sialidase  (New England Biolabs)


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    Name:
    O Glycosidase and Neuraminidase Bundle
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    O Glycosidase and Neuraminidase Bundle
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    E0540S
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    Category:
    Glycosidases
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    Structured Review

    New England Biolabs sialidase
    O Glycosidase and Neuraminidase Bundle
    O Glycosidase and Neuraminidase Bundle
    https://www.bioz.com/result/sialidase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sialidase - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells"

    Article Title: A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0128190

    PVL binding of tumor cell lines. A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml -1 for 30 mn; red line: rPVL-alexa 488 5 μg ml -1 in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml -1 after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml -1 rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.
    Figure Legend Snippet: PVL binding of tumor cell lines. A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml -1 for 30 mn; red line: rPVL-alexa 488 5 μg ml -1 in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml -1 after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml -1 rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.

    Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Labeling, Staining

    2) Product Images from "Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure"

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0150210

    Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p
    Figure Legend Snippet: Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p

    Techniques Used: Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Recombinant, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page

    Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p
    Figure Legend Snippet: Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p

    Techniques Used: Activity Assay, Aqueous Normal-phase Chromatography, Expressing, Real-time Polymerase Chain Reaction, Synthesized, Staining

    3) Product Images from "Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus"

    Article Title: Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02109-8

    Rg CBM40 binding to mucus-producing cells and intestinal tissue sections. a Immunostaining pattern for Rg CBM40 on LS174T cells correlated with mucin (MUC2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). b Immunostaining pattern for Rg CBM40 on cryosections of mouse colon correlated with mucin (Muc2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). Cell nuclei were counterstained with DAPI, shown in blue. c Sialidase pre-treatment of mouse colonic cryosections markedly reduced the binding of Rg CBM40 and SNA lectin. Cell nuclei were counterstained with DAPI, shown in blue. d Rg CBM40 competition assay with SNA on cryosections of mouse colon. Rg CBM40 is shown in green. Cell nuclei were counterstained with DAPI, shown in blue. No Rg CBM40 specific staining was detectable when SNA was present. e R. gnavus binding competition assay with SNA on cryosections of mouse colon. R. gnavus ATCC 29149 was incubated on sequential cryosections of mouse colon with or without SNA treatment and is shown in red. The mucus layer is shown in green. Sequential sections were required as both antibodies were raised in the same species. Cell nuclei were counterstained with DAPI, shown in blue. No R.gnavus staining was detectable when SNA was present. Appropriate primary antibody and secondary antibody only controls are also shown underneath each panel, showing some background staining. Scale bar: 20 μm
    Figure Legend Snippet: Rg CBM40 binding to mucus-producing cells and intestinal tissue sections. a Immunostaining pattern for Rg CBM40 on LS174T cells correlated with mucin (MUC2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). b Immunostaining pattern for Rg CBM40 on cryosections of mouse colon correlated with mucin (Muc2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). Cell nuclei were counterstained with DAPI, shown in blue. c Sialidase pre-treatment of mouse colonic cryosections markedly reduced the binding of Rg CBM40 and SNA lectin. Cell nuclei were counterstained with DAPI, shown in blue. d Rg CBM40 competition assay with SNA on cryosections of mouse colon. Rg CBM40 is shown in green. Cell nuclei were counterstained with DAPI, shown in blue. No Rg CBM40 specific staining was detectable when SNA was present. e R. gnavus binding competition assay with SNA on cryosections of mouse colon. R. gnavus ATCC 29149 was incubated on sequential cryosections of mouse colon with or without SNA treatment and is shown in red. The mucus layer is shown in green. Sequential sections were required as both antibodies were raised in the same species. Cell nuclei were counterstained with DAPI, shown in blue. No R.gnavus staining was detectable when SNA was present. Appropriate primary antibody and secondary antibody only controls are also shown underneath each panel, showing some background staining. Scale bar: 20 μm

    Techniques Used: Binding Assay, Immunostaining, Staining, Competitive Binding Assay, Incubation

    ELISA of Rg CBM40 binding to purified mucins. a Rg CBM40 binding to a range of purified mucins; mucin 2 (MUC2) and mixed mucins (mucins) from human cell line LS174T, purified pig gastric mucin (pPGM), and murine mucins from germ free (GF), wild type (WT), and C3GnT −/− mice. b Correlation of Rg CBM40 binding with % sialylated structure for each mucin tested. The % sialylated structures was determined by MS. c Rg CBM40 binding to LS174T MUC2 which has been treated chemically (TFA) or enzymatically with a sialidase from Clostridium perfringens ( Cp ), Salmonella typhimurium ( St ), Akkermansia muciniphila ( Am ) or Ruminococcus gnavus ( Rg ) d Rg CBM40 binding to LS174T MUC2 in competition with sugars. Rg CBM40 has been preincubated with the indicated sugars. In all cases, Rg CBM40 was incubated with immobilised mucins and binding detected using an anti-sialidase primary antibody and an anti-rabbit secondary antibody conjugated to horseradish peroxidase. The enzyme was incubated with TMB and the absorbance at 450 nm (A450) measured. The error bars show the standard error of the mean (SEM) of three replicates. P values are indicated; NS-not significant, * p
    Figure Legend Snippet: ELISA of Rg CBM40 binding to purified mucins. a Rg CBM40 binding to a range of purified mucins; mucin 2 (MUC2) and mixed mucins (mucins) from human cell line LS174T, purified pig gastric mucin (pPGM), and murine mucins from germ free (GF), wild type (WT), and C3GnT −/− mice. b Correlation of Rg CBM40 binding with % sialylated structure for each mucin tested. The % sialylated structures was determined by MS. c Rg CBM40 binding to LS174T MUC2 which has been treated chemically (TFA) or enzymatically with a sialidase from Clostridium perfringens ( Cp ), Salmonella typhimurium ( St ), Akkermansia muciniphila ( Am ) or Ruminococcus gnavus ( Rg ) d Rg CBM40 binding to LS174T MUC2 in competition with sugars. Rg CBM40 has been preincubated with the indicated sugars. In all cases, Rg CBM40 was incubated with immobilised mucins and binding detected using an anti-sialidase primary antibody and an anti-rabbit secondary antibody conjugated to horseradish peroxidase. The enzyme was incubated with TMB and the absorbance at 450 nm (A450) measured. The error bars show the standard error of the mean (SEM) of three replicates. P values are indicated; NS-not significant, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Mouse Assay, Mass Spectrometry, Incubation

    4) Product Images from "Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells"

    Article Title: Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.115998

    Partial MALDI-TOF MS profiles of the permethylated N -linked glycans derived from SRECs after digestion with sialidase S or sialidase A. Data were obtained from the 50% acetonitrile fraction and all molecular ions are present in sodiated form ([M + Na] + ). Sialylated species are annotated in red (see supplemental Table S1 ).
    Figure Legend Snippet: Partial MALDI-TOF MS profiles of the permethylated N -linked glycans derived from SRECs after digestion with sialidase S or sialidase A. Data were obtained from the 50% acetonitrile fraction and all molecular ions are present in sodiated form ([M + Na] + ). Sialylated species are annotated in red (see supplemental Table S1 ).

