sialidase  (New England Biolabs)


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    Name:
    O Glycosidase Neuraminidase Bundle
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    Catalog Number:
    E0540
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    Applications:
    Proteomics & Glycomics
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    Structured Review

    New England Biolabs sialidase
    O Glycosidase Neuraminidase Bundle

    https://www.bioz.com/result/sialidase/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    sialidase - by Bioz Stars, 2021-07
    86/100 stars

    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 "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

    4) 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

    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 "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 ). 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 ). EU , emission units.

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

    7) Product Images from "Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration"

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

    Journal: eLife

    doi: 10.7554/eLife.61552

    Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

    Sialidase treatment studies. ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in Figure 2F (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in Figure 2G (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.
    Figure Legend Snippet: Sialidase treatment studies. ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in Figure 2F (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in Figure 2G (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

    Techniques Used: Incubation, Cytometry, Binding Assay, Fluorescence, Mutagenesis, Expressing

    Lectin binding to wild-type and glycogene-KO 293 T cells. A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.
    Figure Legend Snippet: Lectin binding to wild-type and glycogene-KO 293 T cells. A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.

    Techniques Used: Binding Assay, Plasmid Preparation, Flow Cytometry

    8) 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

    9) 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

    10) 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

    11) 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

    N -glycosylation of Etanercept TNFR and Fc domains. a Deconvoluted spectrum of dimeric TNFR digested with sialidase and O ). The most probable glycan structures lacking sialic acids are annotated. b ). The most probable N -glycoforms and C-terminal lysine variants are annotated. Asterisks indicate Na +
    Figure Legend Snippet: N -glycosylation of Etanercept TNFR and Fc domains. a Deconvoluted spectrum of dimeric TNFR digested with sialidase and O ). The most probable glycan structures lacking sialic acids are annotated. b ). The most probable N -glycoforms and C-terminal lysine variants are annotated. Asterisks indicate Na +

    Techniques Used:

    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

    12) 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

    13) Product Images from "Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration"

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

    14) 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 ). 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 ). EU , emission units.

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

    15) Product Images from "Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration"

    Article Title: Inhibition of SARS-CoV-2 viral entry upon blocking N- and O-glycan elaboration

    Journal: eLife

    doi: 10.7554/eLife.61552

    Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model. ( A ) ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293 T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. ( B ) SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral entry ( B ) assay are listed. ( C ) Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

    Sialidase treatment studies. ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in Figure 2F (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in Figure 2G (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.
    Figure Legend Snippet: Sialidase treatment studies. ( A ) Sialidase protocol validation. All lectins were directly conjugated with Alexa dyes. They were incubated with cells at 1–5 µg/mL for 15 min before a quick wash and cytometry measurement. Compared to untreated control (left), sialidase treatment (right) decreased SNA lectin binding to α2,6 sialylated structures by 15-fold and increased ECL binding to desialylated lactosamine chains (Galβ1,4GlcNAcβ) by an order of magnitude. ( B ) Pseudovirus assay. DsRed fluorescence in HEK293T and stable 293T/ACE2 cells upon addition of VSVG, Spike-WT and Spike-mutant pseudotyped virus. ( C ) Sialidase treatment of pseudovirus. % DsRed positive cell data are shown for study in Figure 2F (main manuscript). Viral entry was sialidase independent. ( D ) Sialidase treatment of HEK/ACE2 cells. Pseudovirus expressing VSVG, Spike-WT and Spike-mutant were added to cells under conditions described in Figure 2G (main manuscript). All error bars are standard deviations. Data are representative of 3 independent runs.

    Techniques Used: Incubation, Cytometry, Binding Assay, Fluorescence, Mutagenesis, Expressing

