recombinant glycerol free pngase f  (New England Biolabs)


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    Structured Review

    New England Biolabs recombinant glycerol free pngase f
    Genetic ablation of host  N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the  N- glycosylation pathway targeted in this study. The precursor  N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The  N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text.  N- glycans are represented using CFG nomenclature. Blue square,  N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion  N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2  siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2  infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 
    Recombinant Glycerol Free Pngase F, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant glycerol free pngase f/product/New England Biolabs
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    recombinant glycerol free pngase f - by Bioz Stars, 2022-10
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    Images

    1) Product Images from "Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection"

    Article Title: Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection

    Journal: mBio

    doi: 10.1128/mbio.03718-21

    Genetic ablation of host  N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the  N- glycosylation pathway targeted in this study. The precursor  N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The  N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text.  N- glycans are represented using CFG nomenclature. Blue square,  N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion  N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2  siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2  infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 
    Figure Legend Snippet: Genetic ablation of host N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the N- glycosylation pathway targeted in this study. The precursor N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text. N- glycans are represented using CFG nomenclature. Blue square, N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2 siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2 infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 

    Techniques Used: Infection, Synthesized, Modification, Transfection, Staining

    SARS-CoV-2 surface N- glycans are essential for infection. (A) Infective particles in infected Vero E6 supernatants (MOI of 0.001, 48 h) treated with 0, 0.1, 5, and 75 μM NGI-1. (B) Total SARS-CoV-2 RNA in supernatants in panel A. (C) Proportion of infected Vero E6 cells treated with 0, 0.1, 5, and 75 μM NGI-1 and infected with virus from either untreated (DMSO) or 5 μM NGI-1-treated cell supernatants (MOI of 0.05, 24 h); inoculum normalized by total viral RNA. (D) Western blot using anti-spike (green) and anti-M (red) antibodies on lysed native purified virions (C) of mock treated (P-), PNGase F-treated (P+), and heat-inactivated PNGase F-treated (P IN ) cells or supernatant from uninfected cells (Un). Treatment legend (top), apparent molecular mass (left), and band identity (right) are given. Full-length spike (S0), spike aggregates (SA), and glycosylated (g) and unglycosylated (Ug) M protein are shown; asterisks highlight downshifted bands. (E) Spike protein schematic highlighting all N- glycosylation sites and asparagines (N) converted to aspartic acid (boldface red D) after PNGase F deglycosylation as found by mass spectrometry and spike subunits (S1, S2), N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), ands transmembrane domain (TM). (F) Whole-well scans of confluent Vero E6 cells infected with SARS-CoV-2 supernatant (SN) or purified virions analyzed in panels D (MOI of 0.05, 24 h). Anti-spike antibodies, green; scale bar, 5 mm. (G) Representative images of Vero E6 cells in panel F. Anti-spike, green; merged with DAPI counterstain, magenta. Scale bar, 200 μm. (H) Percentage of infected cells in panel F, including purified virions coinoculated with active PNGase F (P CO ) and cells either pretreated (P+ cell ) or not treated (P- cell ) with active PNGase F prior to inoculation with native virions, normalized to SN. Bars indicate mean values, error bars show +SD, and asterisks indicate significance ( P
    Figure Legend Snippet: SARS-CoV-2 surface N- glycans are essential for infection. (A) Infective particles in infected Vero E6 supernatants (MOI of 0.001, 48 h) treated with 0, 0.1, 5, and 75 μM NGI-1. (B) Total SARS-CoV-2 RNA in supernatants in panel A. (C) Proportion of infected Vero E6 cells treated with 0, 0.1, 5, and 75 μM NGI-1 and infected with virus from either untreated (DMSO) or 5 μM NGI-1-treated cell supernatants (MOI of 0.05, 24 h); inoculum normalized by total viral RNA. (D) Western blot using anti-spike (green) and anti-M (red) antibodies on lysed native purified virions (C) of mock treated (P-), PNGase F-treated (P+), and heat-inactivated PNGase F-treated (P IN ) cells or supernatant from uninfected cells (Un). Treatment legend (top), apparent molecular mass (left), and band identity (right) are given. Full-length spike (S0), spike aggregates (SA), and glycosylated (g) and unglycosylated (Ug) M protein are shown; asterisks highlight downshifted bands. (E) Spike protein schematic highlighting all N- glycosylation sites and asparagines (N) converted to aspartic acid (boldface red D) after PNGase F deglycosylation as found by mass spectrometry and spike subunits (S1, S2), N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), ands transmembrane domain (TM). (F) Whole-well scans of confluent Vero E6 cells infected with SARS-CoV-2 supernatant (SN) or purified virions analyzed in panels D (MOI of 0.05, 24 h). Anti-spike antibodies, green; scale bar, 5 mm. (G) Representative images of Vero E6 cells in panel F. Anti-spike, green; merged with DAPI counterstain, magenta. Scale bar, 200 μm. (H) Percentage of infected cells in panel F, including purified virions coinoculated with active PNGase F (P CO ) and cells either pretreated (P+ cell ) or not treated (P- cell ) with active PNGase F prior to inoculation with native virions, normalized to SN. Bars indicate mean values, error bars show +SD, and asterisks indicate significance ( P