    Techniques Used: Mass Spectrometry, Derivative Assay

    Sialidase treatment of SRECs prior to virus infection. The data shown are the mean ± S.E. of three independent experiments performed in triplicate. *, p
    Figure Legend Snippet: Sialidase treatment of SRECs prior to virus infection. The data shown are the mean ± S.E. of three independent experiments performed in triplicate. *, p

    Techniques Used: Infection

    5) Product Images from "Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model"

    Article Title: Sialylated Cervical Mucins Inhibit the Activation of Neutrophils to Form Neutrophil Extracellular Traps in Bovine in vitro Model

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02478

    Cervical mucins inhibit the release of NETs induced by 1.5 μM PMA in combination with 3 μM ionomycin by sialic acid on its surface. The experiments were performed with mucins from (A) estrus, (B) luteal, and (C) follicular samples. Untreated mucins, as well as hydrolyzed, C7 modified and neuraminidase treated mucins were applied to stimulated neutrophils in a final concentration of 20 μg/μL. The percentage of activated cells was calculated by counting the segmented nuclei as well as the total cell number. Mean values and standard deviations are displayed in the diagrams ( n = 3 different animals). Paired ANOVA and a multiple-comparison Tukey test were applied. Statistically significant differences are given: ns, not significant, * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. Glycan illustration: Yellow square: N-acetylgalactosamine, Blue square: N-acetylglucosamine, Yellow circle: Galactose, Purple diamond: Sialic acid, rose diamond: C7 modified sialic acid.
    Figure Legend Snippet: Cervical mucins inhibit the release of NETs induced by 1.5 μM PMA in combination with 3 μM ionomycin by sialic acid on its surface. The experiments were performed with mucins from (A) estrus, (B) luteal, and (C) follicular samples. Untreated mucins, as well as hydrolyzed, C7 modified and neuraminidase treated mucins were applied to stimulated neutrophils in a final concentration of 20 μg/μL. The percentage of activated cells was calculated by counting the segmented nuclei as well as the total cell number. Mean values and standard deviations are displayed in the diagrams ( n = 3 different animals). Paired ANOVA and a multiple-comparison Tukey test were applied. Statistically significant differences are given: ns, not significant, * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001. Glycan illustration: Yellow square: N-acetylgalactosamine, Blue square: N-acetylglucosamine, Yellow circle: Galactose, Purple diamond: Sialic acid, rose diamond: C7 modified sialic acid.

    Techniques Used: Modification, Concentration Assay

    6) Product Images from "Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis"

    Article Title: Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0152326

    Glycosylation status of CD43 on leukemia cells is associated with sensitivity to CTL-mediated cytolysis. (A) Gating strategies for FACS-sorting the R54 high and R54 low subpopulations of OVA-expressing MLL/AF9 leukemia cells. (B) FACS analysis of intracellular IFN-γ in OT-1 T cells after co-culture with either R54 high or R54 low MLL/AF9 leukemia cells. IFN-γ expression in CD8 + T cells is shown. (C) 51 Cr cytotoxicity assay with OT-1 T cells, using either R54 high or R54 low leukemia cells as targets. (D) FACS analysis of OVA-IRES-GFP expression levels in MLL/AF9-OVA leukemia clones derived from c-kit + BM cells of the wild type or CD43 -/- mouse (E) 51 Cr cytotoxicity assay with OT-1 T cells, using either the wild type or CD43 -/- leukemia cells as targets (F) 51 Cr cytotoxicity assay with OT-1 T cells, using leukemia cells with or without sialidase treatment (E/T ratio = 1).
    Figure Legend Snippet: Glycosylation status of CD43 on leukemia cells is associated with sensitivity to CTL-mediated cytolysis. (A) Gating strategies for FACS-sorting the R54 high and R54 low subpopulations of OVA-expressing MLL/AF9 leukemia cells. (B) FACS analysis of intracellular IFN-γ in OT-1 T cells after co-culture with either R54 high or R54 low MLL/AF9 leukemia cells. IFN-γ expression in CD8 + T cells is shown. (C) 51 Cr cytotoxicity assay with OT-1 T cells, using either R54 high or R54 low leukemia cells as targets. (D) FACS analysis of OVA-IRES-GFP expression levels in MLL/AF9-OVA leukemia clones derived from c-kit + BM cells of the wild type or CD43 -/- mouse (E) 51 Cr cytotoxicity assay with OT-1 T cells, using either the wild type or CD43 -/- leukemia cells as targets (F) 51 Cr cytotoxicity assay with OT-1 T cells, using leukemia cells with or without sialidase treatment (E/T ratio = 1).

    Techniques Used: CTL Assay, FACS, Expressing, Co-Culture Assay, Cytotoxicity Assay, Clone Assay, Derivative Assay

    Epitopes for R54 and B2 mAbs, but S11 mAb, are sensitive to O-glycosylation inhibitor or sialidase. FACS analysis of binding of each CD43-specific mAb to MLL/AF9 leukemia cells or M1 leukemia cells treated with 1 mM benzyl-GalNac for 24 hours (A) or 250 U/ml sialidase for 1 hour (B).
    Figure Legend Snippet: Epitopes for R54 and B2 mAbs, but S11 mAb, are sensitive to O-glycosylation inhibitor or sialidase. FACS analysis of binding of each CD43-specific mAb to MLL/AF9 leukemia cells or M1 leukemia cells treated with 1 mM benzyl-GalNac for 24 hours (A) or 250 U/ml sialidase for 1 hour (B).

    Techniques Used: FACS, Binding Assay

    7) Product Images from "Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals"

    Article Title: Native mass spectrometry combined with enzymatic dissection unravels glycoform heterogeneity of biopharmaceuticals

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04061-7

    Annotation of Etanercept O -glycoforms. a Deconvoluted spectrum of Etanercept after digestion with PNGase F/sialidase (raw spectrum shown in Fig. 1h ). O -glycoforms, i.e., the number of core 1 units (Hex-HexNAc), as well as lysine variants are annotated. Each symbol indicates a certain number of O -glycan cores. b Deconvoluted spectrum of Etanercept after digestion with PNGase F (raw spectrum shown in Fig. 1f ). The number of O -glycan cores is indicated by a specific symbol in accordance with Fig. 2a. Multiple signals annotated with the same symbol represent sialic acid (Neu5Ac) variants of each O -glycoform. The number of Neu5Ac residues is indicated above each annotated peak. Peak lists with all possible glycoform assignments are available in Supplementary Data 1
    Figure Legend Snippet: Annotation of Etanercept O -glycoforms. a Deconvoluted spectrum of Etanercept after digestion with PNGase F/sialidase (raw spectrum shown in Fig. 1h ). O -glycoforms, i.e., the number of core 1 units (Hex-HexNAc), as well as lysine variants are annotated. Each symbol indicates a certain number of O -glycan cores. b Deconvoluted spectrum of Etanercept after digestion with PNGase F (raw spectrum shown in Fig. 1f ). The number of O -glycan cores is indicated by a specific symbol in accordance with Fig. 2a. Multiple signals annotated with the same symbol represent sialic acid (Neu5Ac) variants of each O -glycoform. The number of Neu5Ac residues is indicated above each annotated peak. Peak lists with all possible glycoform assignments are available in Supplementary Data 1

    Techniques Used:

    N - and O -glycosylation of Etanercept lacking sialic acids. a Deconvoluted spectrum of Etanercept treated with sialidase and O -glycosidase acquired under native conditions (raw spectrum is shown in Supplementary Fig. 8b ). The most probable glycan structures lacking sialic acids are annotated. The six most abundant N -glycoforms are boxed and marked as A to F, respectively. b Deconvoluted spectrum of sialidase-treated Etanercept upon native MS (raw spectrum is shown in Fig. 1d ). The most probable glycoforms are annotated. N -glycan structures are referred to as A to F as specified in Fig. 4a; O -glycoforms are labeled according to Fig. 2a . Peak lists with all possible glycoform assignments are available in Supplementary Data 1
    Figure Legend Snippet: N - and O -glycosylation of Etanercept lacking sialic acids. a Deconvoluted spectrum of Etanercept treated with sialidase and O -glycosidase acquired under native conditions (raw spectrum is shown in Supplementary Fig. 8b ). The most probable glycan structures lacking sialic acids are annotated. The six most abundant N -glycoforms are boxed and marked as A to F, respectively. b Deconvoluted spectrum of sialidase-treated Etanercept upon native MS (raw spectrum is shown in Fig. 1d ). The most probable glycoforms are annotated. N -glycan structures are referred to as A to F as specified in Fig. 4a; O -glycoforms are labeled according to Fig. 2a . Peak lists with all possible glycoform assignments are available in Supplementary Data 1