    Lectin binding to wild-type and glycogene-KO 293 T cells. A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.
    Figure Legend Snippet: Lectin binding to wild-type and glycogene-KO 293 T cells. A panel of lectins (from Vector Labs) was conjugated with Alexa dyes, either Alexa 405, 488 or 647. The binding of these fluorescent reagents to wild-type 293T, [N] - 293T and [O] - 293 T cells was measured using flow cytometry. The lectins bound: ( A ) N-glycan high-mannose and complex structures [ConA and LCA bind αMan in high-mannose glycans; PHA-L and PHA-E bind complex glycans], ( B ) lactosamine chains primarily on N-linked glycans [RCA, ECL bind terminal Gal or lactose; DSL bind β1,4GlcNAc], and ( C ) O-glycan related structures [PNA binds Galβ1,GalNAc; VVA and SBA bind GalNAcα]. Measurements were made with either untreated or sialidase treated 293 T cells. As seen: i. Knocking out MGAT1 in [N] - 293T reduces lectin binding in panels A and B (see arrow). ii. Knocking out C1GalT1 results in a dramatic decrease in PNA binding and increase in VVA and SBA binding, These data are consistent with the expected changes in lectin profile upon knocking out these N- and O-glycan-specific enzymes.

    Techniques Used: Binding Assay, Plasmid Preparation, Flow Cytometry

    16) Product Images from "Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration"

    Article Title: Inhibition of SARS-CoV-2 viral entry in vitro upon blocking N- and O-glycan elaboration

    Journal: bioRxiv

    doi: 10.1101/2020.10.15.339838

    Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.
    Figure Legend Snippet: Principal findings and conceptual model: A . ACE2-Fc binding was measured to wild-type or glycoEnzyme-KO 293T cells expressing Spike. Sialidase treatment of cells was performed in some cases. Similar studies also measured S1-Fc and RBD-Fc binding to cell-surface expressed ACE2. B . SARS-CoV-2 pseudovirus (bearing Spike-WT, Spike-mut, Spike-delta variants) were generated in wild-type or glycoEnzyme-KO 293Ts, in the presence and absence of kifunensine. Main results of binding ( A ) and viral-entry ( B ) assay are listed. C . Conceptual model shows that kifunensine can induce S1-S2 site proteolysis on Spike-WT and Spike-mut virus, but not Spike-delta virus. This proteolysis reduces RBD presentation and attenuates viral entry into 293T/ACE2. Without affecting S1-S2 cleavage, kifunensine also partially reduced Spike-delta pseudovirus entry function. The data suggest additional roles for Spike N-glycans during viral entry.

    Techniques Used: Binding Assay, Expressing, Generated

    17) Product Images from "SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors"

    Article Title: SARS-CoV-2 Spike Protein Interacts with Multiple Innate Immune Receptors

    Journal: bioRxiv

    doi: 10.1101/2020.07.29.227462

    Major N-glycans and O-glycopeptides identified in the recombinant full-length SARS-CoV-2 S. Schematic representation of SARS-CoV-2 S shown in the middle. The positions of N-glycosylation sites are shown on top. Protein domains in the illustration are: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). The cleavage sites of S1/S2 and S2’ are labelled. Major N-glycan structures detected by mass spectrometry were categorized by their epitopes on the non-reducing terminal and shown on top. Cartoon symbols above a curly parenthesis indicates sequences corresponding to these compositions cannot be unequivocally defined. The structures presented are only the major glycans on the recombinant full-length S, S1, and S2. A full list of glycans can be found in Supplementary Table 1 . The O-glycopeptides detected in the full-length S protein are presented at the bottom. The identified O-glycosylation sites are marked on the protein and the O-glycans on each specific site are listed below each site. The LC-MS/MS spectrum of each O-glycopeptide can be found in Supplementary Fig. 6 . Labelled with asterisk were those found only after neuraminidase treatment.
    Figure Legend Snippet: Major N-glycans and O-glycopeptides identified in the recombinant full-length SARS-CoV-2 S. Schematic representation of SARS-CoV-2 S shown in the middle. The positions of N-glycosylation sites are shown on top. Protein domains in the illustration are: N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), central helix (CH), connector domain (CD), and transmembrane domain (TM). The cleavage sites of S1/S2 and S2’ are labelled. Major N-glycan structures detected by mass spectrometry were categorized by their epitopes on the non-reducing terminal and shown on top. Cartoon symbols above a curly parenthesis indicates sequences corresponding to these compositions cannot be unequivocally defined. The structures presented are only the major glycans on the recombinant full-length S, S1, and S2. A full list of glycans can be found in Supplementary Table 1 . The O-glycopeptides detected in the full-length S protein are presented at the bottom. The identified O-glycosylation sites are marked on the protein and the O-glycans on each specific site are listed below each site. The LC-MS/MS spectrum of each O-glycopeptide can be found in Supplementary Fig. 6 . Labelled with asterisk were those found only after neuraminidase treatment.