    Techniques Used: Infection, Western Blot, Purification, Mass Spectrometry, Binding Assay

    2) Product Images from "Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 β-cells"

    Article Title: Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 β-cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55873-6

    The alternative 90 kDa protein product exhibits a different glycosylation profile. ( A ) Representative western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 1250 U or 2500 U Endo H. ( B ) Representative western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 500 U PNGase F. ( C ). Western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 1250 U Endo H and 500 U PNGase F. ( D ) Representative western blot of subcellular fractionation by sucrose gradient of  wtSorcs1  and  mutSorcs1 -expressing INS1 cells. ( E ) Densitometry analysis of combined bands in subcellular fractionation immunoblots in  wtSorcs1  and  mutSorcs1  INS1 cells. ( F ) Densitometry analysis of 180, 130 and 90 kDa  mutSorcs1  INS1 cell bands in subcellular fractionation immunoblots. *p 
    Figure Legend Snippet: The alternative 90 kDa protein product exhibits a different glycosylation profile. ( A ) Representative western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 1250 U or 2500 U Endo H. ( B ) Representative western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 500 U PNGase F. ( C ). Western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 1250 U Endo H and 500 U PNGase F. ( D ) Representative western blot of subcellular fractionation by sucrose gradient of wtSorcs1 and mutSorcs1 -expressing INS1 cells. ( E ) Densitometry analysis of combined bands in subcellular fractionation immunoblots in wtSorcs1 and mutSorcs1 INS1 cells. ( F ) Densitometry analysis of 180, 130 and 90 kDa mutSorcs1 INS1 cell bands in subcellular fractionation immunoblots. *p 

    Techniques Used: Western Blot, Fractionation, Expressing

    3) Product Images from "Resident microbes shape the vaginal epithelial glycan landscape"