    Techniques Used: Mass Spectrometry, Labeling

    N -glycosylation of Etanercept TNFR and Fc domains. a Deconvoluted spectrum of dimeric TNFR digested with sialidase and O -glycosidase acquired under native conditions (raw spectrum shown in Supplementary Fig. 6c, d ). The most probable glycan structures lacking sialic acids are annotated. b Deconvoluted spectrum of Fc dimer upon native MS (raw spectrum shown in Supplementary Fig. 7 ). The most probable N -glycoforms and C-terminal lysine variants are annotated. Asterisks indicate Na + adducts. Peak lists with all possible glycoform assignments are available in Supplementary Data 1
    Figure Legend Snippet: N -glycosylation of Etanercept TNFR and Fc domains. a Deconvoluted spectrum of dimeric TNFR digested with sialidase and O -glycosidase acquired under native conditions (raw spectrum shown in Supplementary Fig. 6c, d ). The most probable glycan structures lacking sialic acids are annotated. b Deconvoluted spectrum of Fc dimer upon native MS (raw spectrum shown in Supplementary Fig. 7 ). The most probable N -glycoforms and C-terminal lysine variants are annotated. Asterisks indicate Na + adducts. Peak lists with all possible glycoform assignments are available in Supplementary Data 1

    Techniques Used: Mass Spectrometry

    Molecular structure and native mass spectrometry of Etanercept. a Schematic illustration of dimeric Etanercept consisting of a TNFR and an Fc domain. Disulfide bonds in the Fc region are indicated as yellow lines; disulfide bridges in the TNFR domain are not shown. Monosaccharide symbols are listed. Exemplary cleavage sites of IdeS, PNGase F and sialidase are indicated. Native mass spectra of b intact Etanercept ( R set = 17,500 at m/z 200), d Etanercept digested with sialidase or f PNGase F ( R set = 35,000 at m/ z 200), and h a combination of PNGase F/sialidase, respectively ( R set = 70,000 at m/z 200). Charge states are indicated. Zooms into the most abundant charge states are shown in c , e , g , and i
    Figure Legend Snippet: Molecular structure and native mass spectrometry of Etanercept. a Schematic illustration of dimeric Etanercept consisting of a TNFR and an Fc domain. Disulfide bonds in the Fc region are indicated as yellow lines; disulfide bridges in the TNFR domain are not shown. Monosaccharide symbols are listed. Exemplary cleavage sites of IdeS, PNGase F and sialidase are indicated. Native mass spectra of b intact Etanercept ( R set = 17,500 at m/z 200), d Etanercept digested with sialidase or f PNGase F ( R set = 35,000 at m/ z 200), and h a combination of PNGase F/sialidase, respectively ( R set = 70,000 at m/z 200). Charge states are indicated. Zooms into the most abundant charge states are shown in c , e , g , and i

    Techniques Used: Mass Spectrometry

    8) Product Images from "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)"

    Article Title: Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003302

    Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .
    Figure Legend Snippet: Screening for sialidase activity from a hot spring metagenomic library. A , restriction fragment analysis of 12 randomly selected clones from the hot spring metagenomic library were isolated and digested with the rare-cutting endonuclease SbfI. Digested fosmids were separated overnight on a 1% agarose gel along with a λHindIII size marker and a linearized pSMART FOS empty vector control ( lane 14 ; contains one SbfI site). Each clone showed a unique banding pattern, with fragments whose combined sizes indicated the presence of an insert of at least 30–40 kb. B , E. coli cells harboring individual fosmid clones were assayed for sialidase activity with X-Neu5Ac incorporated into agar medium. A single positive clone forming a blue colony is denoted with an arrow. C , lysate from microcultures of E. coli cells harboring individual fosmid clones were assayed for sialidase activity with 4MU-α-Neu5Ac. A single positive clone is denoted with an arrow .

    Techniques Used: Activity Assay, Clone Assay, Isolation, Agarose Gel Electrophoresis, Marker, Plasmid Preparation

    Purification and biochemical characterization of recombinant ORF12p. A , His-tagged ORF12p sialidase was expressed in E. coli and purified using a His-trap column as described under “Experimental procedures.” Shown is SDS–PAGE separation of lysates from uninduced cells ( U ), induced cells ( I ), and nickel-purified ORF12p–His ( P ). B–D , purified ORF12p–His was used to determine the pH ( B ) and temperature ( C ) optima of ORF12p–His and the effect of metal ions on its catalysis ( D ). In these experiments, reactions were performed in triplicate using the substrate 3′-sialyl- N -acetyllactosamine-2AB. Reaction products were analyzed by UPLC–HILIC–FLR and quantitated by peak integration. E , Michaelis–Menten plot of ORF12p catalyzed hydrolysis of 4MU-α-Neu5Ac. The initial velocity was determined in triplicate for each 4MU-α-Neu5Ac concentration.
    Figure Legend Snippet: Purification and biochemical characterization of recombinant ORF12p. A , His-tagged ORF12p sialidase was expressed in E. coli and purified using a His-trap column as described under “Experimental procedures.” Shown is SDS–PAGE separation of lysates from uninduced cells ( U ), induced cells ( I ), and nickel-purified ORF12p–His ( P ). B–D , purified ORF12p–His was used to determine the pH ( B ) and temperature ( C ) optima of ORF12p–His and the effect of metal ions on its catalysis ( D ). In these experiments, reactions were performed in triplicate using the substrate 3′-sialyl- N -acetyllactosamine-2AB. Reaction products were analyzed by UPLC–HILIC–FLR and quantitated by peak integration. E , Michaelis–Menten plot of ORF12p catalyzed hydrolysis of 4MU-α-Neu5Ac. The initial velocity was determined in triplicate for each 4MU-α-Neu5Ac concentration.

    Techniques Used: Purification, Recombinant, SDS Page, Hydrophilic Interaction Liquid Chromatography, Concentration Assay

    Specificity of ORF12p on sialic acid containing substrates using UPLC–HILIC–FLR analysis. A and B , the ability of ORF12p to cleave the fluorescently labeled substrates 3′- or 6′-sialyl- N -acetyllactosamine-2AB. Undigested substrates 3′- or 6′-sialyllactosamine-2AB run at ∼10.6- or 12.3-min retention times, respectively ( A and B , top panels ). Control digestion with the NeuA sialidase shifted both substrate peaks to ∼5.5-min retention time ( A and B , bottom panels ). Digestion of these substrates with 1 unit of ORF12p–His resulted in the same peak shift ( A and B , middle panels ). C , ORF12p's ability to hydrolyze α2–8 Neu5Ac was assessed using a 2AB-labeled GD3 ganglioside headgroup substrate that contains two sialic acid residues linked via an α2–8 bond. Undigested substrate ran at ∼16.5-min retention time with a very minor peak at ∼11-min retention time corresponding to partially degraded substrate comprised of a single α2–6 terminal sialic acid ( C , top panel ). NeuA-treated substrate shifted at ∼5.5 min retention time ( C , bottom panel ). Treatment with 1 unit of ORF12p did not shift the major substrate peak ( C , middle panel ). D and E , activity of ORF12p on biantennary complex N -glycans with terminal sialic acid residues (Neu5Ac, D ; or Neu5Gc, E ). Undigested substrates run at ∼26.6- and 27.9-min retention time, respectively ( D and E , top panels ). NeuA treatment shifted both substrate peaks at ∼23-min retention time ( D and E , bottom panels ). Incubation of the substrates with 1 or 10 units of ORF12p resulted in the same peak shift, but incomplete substrate desialylation was observed resulting in another smaller peak shift at ∼24.9- and 25.6-min retention time, respectively ( D and E , middle panels ). Symbolic representation of glycan structures was drawn following the guidelines of the Consortium for Functional Glycomics ( 50 ). EU , emission units.
    Figure Legend Snippet: Specificity of ORF12p on sialic acid containing substrates using UPLC–HILIC–FLR analysis. A and B , the ability of ORF12p to cleave the fluorescently labeled substrates 3′- or 6′-sialyl- N -acetyllactosamine-2AB. Undigested substrates 3′- or 6′-sialyllactosamine-2AB run at ∼10.6- or 12.3-min retention times, respectively ( A and B , top panels ). Control digestion with the NeuA sialidase shifted both substrate peaks to ∼5.5-min retention time ( A and B , bottom panels ). Digestion of these substrates with 1 unit of ORF12p–His resulted in the same peak shift ( A and B , middle panels ). C , ORF12p's ability to hydrolyze α2–8 Neu5Ac was assessed using a 2AB-labeled GD3 ganglioside headgroup substrate that contains two sialic acid residues linked via an α2–8 bond. Undigested substrate ran at ∼16.5-min retention time with a very minor peak at ∼11-min retention time corresponding to partially degraded substrate comprised of a single α2–6 terminal sialic acid ( C , top panel ). NeuA-treated substrate shifted at ∼5.5 min retention time ( C , bottom panel ). Treatment with 1 unit of ORF12p did not shift the major substrate peak ( C , middle panel ). D and E , activity of ORF12p on biantennary complex N -glycans with terminal sialic acid residues (Neu5Ac, D ; or Neu5Gc, E ). Undigested substrates run at ∼26.6- and 27.9-min retention time, respectively ( D and E , top panels ). NeuA treatment shifted both substrate peaks at ∼23-min retention time ( D and E , bottom panels ). Incubation of the substrates with 1 or 10 units of ORF12p resulted in the same peak shift, but incomplete substrate desialylation was observed resulting in another smaller peak shift at ∼24.9- and 25.6-min retention time, respectively ( D and E , middle panels ). Symbolic representation of glycan structures was drawn following the guidelines of the Consortium for Functional Glycomics ( 50 ). EU , emission units.

