATP in C and D . C , P were also calculated for controls (relative to Mock control).

Article Snippet: To label sphingomyelin, non-permeabilized cells were incubated with sphingomyelin-specific binding protein, lysenin (0.5 µg/mL; Peptide Institute, Inc, Osaka, Japan) in 2% bovine serum albumin in PBS for 4 hours.

Techniques: Activity Assay, Derivative Assay, Transfection, Staining, Confocal Microscopy, Fluorescence, Expressing, Flow Cytometry, Immunostaining, Isolation, Activation Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis

doi: 10.1161/ATVBAHA.122.318443

Figure Lengend Snippet: Increased sphingomyelin (SM) levels and reduced TF (tissue factor) activity in fibroblasts from Niemann-Pick disease (NPD) patients. A , Increased SM levels in fibroblasts from NPD patients. Fibroblasts from 3 healthy individuals (WT) and 3 NPD patients (1 NPD type A and 2 NPD type B) were stained for SM (red) using lysenin. DAPI was used to label nuclei (blue). Representative images from WT and NPD patients’ fibroblasts were shown in the left panel. Quantified values of fluorescence intensity in the plasma membrane were shown in the right panel (number of cells analyzed in each group, 10). B , Flow cytometry analysis of cell surface tissue factor expression in WT and NPD fibroblasts. Intact, nonpermeabilized WT and NPD fibroblasts were labeled with TF mAb. As a control, WT fibroblasts were also stained with control IgG (Con IgG). The immunostained cells were subjected to FACS analysis. Histograms of TF staining of fibroblasts from WT and NPD patients were overlapped. C and D , TF activity on the cell surface ( C ) or in EVs ( D ) of WT and NPD fibroblasts. WT and NPD fibroblasts derived from 3 different subjects were treated with a control vehicle (Control) or ATP (200 µM, 15 min). EVs released into the supernatant medium were isolated. TF activity associated with cells and EVs was determined in a factor X activation assay (n=3). For statistical analysis (for all panels), data from 3 cell lines of the same experimental group were combined and statistical significance was determined by nonparametric Mann-Whitney test.

Article Snippet: To label sphingomyelin, non-permeabilized cells were incubated with sphingomyelin-specific binding protein, lysenin (0.5 µg/mL; Peptide Institute, Inc, Osaka, Japan) in 2% bovine serum albumin in PBS for 4 hours.

Techniques: Activity Assay, Staining, Fluorescence, Flow Cytometry, Expressing, Labeling, Derivative Assay, Isolation, Activation Assay, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Alterations to Sphingomyelin Metabolism Affect Hemostasis and Thrombosis