    Article Title: Resident microbes shape the vaginal epithelial glycan landscape

    Journal: medRxiv

    doi: 10.1101/2022.02.23.22271417

    Sialylated vaginal epithelial N -linked glycans are depleted in BV. ( A ) Schematic for 2-amino benzamide (2AB) profiling of N- linked glycans by high performance anion exchange chromatography (HPAEC). Glycans were released using PNGase-F from protein extracts derived from VECs pools and fluorescently labelled with 2-AB prior to analysis by HPAEC. ( B ) HPAEC profiles of 2-AB labeled N -glycans derived from (i) protein standards, with well-known glycan structures, Rnase B and Bovine Fetuin, (ii) 2 pools of No BV VECs (n=10 specimens/pool), (ii) 2 pools of BV VECs (n=10 specimens/pool). ( C ) HPAEC profiles of N -glycans derived from (i) Rnase B and Bovine Fetuin, and (ii) pools of No BV VECs (same VEC pools as used in B , n=10 specimens/pool) pretreated with commercially available sialidase (+ A.u. sialidase, dotted line) or with buffer alone (- A.u . sialidase, solid line). Structures of different types of N -glycans (high mannose, mono- and di-sialylated) are depicted following the NCBI Symbol Nomenclature for Glycans (yellow circle, galactose; green circle, mannose; blue square, N -acetylglucosamine; purple diamond, sialic acid). A.u. sialidase = sialidase from Arthrobacter ureafaciens. A total of N=40 specimens were used to generate these data. VEC pools generated from these specimens were also used for studies reported in Fig. 2C, 2D and Fig. 4 . See methods for pooling rationale.
    Figure Legend Snippet: Sialylated vaginal epithelial N -linked glycans are depleted in BV. ( A ) Schematic for 2-amino benzamide (2AB) profiling of N- linked glycans by high performance anion exchange chromatography (HPAEC). Glycans were released using PNGase-F from protein extracts derived from VECs pools and fluorescently labelled with 2-AB prior to analysis by HPAEC. ( B ) HPAEC profiles of 2-AB labeled N -glycans derived from (i) protein standards, with well-known glycan structures, Rnase B and Bovine Fetuin, (ii) 2 pools of No BV VECs (n=10 specimens/pool), (ii) 2 pools of BV VECs (n=10 specimens/pool). ( C ) HPAEC profiles of N -glycans derived from (i) Rnase B and Bovine Fetuin, and (ii) pools of No BV VECs (same VEC pools as used in B , n=10 specimens/pool) pretreated with commercially available sialidase (+ A.u. sialidase, dotted line) or with buffer alone (- A.u . sialidase, solid line). Structures of different types of N -glycans (high mannose, mono- and di-sialylated) are depicted following the NCBI Symbol Nomenclature for Glycans (yellow circle, galactose; green circle, mannose; blue square, N -acetylglucosamine; purple diamond, sialic acid). A.u. sialidase = sialidase from Arthrobacter ureafaciens. A total of N=40 specimens were used to generate these data. VEC pools generated from these specimens were also used for studies reported in Fig. 2C, 2D and Fig. 4 . See methods for pooling rationale.

    Techniques Used: Chromatography, Derivative Assay, Labeling, Generated

    4) Product Images from "Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens"

    Article Title: Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1016/j.mcpro.2022.100201

    MALDI-TOF–MS of  Brugia malayi  adult worm (mixed sex)  N -linked glycans.  PNGase F-released glycans from  B. malayi  glycoproteins were labeled with 2-AA and analyzed using MALDI-TOF–MS in negative-ion reflectron mode. Monoisotopic masses of measured signals are indicated, and proposed glycan structures for ions with signal-to-noise ratios superior to 4 and intensities above 5000 are depicted using the Consortium for Functional Glycomics (CFG) nomenclature (see  symbol key inset ). Compositions and structures were deduced using a panel of glycan sequencing techniques in combination with MALDI-TOF–MS/MS fragmentation and information from the literature. 2-AA, 2-aminobenzoic acid; MS, mass spectrometry; MS/MS, tandem MS; PNGase F, peptide: N -glycosidase F.
    Figure Legend Snippet: MALDI-TOF–MS of Brugia malayi adult worm (mixed sex) N -linked glycans. PNGase F-released glycans from B. malayi glycoproteins were labeled with 2-AA and analyzed using MALDI-TOF–MS in negative-ion reflectron mode. Monoisotopic masses of measured signals are indicated, and proposed glycan structures for ions with signal-to-noise ratios superior to 4 and intensities above 5000 are depicted using the Consortium for Functional Glycomics (CFG) nomenclature (see symbol key inset ). Compositions and structures were deduced using a panel of glycan sequencing techniques in combination with MALDI-TOF–MS/MS fragmentation and information from the literature. 2-AA, 2-aminobenzoic acid; MS, mass spectrometry; MS/MS, tandem MS; PNGase F, peptide: N -glycosidase F.

    Techniques Used: Labeling, Functional Assay, Sequencing, Tandem Mass Spectroscopy, Mass Spectrometry

    5) Product Images from "Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay"

    Article Title: Systems-wide analysis of glycoprotein conformational changes by limited deglycosylation assay