    Techniques Used: Hydrophilic Interaction Liquid Chromatography, Labeling, Activity Assay, Incubation, Functional Assay

    Identification of the sialidase-encoding ORF on fosmid G7 and its in vitro expression. A , a map of fosmid G7 transposon insertion sites ( red lines ) in mutants with abolished sialidase activity. B , SDS–PAGE of ORF9 and ORF12 proteins expressed in vitro using the PURExpress system. C , sialidase activity produced in PURExpress reaction mixtures was assessed using the substrate 4MU-α-Neu5Ac as described under “Experimental procedures.” D , the deduced amino acid sequence of ORF12p. The nucleotide sequence and the deduced protein sequence for ORF12 are annotated in the fosmid G7 sequence record (GenBank TM accession number MH016668 ).
    Figure Legend Snippet: Identification of the sialidase-encoding ORF on fosmid G7 and its in vitro expression. A , a map of fosmid G7 transposon insertion sites ( red lines ) in mutants with abolished sialidase activity. B , SDS–PAGE of ORF9 and ORF12 proteins expressed in vitro using the PURExpress system. C , sialidase activity produced in PURExpress reaction mixtures was assessed using the substrate 4MU-α-Neu5Ac as described under “Experimental procedures.” D , the deduced amino acid sequence of ORF12p. The nucleotide sequence and the deduced protein sequence for ORF12 are annotated in the fosmid G7 sequence record (GenBank TM accession number MH016668 ).

    Techniques Used: In Vitro, Expressing, Activity Assay, SDS Page, Produced, Sequencing

    9) Product Images from "Sialylation regulates myofibroblast differentiation of human skin fibroblasts"

    Article Title: Sialylation regulates myofibroblast differentiation of human skin fibroblasts

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-017-0534-1

    Defect in raft localization of CD44 during the process to senescence was restored by treatment with a sialidase inhibitor. a Cell surface glycans in early passage ( EP ), late passage ( LP ), and zanamivir (2 mM)-treated LP fibroblasts were analyzed by FACS using lectins. Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown. Results are presented as means ± SD from three independent experiments. b Total cell lysates from EP, LP, and zanamivir-treated LP fibroblasts were pulled down with ECA. Immunoblotting of CD44 was performed on ECA-binding proteins ( left ). In addition, immunoprecipitation ( IP ) of CD44, followed by immunoblotting of ECA, MAL-II, and CD44, was performed ( right ). Representative images are shown. c Immunocytochemical staining was performed in EP, LP, and zanamivir-treated LP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). d The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( c ), from two independent experiments (total of six fields); *** P
    Figure Legend Snippet: Defect in raft localization of CD44 during the process to senescence was restored by treatment with a sialidase inhibitor. a Cell surface glycans in early passage ( EP ), late passage ( LP ), and zanamivir (2 mM)-treated LP fibroblasts were analyzed by FACS using lectins. Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown. Results are presented as means ± SD from three independent experiments. b Total cell lysates from EP, LP, and zanamivir-treated LP fibroblasts were pulled down with ECA. Immunoblotting of CD44 was performed on ECA-binding proteins ( left ). In addition, immunoprecipitation ( IP ) of CD44, followed by immunoblotting of ECA, MAL-II, and CD44, was performed ( right ). Representative images are shown. c Immunocytochemical staining was performed in EP, LP, and zanamivir-treated LP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). d The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( c ), from two independent experiments (total of six fields); *** P

    Techniques Used: FACS, Binding Assay, Immunoprecipitation, Staining

    Age-dependent reduction of myofibroblast differentiation was restored by a sialidase inhibitor. a , b Western blot analysis of phosphorylated extracellular signal-related kinase ( pERK ) was performed in early passage ( EP ), late passage ( LP ), and zanamivir (2 mM)-treated LP fibroblasts. The histogram ( b ) shows mean densitometric readings ± SD for the phosphorylated proteins normalized to the loading controls. Values were obtained from three independent experiments. *P
    Figure Legend Snippet: Age-dependent reduction of myofibroblast differentiation was restored by a sialidase inhibitor. a , b Western blot analysis of phosphorylated extracellular signal-related kinase ( pERK ) was performed in early passage ( EP ), late passage ( LP ), and zanamivir (2 mM)-treated LP fibroblasts. The histogram ( b ) shows mean densitometric readings ± SD for the phosphorylated proteins normalized to the loading controls. Values were obtained from three independent experiments. *P

    Techniques Used: Western Blot

    Myofibroblast differentiation was inhibited by sialidase. a , b Cell surface glycans in control ( Ctr ; non-treated EP fibroblasts) and 100 U/ml sialidase-treated EP fibroblasts were analyzed by FACS using lectins. Three independent experiments were performed and representative results are shown ( a ). Controls are presented in gray . Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown ( b ). Results are presented as means ± SD from three independent experiments. c , d Western blot analysis of α-smooth muscle actin (α -SMA ) was performed 3 days after myofibroblast differentiation in control and sialidase-treated EP fibroblasts. The histogram ( d ) shows the mean densitometric analysis ± SD of α-SMA normalized to the loading control (β-actin). The values were obtained from three independent experiments. e Immunocytochemical staining was performed in control and sialidase-treated EP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). f The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( e ), from two independent experiments (total of six fields); *** P
    Figure Legend Snippet: Myofibroblast differentiation was inhibited by sialidase. a , b Cell surface glycans in control ( Ctr ; non-treated EP fibroblasts) and 100 U/ml sialidase-treated EP fibroblasts were analyzed by FACS using lectins. Three independent experiments were performed and representative results are shown ( a ). Controls are presented in gray . Mean fluorescent intensities ( MFIs ) relative to those of control cells are shown ( b ). Results are presented as means ± SD from three independent experiments. c , d Western blot analysis of α-smooth muscle actin (α -SMA ) was performed 3 days after myofibroblast differentiation in control and sialidase-treated EP fibroblasts. The histogram ( d ) shows the mean densitometric analysis ± SD of α-SMA normalized to the loading control (β-actin). The values were obtained from three independent experiments. e Immunocytochemical staining was performed in control and sialidase-treated EP fibroblasts after transforming growth factor ( TGF )-β1 treatment. Representative images are shown (GM1, red ; CD44, green ; DAPI, blue ; colocalization, yellow ). f The histogram shows the mean ± SD percentage of GM1-CD44 colocalized cells colored yellow , as shown in ( e ), from two independent experiments (total of six fields); *** P

    Techniques Used: FACS, Western Blot, Staining

    10) Product Images from "B-cell maturation antigen is modified by a single N-glycan chain that modulates ligand binding and surface retention"

    Article Title: B-cell maturation antigen is modified by a single N-glycan chain that modulates ligand binding and surface retention