    Techniques Used: Recombinant, Binding Assay, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

    18) 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

    19) 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

    20) Product Images from "The CA19-9 and Sialyl-TRA Antigens Define Separate Subpopulations of Pancreatic Cancer Cells"

    Article Title: The CA19-9 and Sialyl-TRA Antigens Define Separate Subpopulations of Pancreatic Cancer Cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04164-z

    The CA19-9 and sialyl-TRA (sTRA) antigens. ( A ) In order to detect sTRA, we treat the sample with sialidase prior to applying the TRA-1-60 monoclonal antibody (mAb). ( B ) The CA19-9 and sTRA glycans have similar structures except for the presence of fucose in the CA19-9 antigen. ( C ) The treatment of pancreatic cancer tissue with sialidase leads to increased binding of the TRA-1-60 mAb, revealing the presence of sTRA in the tissue. The overlap with the CA19-9 antigen is not known. ( D ) The schematic shows the process we used for multimarker immunofluorescence. Between the second and third rounds, we treated the tissue with sialidase to remove sialic acid from sTRA, enabling detection by the TRA-1-60 mAb.
    Figure Legend Snippet: The CA19-9 and sialyl-TRA (sTRA) antigens. ( A ) In order to detect sTRA, we treat the sample with sialidase prior to applying the TRA-1-60 monoclonal antibody (mAb). ( B ) The CA19-9 and sTRA glycans have similar structures except for the presence of fucose in the CA19-9 antigen. ( C ) The treatment of pancreatic cancer tissue with sialidase leads to increased binding of the TRA-1-60 mAb, revealing the presence of sTRA in the tissue. The overlap with the CA19-9 antigen is not known. ( D ) The schematic shows the process we used for multimarker immunofluorescence. Between the second and third rounds, we treated the tissue with sialidase to remove sialic acid from sTRA, enabling detection by the TRA-1-60 mAb.

    Techniques Used: Binding Assay, Immunofluorescence

    21) 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 Snippet: Total and biotinylated PODXL were detected by Western blot analysis as above. .. Deglycosylation Cell lysates were either mock treated or treated with peptide N-glycosidase F, neuraminidase, or O-glycosidase and neuraminidase (PNGase F; O-Glycosidase & Neuraminidase Bundle; New England Biolabs) as per the manufacturer’s recommendations, followed by SDS-PAGE and Western blot analysis. .. Deglycosylation Cell lysates were either mock treated or treated with peptide N -glycosidase F, neuraminidase, or O-glycosidase and neuraminidase (PNGase F; O-Glycosidase & Neuraminidase Bundle; New England Biolabs) as per the manufacturer’s recommendations, followed by SDS-PAGE and Western blot analysis.

    Article Title: FXYD5 Is an Essential Mediator of the Inflammatory Response during Lung Injury
    Article Snippet: Immunoblots were quantified by densitometry using Image J 1.46r (National Institutes of Health, Bethesda, MD). .. Where indicated, surface biotinylated proteins were treated with O-glycosidase and Neuraminidase Bundle according to the manufacturer’s instructions (New England Biolabs, Inc.) prior to loading on SDS-PAGE as we previously described ( ). .. Interferon α (IFN-α, Biolegend) and TNF-α (Biolegend) were added to A549 cells for up to 24 and 2 h, respectively, as described for LPS treatment.

    Western Blot:

    Article Title: Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis
    Article Snippet: Total and biotinylated PODXL were detected by Western blot analysis as above. .. Deglycosylation Cell lysates were either mock treated or treated with peptide N-glycosidase F, neuraminidase, or O-glycosidase and neuraminidase (PNGase F; O-Glycosidase & Neuraminidase Bundle; New England Biolabs) as per the manufacturer’s recommendations, followed by SDS-PAGE and Western blot analysis. .. Deglycosylation Cell lysates were either mock treated or treated with peptide N -glycosidase F, neuraminidase, or O-glycosidase and neuraminidase (PNGase F; O-Glycosidase & Neuraminidase Bundle; New England Biolabs) as per the manufacturer’s recommendations, followed by SDS-PAGE and Western blot analysis.