    Journal: bioRxiv

    doi: 10.1101/2021.06.04.447131

    3D structure of basal cell adhesion molecule (Lutheran blood group) (A0A1D5Q3L5/BCAM) showing identified N-glycosites. A ) Cartoon representation of PVR illustrating the ratios of the intensities of FNGPs. α-helix are in red, β-sheet in blue, and loops in grey. The N-glycosites are represented in red, and sticks represent the side chains. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, and III represents the ratio of II / I , * represents ratios whose DTT+PNGase F is 0, representing deglycopeptides exclusively missing in DTT + PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent sites with more and less exposure, respectively. The SASA (in Å) are shown in parenthesis. C ) Localization probabilities of the BCAM amino acids calculated using ODiNPred. The N-glycosites of interest are highlighted.
    Figure Legend Snippet: 3D structure of basal cell adhesion molecule (Lutheran blood group) (A0A1D5Q3L5/BCAM) showing identified N-glycosites. A ) Cartoon representation of PVR illustrating the ratios of the intensities of FNGPs. α-helix are in red, β-sheet in blue, and loops in grey. The N-glycosites are represented in red, and sticks represent the side chains. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, and III represents the ratio of II / I , * represents ratios whose DTT+PNGase F is 0, representing deglycopeptides exclusively missing in DTT + PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent sites with more and less exposure, respectively. The SASA (in Å) are shown in parenthesis. C ) Localization probabilities of the BCAM amino acids calculated using ODiNPred. The N-glycosites of interest are highlighted.

    Techniques Used:

    Optimization of the LDA method using RNase B and fetuin. Experimental conditions for optimal limited deglycosylation of bovine RNase B and fetuin by PNGase F were determined. The optimal A ) NP-40 concentration, B ) temperature, C ) and D ) PNGase F:glycoprotein ratio for RNase B and fetuin, respectively, and, E ) and F ) the optimal time for the deglycosylation of RNase B and fetuin, respectively, were assayed by differential migration of the glycoproteins in 15% SDS-PAGE. Fully deglycosylated (+) and fully glycosylated (−) proteins were included for each optimization reaction, denoted by FD and FG, respectively.
    Figure Legend Snippet: Optimization of the LDA method using RNase B and fetuin. Experimental conditions for optimal limited deglycosylation of bovine RNase B and fetuin by PNGase F were determined. The optimal A ) NP-40 concentration, B ) temperature, C ) and D ) PNGase F:glycoprotein ratio for RNase B and fetuin, respectively, and, E ) and F ) the optimal time for the deglycosylation of RNase B and fetuin, respectively, were assayed by differential migration of the glycoproteins in 15% SDS-PAGE. Fully deglycosylated (+) and fully glycosylated (−) proteins were included for each optimization reaction, denoted by FD and FG, respectively.

    Techniques Used: Concentration Assay, Migration, SDS Page

    Limited deglycosylation of LLC-MK2 cells treated with or without 3 mM DTT. The mass spectrometry analysis showing A ) number of identified and quantified deaminated peptides, B ) Venn diagram showing shared proteins from proteome and deglycoproteome analysis, C ) the PCA D ) heatmap of deamidated peptides, E ) total ion intensities of the deamidated deglycopeptides from control, control + PNGase F, DTT, and DTT + PNGase F fractions.
    Figure Legend Snippet: Limited deglycosylation of LLC-MK2 cells treated with or without 3 mM DTT. The mass spectrometry analysis showing A ) number of identified and quantified deaminated peptides, B ) Venn diagram showing shared proteins from proteome and deglycoproteome analysis, C ) the PCA D ) heatmap of deamidated peptides, E ) total ion intensities of the deamidated deglycopeptides from control, control + PNGase F, DTT, and DTT + PNGase F fractions.