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1309417110

    Sialylation influences surface level of BCMA. ( A ) Histograms show that removal of sialic acid results in the increase of surface BCMA. H929 or RPMI8226 cells treated with or without sialidase were subjected to FACS analysis with APC-conjugated anti-BCMA antibody. ( B ) Removal of sialic acid results in the accumulation of preexisting BCMA on the cell surface. H929 or RPMI8226 cells were treated with CHX in the absence or presence of sialidase, followed by FACS analysis with APC-conjugated anti-BCMA antibody. ( Right ) Relative level of BCMA on H929 and RPMI8226 cell surface following treatment with sialidase ( C ) Removal of N -glycans by PNGase F abolishes the effect of sialidase on surface BCMA. CHX-treated H929 or RPMI8226 cells were treated with or without PNGase F in the presence of sialidase. The level of preexisting BCMA on the cell surface was detected by APC-conjugated anti-BCMA antibody by FACS analysis. ( D ) The level of N42A BCMA expressed on RPMI8226 cells did not change after sialidase treatment. RPMI8226 cells transfected with mock control and N42A-BCMA expression vector were treated with sialidase in the presence of CHX, followed by FACS analysis with APC-conjugated anti-BCMA antibody. ( A – D ) Each histogram is based on data from three independent experiments; the number in the histogram indicates the mean of fluorescence. ( Right ) Statistical analysis of three independent experiments. Results are mean ± SEM. * P
    Figure Legend Snippet: Sialylation influences surface level of BCMA. ( A ) Histograms show that removal of sialic acid results in the increase of surface BCMA. H929 or RPMI8226 cells treated with or without sialidase were subjected to FACS analysis with APC-conjugated anti-BCMA antibody. ( B ) Removal of sialic acid results in the accumulation of preexisting BCMA on the cell surface. H929 or RPMI8226 cells were treated with CHX in the absence or presence of sialidase, followed by FACS analysis with APC-conjugated anti-BCMA antibody. ( Right ) Relative level of BCMA on H929 and RPMI8226 cell surface following treatment with sialidase ( C ) Removal of N -glycans by PNGase F abolishes the effect of sialidase on surface BCMA. CHX-treated H929 or RPMI8226 cells were treated with or without PNGase F in the presence of sialidase. The level of preexisting BCMA on the cell surface was detected by APC-conjugated anti-BCMA antibody by FACS analysis. ( D ) The level of N42A BCMA expressed on RPMI8226 cells did not change after sialidase treatment. RPMI8226 cells transfected with mock control and N42A-BCMA expression vector were treated with sialidase in the presence of CHX, followed by FACS analysis with APC-conjugated anti-BCMA antibody. ( A – D ) Each histogram is based on data from three independent experiments; the number in the histogram indicates the mean of fluorescence. ( Right ) Statistical analysis of three independent experiments. Results are mean ± SEM. * P

    Techniques Used: FACS, Transfection, Expressing, Plasmid Preparation, Fluorescence

    Sialylation affects BCMA ligand-mediated protection from apoptosis induced by DEX. RPMI8226 cells pretreated with or without sialidase were treated with DEX in the absence or presence of APRIL or BAFF. Three days later, cells were subjected to annexin V staining by FACS analysis. ( A ) One representative result of three independent experiments is shown; the number in the dot plot indicates the percentage of annexin V–positive cells. ( B ) The mean value ± SEM of three independent experiments from A . ( C ) Percentage of rescue of apoptosis as determined by (% of apoptosis caused by DEX–% of apoptosis induced by DEX in the presence of BCMA ligand)/(% of apoptosis induced by DEX). ( D and E ) Histograms of FACS analysis show that removal of sialylation increases the binding of ligands for BCMA with cell surface. H929 or RPMI8226 cells were pretreated with sialidase and then incubated with Fc-APRIL ( D ) or Fc-BAFF ( E ), followed by detection with FACS analysis. The number in the histogram indicates the mean of fluorescence. Bar graphs below D and E show statistical analysis of ligand binding after treatment of cells with sialidase in three independent experiments. Results are mean ± SEM. * P
    Figure Legend Snippet: Sialylation affects BCMA ligand-mediated protection from apoptosis induced by DEX. RPMI8226 cells pretreated with or without sialidase were treated with DEX in the absence or presence of APRIL or BAFF. Three days later, cells were subjected to annexin V staining by FACS analysis. ( A ) One representative result of three independent experiments is shown; the number in the dot plot indicates the percentage of annexin V–positive cells. ( B ) The mean value ± SEM of three independent experiments from A . ( C ) Percentage of rescue of apoptosis as determined by (% of apoptosis caused by DEX–% of apoptosis induced by DEX in the presence of BCMA ligand)/(% of apoptosis induced by DEX). ( D and E ) Histograms of FACS analysis show that removal of sialylation increases the binding of ligands for BCMA with cell surface. H929 or RPMI8226 cells were pretreated with sialidase and then incubated with Fc-APRIL ( D ) or Fc-BAFF ( E ), followed by detection with FACS analysis. The number in the histogram indicates the mean of fluorescence. Bar graphs below D and E show statistical analysis of ligand binding after treatment of cells with sialidase in three independent experiments. Results are mean ± SEM. * P

    Techniques Used: Staining, FACS, Binding Assay, Incubation, Fluorescence, Ligand Binding Assay

    Glycans on BCMA are terminally modified by sialic acid. ( A and B ) FACS shows the binding with SNA ( A ) or MAL ( B ) on H929 and RPMI8226 cells pretreated with or without sialidase. ( C and D ) ELISA shows the binding of lectins with BCMA. Full-length BCMA purified from transfectants was pretreated with or without sialidase and added to anti-FLAG antibody coated plates, followed by the biotinylated SNA ( C ) or MAL ( D ). ( E and F ) ELISA shows the binding of BCMA to lectins. Biotinylated SNA ( E ) or MAL ( F ) was bound to streptavidin-coated plates before mixing with sialidase-treated or untreated full-length BCMA. Results in A and B are representative of three or four independent experiments, and the number in the histogram indicates the mean of fluorescence. Results in C − F represent mean ± SEM of three or four independent experiments. * P
    Figure Legend Snippet: Glycans on BCMA are terminally modified by sialic acid. ( A and B ) FACS shows the binding with SNA ( A ) or MAL ( B ) on H929 and RPMI8226 cells pretreated with or without sialidase. ( C and D ) ELISA shows the binding of lectins with BCMA. Full-length BCMA purified from transfectants was pretreated with or without sialidase and added to anti-FLAG antibody coated plates, followed by the biotinylated SNA ( C ) or MAL ( D ). ( E and F ) ELISA shows the binding of BCMA to lectins. Biotinylated SNA ( E ) or MAL ( F ) was bound to streptavidin-coated plates before mixing with sialidase-treated or untreated full-length BCMA. Results in A and B are representative of three or four independent experiments, and the number in the histogram indicates the mean of fluorescence. Results in C − F represent mean ± SEM of three or four independent experiments. * P

    Techniques Used: Modification, FACS, Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Fluorescence

    11) Product Images from "Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex"

    Article Title: Determination of major sialylated N-glycans and identification of branched sialylated N-glycans that dynamically change their content during development in the mouse cerebral cortex