    Incubation:

    Article Title: The half-life of the bone-derived hormone osteocalcin is regulated through O-glycosylation in mice, but not in humans
    Article Snippet: In-vitro de-glycosylation assay was performed on 10 μg of bone homogenate. .. Briefly, proteins were denatured in denaturing buffer (0.5% SDS, 40 mM DTT) at 95°C for 5 min and incubated with 80000 units of O-glycosidase and 100 units of neuraminidases for 4 hr at 37 °C following the NEB kit protocol (E0540S; NEB). .. Samples were resolved on 15% Tris-tricine SDS-PAGE gel and blotted using anti-Cterm OCN goat antibody.

    Expressing:

    Article Title: Endoglycan plays a role in axon guidance by modulating cell adhesion
    Article Snippet: .. Both commissural and motoneurons were tested for adhesive strength after HEK cells expressing Endoglycan were treated with O-glycosidase (8’000 U/ml) or α2–3,6,8 Neuraminidase (5 U/ml; NEB Cat# E0540S, kit with both enzymes) for 2 hr before commissural neurons or motoneurons were added ( ; ). .. Growth cone blasting assay Commissural neurons were dissected from the most dorsal region of spinal cords of HH25/26 embryos that were unilaterally electroporated in ovo at HH17-18 with a plasmid encoding mRFP under the β-actin promotor (30 ng/μl) or co-electroporated with a plasmid encoding the open-reading frame of Endoglycan under the β-actin promotor (300 ng/μl).

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    Article Snippet: Enzymes: Peptide- N -Glycosidase F (PNGase F) from Flavobacterium meningosepticum (New England BioLabs), endoglycosidase H (Endo H) from Streptomyces plicatus (Glyco-Prozyme Inc.), Jack bean α-Mannosidase (Sigma-Aldrich) and O -glycosidase & Neuraminidase Bundle (New England BioLabs).

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    New England Biolabs pngase f native
    Altered mobility of α-DG in COG-deficient CHO cells is a function of COG-dependent defects in O- glycosylation. A , representative Western blotting of WT CHO and ldlB cell lysates with or without <t>PNGase</t> F digestion ( n = 2). The cells were treated with or without V. cholerae neuraminidase (120 milliunits/ml) prior to lysis. B , cells were cultured with increasing concentrations of a furin inhibitor, and Western blotting analysis was performed on lysates using an anti-DG (core) antibody ( n = 2). Furin inhibition results in a comparable increase in the apparent molecular weight of α-DG in both WT CHO and ldlB cells. C , cells were treated with two different amounts (60 or 120 milliunits/ml) of V. cholerae neuraminidase, and Western blotting analysis was performed. D , Western blotting of α-DG in WT, ldlB, and cog1 -corrected ldlB cells treated with or without V. cholerae neuraminidase ( n = 2). IB , immunoblot.
    Pngase F Native, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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 .
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    New England Biolabs α2 3 specific neuraminidase
    N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with <t>α2-3</t> linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.
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    New England Biolabs sialidase digestion
    LC-MS/MS analysis of Peptide C and the equivalent peptides after <t>sialidase</t> 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.
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    Altered mobility of α-DG in COG-deficient CHO cells is a function of COG-dependent defects in O- glycosylation. A , representative Western blotting of WT CHO and ldlB cell lysates with or without PNGase F digestion ( n = 2). The cells were treated with or without V. cholerae neuraminidase (120 milliunits/ml) prior to lysis. B , cells were cultured with increasing concentrations of a furin inhibitor, and Western blotting analysis was performed on lysates using an anti-DG (core) antibody ( n = 2). Furin inhibition results in a comparable increase in the apparent molecular weight of α-DG in both WT CHO and ldlB cells. C , cells were treated with two different amounts (60 or 120 milliunits/ml) of V. cholerae neuraminidase, and Western blotting analysis was performed. D , Western blotting of α-DG in WT, ldlB, and cog1 -corrected ldlB cells treated with or without V. cholerae neuraminidase ( n = 2). IB , immunoblot.