    Techniques Used: Mass Spectrometry

    3D structure of GALNT10 showing N128 and N575 N-glycosylation sites undergoing structural modulation upon DTT treatment. A ) Cartoon representation of modelled 3D structure of  M. mulatta  N-acetylgalactosaminyltransferase illustrating the ratios of the intensities of FNGPs upon LDA; α-helix are in firebrick red, β-sheet in slate blue, and loops in green.  I  represents the intensity ratios of CTRL+PNGase F/CTRL,  II  represents the ratio between DTT+PNGase F/DTT,  III  represents the ratio of  II / I ,  *  represents ratios whose DTT+PNGase F/DTT is 0, to denote FNGPs exclusively missing in DTT+PNGase F fractions.  B ) Surface representation showing the SASA of the N-glycosites calculated using PyMol.  C ) 3D structure alignment of modelled  M. mulatta  (red) GALNT10 and determined 3D structures of homologs from  H. sapiens  (green) and  M. musculus  (blue) illustrating the conserved localization of the N-glycosite undergoing pronounced structural conformational modulation upon DTT treatment. The N-glycosites are represented using sticks to show the side chains of the glycosylated Asn.  D ) Localization probabilities of the GALNT amino acids calculated using ODiNPred are shown. The probabilities for the N-glycosites to fall into disordered regions have been indicated.
    Figure Legend Snippet: 3D structure of GALNT10 showing N128 and N575 N-glycosylation sites undergoing structural modulation upon DTT treatment. A ) Cartoon representation of modelled 3D structure of M. mulatta N-acetylgalactosaminyltransferase illustrating the ratios of the intensities of FNGPs upon LDA; α-helix are in firebrick red, β-sheet in slate blue, and loops in green. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, III represents the ratio of II / I , * represents ratios whose DTT+PNGase F/DTT is 0, to denote FNGPs exclusively missing in DTT+PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosites calculated using PyMol. C ) 3D structure alignment of modelled M. mulatta (red) GALNT10 and determined 3D structures of homologs from H. sapiens (green) and M. musculus (blue) illustrating the conserved localization of the N-glycosite undergoing pronounced structural conformational modulation upon DTT treatment. The N-glycosites are represented using sticks to show the side chains of the glycosylated Asn. D ) Localization probabilities of the GALNT amino acids calculated using ODiNPred are shown. The probabilities for the N-glycosites to fall into disordered regions have been indicated.

    Techniques Used:

    Lectin blot analysis of LLC-MK2 cells incubated with or without 3 mM DTT for 30 min followed by deglycosylation under native conditions. A)  ConA lectin blot following LDA.  B ) The combined intensities for each condition are summarized, and the ratios between +PNGase F/−PNGase F for both control and DTT treated fractions. Results are displayed as mean ± SEM; *p
    Figure Legend Snippet: Lectin blot analysis of LLC-MK2 cells incubated with or without 3 mM DTT for 30 min followed by deglycosylation under native conditions. A) ConA lectin blot following LDA. B ) The combined intensities for each condition are summarized, and the ratios between +PNGase F/−PNGase F for both control and DTT treated fractions. Results are displayed as mean ± SEM; *p

    Techniques Used: Incubation

    3D structure of ephrin-A5 (F7GZC7) showing the mapped N-glycosylation site. A ) Cartoon representation of the modelled 3D structure of ephrin-A5 illustrating the ratios of the MS/MS intensities of FNGPs between PNGase F-treated and -untreated cell extracts. α-helix are in firebrick red, β-sheet in slate blue, and loops in green.  I  represents the intensity ratios of CTRL+PNGase F/CTRL,  II  represents the ratio between DTT+PNGase F/DTT, and  III  represents the ratio of  II / I ,  *  represents ratios with DTT+PNGase F equal to 0, indicating deglycopeptides exclusively missing in DTT+PNGase F fractions.  B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent N-glycosites with more and less surface exposure, respectively. The SASA of the mapped N-glycosite (in Å) is shown in parenthesis.  C ) 3D structure alignment of modelled  M. mulatta  (red) ephrin-A5 and determined 3D structures of homologs from  H. sapiens  (green) and  M. musculus  (blue) illustrating the conserved localization of the N-glycosite undergoing pronounced structural conformational modulation upon DTT treatment. The N-glycosites are represented using sticks to show the side chains of the glycosylated Asn.  D ) Localization probabilities of ephrin-5A amino acids calculated using ODiNPred, with the mapped N-glycosite (N37) highlighted.
    Figure Legend Snippet: 3D structure of ephrin-A5 (F7GZC7) showing the mapped N-glycosylation site. A ) Cartoon representation of the modelled 3D structure of ephrin-A5 illustrating the ratios of the MS/MS intensities of FNGPs between PNGase F-treated and -untreated cell extracts. α-helix are in firebrick red, β-sheet in slate blue, and loops in green. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, and III represents the ratio of II / I , * represents ratios with DTT+PNGase F equal to 0, indicating deglycopeptides exclusively missing in DTT+PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent N-glycosites with more and less surface exposure, respectively. The SASA of the mapped N-glycosite (in Å) is shown in parenthesis. C ) 3D structure alignment of modelled M. mulatta (red) ephrin-A5 and determined 3D structures of homologs from H. sapiens (green) and M. musculus (blue) illustrating the conserved localization of the N-glycosite undergoing pronounced structural conformational modulation upon DTT treatment. The N-glycosites are represented using sticks to show the side chains of the glycosylated Asn. D ) Localization probabilities of ephrin-5A amino acids calculated using ODiNPred, with the mapped N-glycosite (N37) highlighted.