    Journal: Glycoconjugate Journal

    doi: 10.1007/s10719-014-9566-2

    Isolation of di-sialylated A2G’2F a separation by Mono Q HPLC of N-glycans from 12w mouse brains. N, S1-S4 indicate the elution positions of neutral, monosialo, disialo, trisialo and tetrasialo PA-N-glycans, respectively. ( a ) N-glycans derived from 12w cerebral cortex were applied again to Mono Q HPLC and the S2 fraction ( indicated by oblique lines ) was collected. ( b ) After sialylated N-glycans from the S2 fraction in Fig. 5a- a were treated with α2,3-sialidase, the sample was applied again to Mono Q HPLC. The S2 fraction ( indicated by oblique lines ) was collected b the α2,3-sialidase-resistant S2 fraction in Fig. 5a- b was applied to an ODS column. There were some major peaks, and the peak 1 was identified as sialylated A2G’2F. The fraction indicated by oblique lines was collected. c N-glycans from the peak 1 in Fig. 5b were treated with β1,3-galactosidase and applied again to an ODS column. The peak 2 was collected for further analysis. Results are representative of more than three independent experiments
    Figure Legend Snippet: Isolation of di-sialylated A2G’2F a separation by Mono Q HPLC of N-glycans from 12w mouse brains. N, S1-S4 indicate the elution positions of neutral, monosialo, disialo, trisialo and tetrasialo PA-N-glycans, respectively. ( a ) N-glycans derived from 12w cerebral cortex were applied again to Mono Q HPLC and the S2 fraction ( indicated by oblique lines ) was collected. ( b ) After sialylated N-glycans from the S2 fraction in Fig. 5a- a were treated with α2,3-sialidase, the sample was applied again to Mono Q HPLC. The S2 fraction ( indicated by oblique lines ) was collected b the α2,3-sialidase-resistant S2 fraction in Fig. 5a- b was applied to an ODS column. There were some major peaks, and the peak 1 was identified as sialylated A2G’2F. The fraction indicated by oblique lines was collected. c N-glycans from the peak 1 in Fig. 5b were treated with β1,3-galactosidase and applied again to an ODS column. The peak 2 was collected for further analysis. Results are representative of more than three independent experiments

    Techniques Used: Isolation, High Performance Liquid Chromatography, Derivative Assay

    Schematic drawing of the HPLC chart. The black line indicates the chromatogram of neutral and desialylated PA-oligosaccharides and the gray line indicates that of neutral sugar chains. From the composite images of two chromatograms, sialylated sugar chains can be calculated as the difference in the peak areas (C α 2,3/6/8 value). Dotted line shows a chromatogram obtained after α2,3-sialidase treatment of the sample. α2,3-sialidase sensitive portion (C α2,3 value) can be obtained from the difference in the peak areas under the dotted and gray lines
    Figure Legend Snippet: Schematic drawing of the HPLC chart. The black line indicates the chromatogram of neutral and desialylated PA-oligosaccharides and the gray line indicates that of neutral sugar chains. From the composite images of two chromatograms, sialylated sugar chains can be calculated as the difference in the peak areas (C α 2,3/6/8 value). Dotted line shows a chromatogram obtained after α2,3-sialidase treatment of the sample. α2,3-sialidase sensitive portion (C α2,3 value) can be obtained from the difference in the peak areas under the dotted and gray lines

    Techniques Used: High Performance Liquid Chromatography

    12) Product Images from "Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus"

    Article Title: Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus

    Journal: Nature Communications

    doi: 10.1038/s41467-017-02109-8

    Rg CBM40 binding to mucus-producing cells and intestinal tissue sections. a Immunostaining pattern for Rg CBM40 on LS174T cells correlated with mucin (MUC2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). b Immunostaining pattern for Rg CBM40 on cryosections of mouse colon correlated with mucin (Muc2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). Cell nuclei were counterstained with DAPI, shown in blue. c Sialidase pre-treatment of mouse colonic cryosections markedly reduced the binding of Rg CBM40 and SNA lectin. Cell nuclei were counterstained with DAPI, shown in blue. d Rg CBM40 competition assay with SNA on cryosections of mouse colon. Rg CBM40 is shown in green. Cell nuclei were counterstained with DAPI, shown in blue. No Rg CBM40 specific staining was detectable when SNA was present. e R. gnavus binding competition assay with SNA on cryosections of mouse colon. R. gnavus ATCC 29149 was incubated on sequential cryosections of mouse colon with or without SNA treatment and is shown in red. The mucus layer is shown in green. Sequential sections were required as both antibodies were raised in the same species. Cell nuclei were counterstained with DAPI, shown in blue. No R.gnavus staining was detectable when SNA was present. Appropriate primary antibody and secondary antibody only controls are also shown underneath each panel, showing some background staining. Scale bar: 20 μm
    Figure Legend Snippet: Rg CBM40 binding to mucus-producing cells and intestinal tissue sections. a Immunostaining pattern for Rg CBM40 on LS174T cells correlated with mucin (MUC2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). b Immunostaining pattern for Rg CBM40 on cryosections of mouse colon correlated with mucin (Muc2) and lectin (SNA) staining, all shown in green. No staining was observed in Rg CBM40-free sample (Blank). Cell nuclei were counterstained with DAPI, shown in blue. c Sialidase pre-treatment of mouse colonic cryosections markedly reduced the binding of Rg CBM40 and SNA lectin. Cell nuclei were counterstained with DAPI, shown in blue. d Rg CBM40 competition assay with SNA on cryosections of mouse colon. Rg CBM40 is shown in green. Cell nuclei were counterstained with DAPI, shown in blue. No Rg CBM40 specific staining was detectable when SNA was present. e R. gnavus binding competition assay with SNA on cryosections of mouse colon. R. gnavus ATCC 29149 was incubated on sequential cryosections of mouse colon with or without SNA treatment and is shown in red. The mucus layer is shown in green. Sequential sections were required as both antibodies were raised in the same species. Cell nuclei were counterstained with DAPI, shown in blue. No R.gnavus staining was detectable when SNA was present. Appropriate primary antibody and secondary antibody only controls are also shown underneath each panel, showing some background staining. Scale bar: 20 μm

    Techniques Used: Binding Assay, Immunostaining, Staining, Competitive Binding Assay, Incubation

    ELISA of Rg CBM40 binding to purified mucins. a Rg CBM40 binding to a range of purified mucins; mucin 2 (MUC2) and mixed mucins (mucins) from human cell line LS174T, purified pig gastric mucin (pPGM), and murine mucins from germ free (GF), wild type (WT), and C3GnT −/− mice. b Correlation of Rg CBM40 binding with % sialylated structure for each mucin tested. The % sialylated structures was determined by MS. c Rg CBM40 binding to LS174T MUC2 which has been treated chemically (TFA) or enzymatically with a sialidase from Clostridium perfringens ( Cp ), Salmonella typhimurium ( St ), Akkermansia muciniphila ( Am ) or Ruminococcus gnavus ( Rg ) d Rg CBM40 binding to LS174T MUC2 in competition with sugars. Rg CBM40 has been preincubated with the indicated sugars. In all cases, Rg CBM40 was incubated with immobilised mucins and binding detected using an anti-sialidase primary antibody and an anti-rabbit secondary antibody conjugated to horseradish peroxidase. The enzyme was incubated with TMB and the absorbance at 450 nm (A450) measured. The error bars show the standard error of the mean (SEM) of three replicates. P values are indicated; NS-not significant, * p
    Figure Legend Snippet: ELISA of Rg CBM40 binding to purified mucins. a Rg CBM40 binding to a range of purified mucins; mucin 2 (MUC2) and mixed mucins (mucins) from human cell line LS174T, purified pig gastric mucin (pPGM), and murine mucins from germ free (GF), wild type (WT), and C3GnT −/− mice. b Correlation of Rg CBM40 binding with % sialylated structure for each mucin tested. The % sialylated structures was determined by MS. c Rg CBM40 binding to LS174T MUC2 which has been treated chemically (TFA) or enzymatically with a sialidase from Clostridium perfringens ( Cp ), Salmonella typhimurium ( St ), Akkermansia muciniphila ( Am ) or Ruminococcus gnavus ( Rg ) d Rg CBM40 binding to LS174T MUC2 in competition with sugars. Rg CBM40 has been preincubated with the indicated sugars. In all cases, Rg CBM40 was incubated with immobilised mucins and binding detected using an anti-sialidase primary antibody and an anti-rabbit secondary antibody conjugated to horseradish peroxidase. The enzyme was incubated with TMB and the absorbance at 450 nm (A450) measured. The error bars show the standard error of the mean (SEM) of three replicates. P values are indicated; NS-not significant, * p

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Mouse Assay, Mass Spectrometry, Incubation

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    Article Title: Influenza binds phosphorylated glycans from human lung
    Article Snippet: For Alexa Fluor 488 fluorescence, 495 nm (ex) and 519 nm (em) were used. .. For enzymatic treatment of the microarrays, each array was rehydrated for 5 min in TSM buffer and then incubated with Neuraminidase A (New England BioLabs) diluted 1:6 in 1× GlycoBuffer for 18 hours rocking at room temperature. .. Following incubation, the array was washed four times each with TSMW (20 mM tris-HCl, 150 mM NaCl, 0.2 mM CaCl2 , 0.2 mM MgCl2 , and 0.05% Tween-20), TSM, and water.