    Journal: The Journal of Biological Chemistry

    Article Title: Defective mucin-type glycosylation on α-dystroglycan in COG-deficient cells increases its susceptibility to bacterial proteases

    doi: 10.1074/jbc.RA118.003014

    Figure Lengend Snippet: Altered mobility of α-DG in COG-deficient CHO cells is a function of COG-dependent defects in O- glycosylation. A , representative Western blotting of WT CHO and ldlB cell lysates with or without PNGase F digestion ( n = 2). The cells were treated with or without V. cholerae neuraminidase (120 milliunits/ml) prior to lysis. B , cells were cultured with increasing concentrations of a furin inhibitor, and Western blotting analysis was performed on lysates using an anti-DG (core) antibody ( n = 2). Furin inhibition results in a comparable increase in the apparent molecular weight of α-DG in both WT CHO and ldlB cells. C , cells were treated with two different amounts (60 or 120 milliunits/ml) of V. cholerae neuraminidase, and Western blotting analysis was performed. D , Western blotting of α-DG in WT, ldlB, and cog1 -corrected ldlB cells treated with or without V. cholerae neuraminidase ( n = 2). IB , immunoblot.

    Article Snippet: A. ureafaciens neuraminidase (P0722) PNGase F (P0704S) were purchased from New England Biolabs.

    Techniques: Western Blot, Lysis, Cell Culture, Inhibition, Molecular Weight

    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:

    N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Journal: Frontiers in Oncology

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    doi: 10.3389/fonc.2018.00070

    Figure Lengend Snippet: N -glycosylation changes in squamous cell carcinoma (SCC). (A) Supervised hierarchical cluster analysis of healthy and tumor skin. Rows display each of the 15 patient glycan data (T, tumor tissue; C, healthy control). Columns indicate the N -glycan ID. Five clusters can be observed: in healthy tissue N -glycans with α2-3 linked N -acetylneuraminic acid (NeuAc) appeared to be present in increased levels whereas α2-6-NeuAc and oligomannose N -glycan levels were higher in tumor tissue. (B) Statistical evaluation of sialylated and oligomannose N -glycans uncovered significant changes [ p ≤ 0.04, using a t -test, indicated by an asterisk (*)]. Oligomannose N -glycans were upregulated whereas α2-3 linked NeuAc carrying N -glycans were down regulated in SCC.

    Article Snippet: The glycan sample was reconstituted in 10 μL 25 mM ammonium acetate buffer (pH 5.5) and 25 U of a α2-3-specific neuraminidase (NEB, Ipswitch, MA, USA) were added.

    Techniques:

    Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Journal: Frontiers in Oncology

    Article Title: Alterations of the Human Skin N- and O-Glycome in Basal Cell Carcinoma and Squamous Cell Carcinoma

    doi: 10.3389/fonc.2018.00070

    Figure Lengend Snippet: Healthy human skin N -glycome. (A) Bean diagram representing the 10 most abundant N -glycans determined from 14 patients. Green bars indicate individual data points, the black line represents the median, and the gray area depicts the data density. Columns indicate the glycan structures given by their glycan ID (Table S1 in Supplementary Material). (B) Relative N -glycan class abundances found in healthy skin biopsies. Sialylated and fucosylated structures were the major components representing the human skin N -glycome. (C) Relative abundances of different structure features found on sialylated N -glycans. Blue bars represent sialylated N -glycans with and without core fucose depending on their sialic acid linkage, showing that core fucosylation was a more abundant feature on N -glycans carrying one or two α2-3 linked N -acetylneuraminic acid (NeuAc) residues (if both, α2-3 and α2-6 linkages were present on one N- glycan, this N -glycan was considered in both linkage categories). Overall, α2-6-linked NeuAc was a slightly more abundant feature compared with α2-3-linked NeuAc. Most of the sialylated N -glycans carried two NeuAc residues. Tri-antennary species were below 3% and rather low abundant using porous graphitized carbon, while multiplexed capillary gel electrophoresis with laser induced fluorescence detection (xCGE-LIF) detected slightly higher levels of tri- and tetra-antennary N -glycans (see Supplementary file “xCGE-LIF quant.xlsx” in Supplementary Material).

    Article Snippet: The glycan sample was reconstituted in 10 μL 25 mM ammonium acetate buffer (pH 5.5) and 25 U of a α2-3-specific neuraminidase (NEB, Ipswitch, MA, USA) were added.

    Techniques: Nucleic Acid Electrophoresis, Fluorescence

    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