    Techniques Used: Tandem Mass Spectroscopy

    Optimized LDA applied to LLC-MK2 cellular glycoproteins. A ) Lectin blotting using Con A on LLC-MK2 cells following LDA using optimized conditions previously determined on standard glycoproteins. B ) Intensity and ratios between +PNGase F/−PNGase F calculated using ImageLab are shown. - P = negative control not treated with PNGase F; +P = PNGase F treated.
    Figure Legend Snippet: Optimized LDA applied to LLC-MK2 cellular glycoproteins. A ) Lectin blotting using Con A on LLC-MK2 cells following LDA using optimized conditions previously determined on standard glycoproteins. B ) Intensity and ratios between +PNGase F/−PNGase F calculated using ImageLab are shown. - P = negative control not treated with PNGase F; +P = PNGase F treated.

    Techniques Used: Negative Control

    3D structure of the uncharacterized protein (A0A5F8ABK3/PVR) showing N-glycosites of interest. A ) Cartoon representation of PVR illustrating the ratios of the intensities of FNGPs. α-helix are in red, β-sheet in blue, and loops in grey. The N-glycosites are in red, and sticks used to represent the side chains. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, and III represents the ratio of II / I , * represents ratios whose DTT+PNGase F is 0, representing deglycopeptides exclusively missing in DTT + PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent sites with more and less exposure, respectively. The SASA (in Å) are shown in parenthesis. C ) Structure alignment of the modelled M. mulatta PVR and the determined structure of the human homolog (QARQ) deposited in PDB. N278 is indicated on both proteins. D ) Localization probabilities of the PVR amino acids calculated using ODiNPred. N-glycosites of interest are highlighted.
    Figure Legend Snippet: 3D structure of the uncharacterized protein (A0A5F8ABK3/PVR) showing N-glycosites of interest. A ) Cartoon representation of PVR illustrating the ratios of the intensities of FNGPs. α-helix are in red, β-sheet in blue, and loops in grey. The N-glycosites are in red, and sticks used to represent the side chains. I represents the intensity ratios of CTRL+PNGase F/CTRL, II represents the ratio between DTT+PNGase F/DTT, and III represents the ratio of II / I , * represents ratios whose DTT+PNGase F is 0, representing deglycopeptides exclusively missing in DTT + PNGase F fractions. B ) Surface representation showing the SASA of the N-glycosite calculated using PyMol, where the warmer and cooler colors represent sites with more and less exposure, respectively. The SASA (in Å) are shown in parenthesis. C ) Structure alignment of the modelled M. mulatta PVR and the determined structure of the human homolog (QARQ) deposited in PDB. N278 is indicated on both proteins. D ) Localization probabilities of the PVR amino acids calculated using ODiNPred. N-glycosites of interest are highlighted.

    Techniques Used:

    Experimental workflow of the limited deglycosylation assay (LDA). LDA probes glycoprotein conformational changes on a global scale following perturbation-induced structural modulation. Cells treated or not with ER stress inducers are lysed in native conditions and subjected to PNGase F treatment (4°C for 16 h). The resulting proteins are analyzed by GeLC approach using SDS-PAGE and in-gel digestion of the entire lane. The tryptic glycopeptides are enriched using HILIC SPE and subsequently deglycosylated by overnight PNGase F treatment. Quantitative nLC-MS/MS analysis was performed to identify differentially formerly N-glycopeptides (FNGPs).
    Figure Legend Snippet: Experimental workflow of the limited deglycosylation assay (LDA). LDA probes glycoprotein conformational changes on a global scale following perturbation-induced structural modulation. Cells treated or not with ER stress inducers are lysed in native conditions and subjected to PNGase F treatment (4°C for 16 h). The resulting proteins are analyzed by GeLC approach using SDS-PAGE and in-gel digestion of the entire lane. The tryptic glycopeptides are enriched using HILIC SPE and subsequently deglycosylated by overnight PNGase F treatment. Quantitative nLC-MS/MS analysis was performed to identify differentially formerly N-glycopeptides (FNGPs).