    Article Title: Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells
    Article Snippet: Fluorescence intensity was measured with a FACSCalibur flow cytometer (BD Biosciences), and infected cells were quantified with FLOWJO 8.5.3 (Treestar, Ashland, OR). .. Sialidase Treatment of Live SRECs Prior to Virus Infection As performed previously , confluent SRECs in 24-well plates were incubated for 3 h at 37 °C in BEGM® plus 150 units (as defined by the manufacturer) per well of either α2–3 specific sialidase from Salmonella typhimurium LT2 (New England Biolabs, Beverly, MA), or dual α2–3/6 sialidase from Clostridium perfringens (New England Biolabs). .. Following enzymatic treatment, cells were washed twice with BEGM® and infected as described above.

    Article Title: A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells
    Article Snippet: Sections were observed under a BX41 microscope equipped with a DP-70 digital camera system (Olympus, Tokyo, Japan) or imaged with a NanoZoomer slide-scanner (Hamamatsu, Hamamatsu City). .. For treatment with glycosidases, sections were incubated (after deparaffination and hydrogen peroxide blocking) with 50 U of sialidase (New England Biolabs) or 25 U of ß-D-N-acetyl-hexosaminidase for 2 hours at 37°C. .. Fresh enzymes were then added and the slides were further incubated overnight at 37°C.

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region
    Article Snippet: For alkylation, 8 µL of 300 m M iodoacetic acid (Sigma, MO) was added to the denatured and reduced sample and incubated at room temperature for 15 min in the dark. .. For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA). .. UPLC separation was performed using a Waters Acquity BEH 200SEC column (4.6 mm × 150 mm, 1.7 µm particle size) using buffers of 0.1% TFA, 0.25% formic acid, 15% acetonitrile, and 85% water at a flow rate of 0.4 mL/min.

    Infection:

    Article Title: Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells
    Article Snippet: Fluorescence intensity was measured with a FACSCalibur flow cytometer (BD Biosciences), and infected cells were quantified with FLOWJO 8.5.3 (Treestar, Ashland, OR). .. Sialidase Treatment of Live SRECs Prior to Virus Infection As performed previously , confluent SRECs in 24-well plates were incubated for 3 h at 37 °C in BEGM® plus 150 units (as defined by the manufacturer) per well of either α2–3 specific sialidase from Salmonella typhimurium LT2 (New England Biolabs, Beverly, MA), or dual α2–3/6 sialidase from Clostridium perfringens (New England Biolabs). .. Following enzymatic treatment, cells were washed twice with BEGM® and infected as described above.

    Blocking Assay:

    Article Title: A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells
    Article Snippet: Sections were observed under a BX41 microscope equipped with a DP-70 digital camera system (Olympus, Tokyo, Japan) or imaged with a NanoZoomer slide-scanner (Hamamatsu, Hamamatsu City). .. For treatment with glycosidases, sections were incubated (after deparaffination and hydrogen peroxide blocking) with 50 U of sialidase (New England Biolabs) or 25 U of ß-D-N-acetyl-hexosaminidase for 2 hours at 37°C. .. Fresh enzymes were then added and the slides were further incubated overnight at 37°C.

    Liquid Chromatography with Mass Spectroscopy:

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region
    Article Snippet: For alkylation, 8 µL of 300 m M iodoacetic acid (Sigma, MO) was added to the denatured and reduced sample and incubated at room temperature for 15 min in the dark. .. For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA). .. UPLC separation was performed using a Waters Acquity BEH 200SEC column (4.6 mm × 150 mm, 1.7 µm particle size) using buffers of 0.1% TFA, 0.25% formic acid, 15% acetonitrile, and 85% water at a flow rate of 0.4 mL/min.

    other:

    Article Title: Unravelling the specificity and mechanism of sialic acid recognition by the gut symbiont Ruminococcus gnavus
    Article Snippet: Sialidase from Clostridium perfringens and Salmonella typhimurium LT2 were from New England Biolabs (Ipswich, MA, USA).

    Activity Assay:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: The amplicons were labeled with digoxygenin (DIG Hi prime, Roche Applied Sciences) according to the manufacturer’s instructions. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel. ..

    Agarose Gel Electrophoresis:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: The amplicons were labeled with digoxygenin (DIG Hi prime, Roche Applied Sciences) according to the manufacturer’s instructions. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel. ..

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    New England Biolabs sialidase activity
    Composition of substitution mutations with respect to consensus sequences. Percentages represent the mean across eight M. synoviae strains for nanI , nagA , nanA , nagC , nanE , and nagB genes of the <t>sialidase</t> locus, and three M. hyopneumoniae strains for housekeeping genes dnaA and ftsY .
    Sialidase Activity, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Composition of substitution mutations with respect to consensus sequences. Percentages represent the mean across eight M. synoviae strains for nanI , nagA , nanA , nagC , nanE , and nagB genes of the sialidase locus, and three M. hyopneumoniae strains for housekeeping genes dnaA and ftsY .

    Journal: Microbial pathogenesis

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae

    doi: 10.1016/j.micpath.2008.02.002

    Figure Lengend Snippet: Composition of substitution mutations with respect to consensus sequences. Percentages represent the mean across eight M. synoviae strains for nanI , nagA , nanA , nagC , nanE , and nagB genes of the sialidase locus, and three M. hyopneumoniae strains for housekeeping genes dnaA and ftsY .

    Article Snippet: Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    Techniques:

    PVL binding of tumor cell lines. A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml -1 for 30 mn; red line: rPVL-alexa 488 5 μg ml -1 in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml -1 after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml -1 rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.

    Journal: PLoS ONE

    Article Title: A Recombinant Fungal Lectin for Labeling Truncated Glycans on Human Cancer Cells

    doi: 10.1371/journal.pone.0128190

    Figure Lengend Snippet: PVL binding of tumor cell lines. A. Flow cytometry histograms show rPVL-Alexa 488 binding to a lung immortalized cell line (HBEC-3KT), two lung tumor cell lines (H358 and A549) as well as on a breast tumor cell line (MCF-7). The x axis indicates fluorescence intensity. The y axis indicates cell number. Black line: untreated control cells; blue line rPVL-Alexa 488 5 μg ml -1 for 30 mn; red line: rPVL-alexa 488 5 μg ml -1 in the presence of GlcNAc 100 mM; green line: rPVL-Alexa 488 5 μg ml -1 after sialidase pretreatment. B. Microscopy images of A549 NSCLC cells treated for 30 min at 37°C with 5 μg ml -1 rPVL labeled with Alexa 488 in the presence or absence of 100 mM GlcNAc. Green channel shows rPVL-Alexa 488, blue channel shows nuclei labeled with DAPI staining.

    Article Snippet: For treatment with glycosidases, sections were incubated (after deparaffination and hydrogen peroxide blocking) with 50 U of sialidase (New England Biolabs) or 25 U of ß-D-N-acetyl-hexosaminidase for 2 hours at 37°C.

    Techniques: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Microscopy, Labeling, Staining

    LC-MS/MS analysis of Peptide C and the equivalent peptides after sialidase and O -glycanase treatment. These peptides were derived from both trypsin and endoprotinase Lys C digestion of the reduced/alkylated huLCAT-Fc control and two other linker mutants.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region

    doi: 10.1002/pro.2373

    Figure Lengend Snippet: LC-MS/MS analysis of Peptide C and the equivalent peptides after sialidase and O -glycanase treatment. These peptides were derived from both trypsin and endoprotinase Lys C digestion of the reduced/alkylated huLCAT-Fc control and two other linker mutants.