doi: 10.1161/ATVBAHA.122.318443

Figure Lengend Snippet: ASMase (acid sphingomyelinase) or SMS2 (sphingomyelin synthase 2) deficiency alters TF (tissue factor) activity in vivo in mononuclear cells by modulating the encryption and decryption of TF. A , Sphingomyelin (SM) levels in peripheral blood mononuclear cells (PBMCs) isolated from WT, ASMase −/− , and SMS2 −/− mice. PBMCs were isolated from WT, ASMase −/− , or SMS2 −/− mice by Ficoll gradient centrifugation. Cells were fixed, and SM was stained by lysenin binding as described in Methods. Immunostained cells were analyzed by fluorescence microscopy, and the fluorescence intensity of SM (red) staining in the plasma membrane was quantified using Image J (visible cells in the image were monocytes; other mononuclear cells were much smaller and stained weakly, and thus not readily visible; n=30 to 80 cells). DAPI was used to label nuclei (blue). The left panel shows representative images, and the right panel shows quantified data. B , Cell surface tissue factor expression levels in PBMCs. WT, ASMase −/− , and SMS2 −/− mice were administered with LPS (5 mg/kg, IP) to induce de novo synthesis of TF or left untreated (NT). After 4 hours of LPS administration, blood was collected, and PMBCs were isolated. Nonpermeabilized PBMCs were fixed, immunostained for TF, and subjected to flow cytometry. As a control, PBMCs from LPS administered WT mice were stained with control IgG (Con IgG). Histograms of TF immunostaining of PBMCs from untreated WT, ASMase −/− and SMS2 −/− mice, and control IgG stained PBMCs of LPS-treated WT mice were overlapped (far left histogram). Histograms of TF immunostaining of PBMCs from LPS administered WT and ASMase −/− were also overlapped (far right histogram). Histogram of TF immunostaining of PBMCs from LPS-treated SMS2 −/− showed a slight shift to the left compared with histograms of WT and ASMase −/− mice. C , TF activity levels in PBMCs of WT, ASMase −/− and SMS2 −/− mice (number of mice/group=8). WT, ASMase −/− , and SMS2 −/− mice were administered with saline (control), LPS (5 mg/kg, 4 hours) or LPS (5 mg/kg, 4 hours) plus ATP (50 mg/kg, 15 minutes, following 4 hours of LPS). At the end of the experimental treatment period, blood was collected, and PBMCs were isolated by Ficoll gradient centrifugation. TF activity associated with PBMCs was measured in a factor X activation assay. D , TF activity associated with EVs in plasma (number of mice/group=8). WT, ASMase −/− , and SMS2 −/− mice were treated with saline, LPS, or LPS+ATP as described above. EVs were isolated from the plasma, and TF activity associated with EVs was measured in a factor X activation assay. E , Thrombin generation in the plasma of WT, ASMase −/− , and SMS2 −/− mice. WT, ASMase −/− , and SMS2 −/− mice were treated with saline, LPS, or LPS+ATP as described above. Thrombin generation in murine blood was measured as levels of thrombin: antithrombin (TAT) complexes formed in the blood (number of mice/group=8). Statistical analysis was performed using 1-way ANOVA followed by Dunnett post hoc test. P were calculated relative to WT in A , and WT within the specific treatment group in C , D , and E .

Article Snippet: To label sphingomyelin, non-permeabilized cells were incubated with sphingomyelin-specific binding protein, lysenin (0.5 µg/mL; Peptide Institute, Inc, Osaka, Japan) in 2% bovine serum albumin in PBS for 4 hours.

Techniques: Activity Assay, In Vivo, Isolation, Gradient Centrifugation, Staining, Binding Assay, Fluorescence, Microscopy, Expressing, Flow Cytometry, Immunostaining, Activation Assay

Journal: The American Journal of Surgical Pathology

Article Title: Liver and Skin Histopathology in Adults with Acid Sphingomyelinase Deficiency (Niemann-Pick Disease Type B)

doi: 10.1097/PAS.0b013e31825793ff

Figure Lengend Snippet: In some biopsies, SM is present in sinusoidal Kupffer cells only (K distribution). In other biopsies, SM has accumulated in sinusoidal Kupffer cells, foci of proliferating portal Kupffer cells and hepatocytes (KH distribution). A. Patient 6: SM is present within enlarged, foamy Kupffer cells (arrows; H&E, 60× objective). B. Patient 1: SM is present within clusters of enlarged foamy Kupffer cells (K) and foamy hepatocytes (H) (H&E, 60× objective). C. In HRLM sections, SM stains purple in sinusoidal Kupffer cells (modified toluidine blue, 100× oil objective). D. In KH distribution, SM is present in both Kupffer cells and hepatocytes (modified toluidine blue, 100× oil objective). E and F. Lysenin affinity staining also highlights the differences in SM accumulation in K versus KH distribution patterns (lysenin affinity stain, 100× oil objective). G. CD68 immunohistochemistry highlights the enlarged Kupffer cells as single cells (10× objective). H. CD68 immunohistochemistry highlights enlarged Kupffer cells as single cells and as clusters proliferating in portal tracts and infiltrating bands of fibrosis (10× objective).

Article Snippet: For SM staining, a 5μg/mL solution of lysenin protein (Peptides International; Louisville, KY) was diluted in TBS with 0.5% BSA (TBS-B, DAKO) and placed on the slides and incubated at 37°C for 1 hour.

Techniques: Modification, Staining, Immunohistochemistry