    Article Snippet: For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Derivative Assay

    MS/MS spectrum of a doubly charged ion, m / z = 1127.53, for Peptide C (peptide sequence: QGPPASPT 407 AS 409 PEPPPPEGS 418 GGGGDK) treated with sialidase and O -glycanase followed by alkaline β-elimination. Y-series ions including [y7], [y10], [y13],

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region

    doi: 10.1002/pro.2373

    Figure Lengend Snippet: MS/MS spectrum of a doubly charged ion, m / z = 1127.53, for Peptide C (peptide sequence: QGPPASPT 407 AS 409 PEPPPPEGS 418 GGGGDK) treated with sialidase and O -glycanase followed by alkaline β-elimination. Y-series ions including [y7], [y10], [y13],

    Article Snippet: For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA).

    Techniques: Mass Spectrometry, Sequencing

    MS/MS ion chromatograms of Peptide C after deglycosylation with both sialidase and O -glycanase. Spectra were selected and analyzed for three doubly charged ions. Panel A: m / z = 1136.53, representing parent peptide with no modification; Panel B: m / z =

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region

    doi: 10.1002/pro.2373

    Figure Lengend Snippet: MS/MS ion chromatograms of Peptide C after deglycosylation with both sialidase and O -glycanase. Spectra were selected and analyzed for three doubly charged ions. Panel A: m / z = 1136.53, representing parent peptide with no modification; Panel B: m / z =

    Article Snippet: For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA).

    Techniques: Mass Spectrometry, Modification

    LC-MS analysis of deglycosylated Peptide C obtained from endoproteinase Lys C digestion of Peptide A. Panel A: Peptide C without treatment; Panel B: Peptide C treated with sialidase; and Panel C: Peptide C treated with sialidase and O -glycanase. These

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region

    doi: 10.1002/pro.2373

    Figure Lengend Snippet: LC-MS analysis of deglycosylated Peptide C obtained from endoproteinase Lys C digestion of Peptide A. Panel A: Peptide C without treatment; Panel B: Peptide C treated with sialidase; and Panel C: Peptide C treated with sialidase and O -glycanase. These

    Article Snippet: For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA).

    Techniques: Liquid Chromatography with Mass Spectroscopy

    ESI-TOF MS analysis of reduced, alkylated, and deglycosylated huLCAT-Fc. Panel A: Sample treated with sialidase and PNGase F. Panel B: Sample treated with sialidase, PNGase F, and O -glycanase.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Recombinant human lecithin-cholesterol acyltransferase Fc fusion: Analysis of N- and O-linked glycans and identification and elimination of a xylose-based O-linked tetrasaccharide core in the linker region

    doi: 10.1002/pro.2373

    Figure Lengend Snippet: ESI-TOF MS analysis of reduced, alkylated, and deglycosylated huLCAT-Fc. Panel A: Sample treated with sialidase and PNGase F. Panel B: Sample treated with sialidase, PNGase F, and O -glycanase.

    Article Snippet: For PNGase F and sialidase digestion, reduced and alkylated huLCAT-Fc was neutralized to pH 7.5, and then 1 µL of PNGase F (New England Biolab, MA) was added, and the sample was incubated at 37°C for 2 h. Then, 2 µL of sialidase (ProZyme, CA) was added and incubated at 37°C for 30 min. For O -glycanase, sialidase, and PNGase F digestion, 4 µL O -glycanase (ProZyme) and 2 µL sialidase were added to 50 µg of reduced and alkylated huLCAT-Fc followed by incubation at 37°C for 3 h, which was then followed by the addition of 2 µL PNGase F and incubation at 37°C for 1 h. Intact masses of various deglycosylated huLCAT-Fc preparations were determined by LC-MS analysis using an Agilent Technologies 6210 ESI time-of-flight mass analyzer in conjunction with a Waters UPLC system (Bedford, MA).

    Techniques: Mass Spectrometry

    Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Altered O -glycosylation on CSRP3 in the LV of DS hypertensive rats. (A) ACA lectin blot analysis and SYPRO Ruby staining of fractions from sialidase-treated LV extracts. Arrow indicates the ACA-positive band, which is observed strongly in fraction 3 of HS ( H ) but weakly in that of the LS ( L ) group. (B) Two-dimensional PAGE images of sialidase-treated LV fraction 3. Proteins transferred to membranes were subjected to SYPRO Ruby staining, and then to ACA lectin blotting. Insets show magnified images of two spots used for protein identification. (C) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of recombinant human CSRP3. Recombinant proteins expressed in E . coli (unglycosylated negative control) and in HEK293 cells (potentially glycosylated reference) were analyzed after treatment with sialidase and O -glycosidase. (D) Relative expression levels of Csrp3 in the LV tissues. qPCR data were normalized to Tbp expression levels. The numbers of examined rats were n = 12 and n = 15 for HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (E) Protein levels of CSRP3 in LV extracts. Densitometry analysis data of western blotting are shown (n = 6). (D,E) The data are presented as the fold change compared with LS rats at 12 weeks. (F) Western blot ( WB ) and ACA lectin blot ( LB ) analyses of CSRP3 from LV extracts of DS rats. CSRP3 in LV extracts was immunoprecipitated, denatured, separated by SDS-PAGE, and analyzed. Recombinant human CSRP3 was used as an experimental control of immunoprecipitation with anti-CSRP3 antibody (+) or normal IgG (-). Lower panel shows densitometry analysis data; the intensity of each band in LB was normalized to that in WB (n = 6). (G) Effects of glycosidases on CSRP3 dimerization. LV extracts from three HS ( H ) or LS ( L ) rats at 16 weeks were treated with three glycosidases as indicated and then analyzed by western blotting for CSRP3. Arrows indicate the bands corresponding to monomers and dimers. Lower panels show densitometry analysis from five experiments; dimer/monomer ratios are presented as the fold change compared with LS rats without glycosidase treatment. (D-G) *, p

    Article Snippet: Protein deglycosylation LV extracts prepared for western blot and lectin blot analyses and recombinant proteins were treated with glycosidases, including sialidase, O -glycosidase, and PNGase F, which were all obtained from New England Biolabs (Beverly, MA).

    Techniques: Staining, Polyacrylamide Gel Electrophoresis, Western Blot, Recombinant, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page

    Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p

    Journal: PLoS ONE

    Article Title: Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure

    doi: 10.1371/journal.pone.0150210

    Figure Lengend Snippet: Altered mucin-type O -glycosylation in the LV of DS hypertensive rats. (A) T-synthase activity in LV extracts. Data were normalized to protein content. (B) Correlation of T-synthase activity with ANP gene expression. ANP gene expression level was quantified by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (C) Correlation of T-synthase activity with ejection fraction. (D) Relative expression levels of glycogenes involved in the early stage of mucin-type O -glycosylation in the LV tissues of DS rats were analyzed by qPCR and normalized to that of Tbp . Data are presented as the fold change compared with LS rats at 12 weeks. (E) Schematic summary of gene expression analysis data shown in (D). Examined glycosyltransferases in the mucin-type O -glycosylation pathway are shown in red (upregulated), blue (downregulated), or black (no change) letters. Relatively rare core structures (core 5, 6, 7, and 8) synthesized from Tn are omitted. The biosynthetic pathway of disialyl-T is upregulated, as indicated with bold arrows. GalNAc, N -acetylgalactosamine; GlcNAc, N -acetylglucosamine; Gal, galactose; NeuAc, N -acetylneuraminic acid. (F) Lectin blot analysis of sialidase-treated LV extracts using ACA. Representative images demonstrate ACA-reactive glycoproteins and SYPRO Ruby-stained total proteins of three individual rats in each group. Lower panel shows densitometry analysis; intensity of each band was normalized to total protein amount. Data are presented as the fold change (n = 6) compared with sialidase-untreated LV extracts of HS rats at 12 weeks. (A,D) The numbers of examined rats were n = 12 and n = 15 for the HS groups at 12 and 16 weeks, respectively; n = 6 for LS groups at each period. (A,D,F) *, p

    Article Snippet: Protein deglycosylation LV extracts prepared for western blot and lectin blot analyses and recombinant proteins were treated with glycosidases, including sialidase, O -glycosidase, and PNGase F, which were all obtained from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Aqueous Normal-phase Chromatography, Expressing, Real-time Polymerase Chain Reaction, Synthesized, Staining