    Techniques Used: SDS Page, Hydrophilic Interaction Liquid Chromatography, Tandem Mass Spectroscopy

    6) Product Images from "Protein N-glycosylation is essential for SARS-CoV-2 infection"

    Article Title: Protein N-glycosylation is essential for SARS-CoV-2 infection

    Journal: bioRxiv

    doi: 10.1101/2021.02.05.429940

    ( A ) Lectin blotting of virion lysates from ‘ Fig. 3D ’ probed with concanavalin A-FITC (ConA); membrane stained with nigrosin as loading control; apparent molecular weight (left). Although most of the bands recognized by the lectin are likely to be residual serum glycoproteins from the culture media and Vero E6 secretome, there is an overall decrease in recognition after PNGase F treatment. ( B ) Total viral RNA quantification by qRT-PCR in supernatants from ‘ Fig. 3E ’. ( C ) Virus titres in supernatants from ‘ Fig. 3E ’ quantified by plaque assay. Bars indicate mean values; error bars show +S.D.; asterisks indicate significance ( p
    Figure Legend Snippet: ( A ) Lectin blotting of virion lysates from ‘ Fig. 3D ’ probed with concanavalin A-FITC (ConA); membrane stained with nigrosin as loading control; apparent molecular weight (left). Although most of the bands recognized by the lectin are likely to be residual serum glycoproteins from the culture media and Vero E6 secretome, there is an overall decrease in recognition after PNGase F treatment. ( B ) Total viral RNA quantification by qRT-PCR in supernatants from ‘ Fig. 3E ’. ( C ) Virus titres in supernatants from ‘ Fig. 3E ’ quantified by plaque assay. Bars indicate mean values; error bars show +S.D.; asterisks indicate significance ( p

    Techniques Used: Staining, Molecular Weight, Quantitative RT-PCR, Plaque Assay

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    New England Biolabs recombinant glycerol free pngase f
    Genetic ablation of host  N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the  N- glycosylation pathway targeted in this study. The precursor  N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The  N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text.  N- glycans are represented using CFG nomenclature. Blue square,  N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion  N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2  siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2  infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 
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    Genetic ablation of host  N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the  N- glycosylation pathway targeted in this study. The precursor  N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The  N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text.  N- glycans are represented using CFG nomenclature. Blue square,  N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion  N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2  siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2  infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 

    Journal: mBio

    Article Title: Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection

    doi: 10.1128/mbio.03718-21

    Figure Lengend Snippet: Genetic ablation of host N- glycosylation reduces SARS-CoV-2 infection. (A) Schematic of the key steps in the N- glycosylation pathway targeted in this study. The precursor N- glycan is first synthesized in the ER and transferred to the SARS-CoV-2 spike protein (red) by the OST’s catalytic subunit, STT3 (isoforms A and B). The N- glycans are further processed by the α-glucosidases I and II (GANAB catalytic subunit) before the glycoprotein is exported to the Golgi apparatus, where the glycans are modified by the α-mannosidase I, GnT-I (MGAT1), and α-mannosidase II. Mature glycosylated virions egress to the extracellular space, where they can be artificially deglycosylated using PNGase F. Glycosylation enzymes, gray; glycosylation inhibitors, red text; glycosylation enzymes targeted by siRNA, boldface text. N- glycans are represented using CFG nomenclature. Blue square, N -acetylglucosamine; green circle, mannose; blue circle, glucose; yellow circle, galactose; magenta diamond, sialic acid; virion N- glycan structures are representative only. (B) Representative whole-well scans of confluent Vero E6 monolayers transfected either with siNT (nontargeting control) or siRNAs targeting STT3-A, STT3-B, STT3A+B, GANAB, and MGAT1 and infected with SARS-CoV-2 (MOI of 0.05, 24 h). Cells immunostained using anti-spike antibodies (green), counterstained with DAPI DNA staining (magenta), and merged. Scale bar, 5 mm. (C) Representative images of Vero E6 and HEK293 ACE-2 siRNA cells infected with SARS-CoV-2 (MOI of 0.05 for 24 h, Vero; MOI of 0.1 for 24 h, HEK293). Anti-spike, gray; merge, green; with DAPI, magenta. Scale bars, 200 μm. (D) Percentage of Vero E6 and HEK293 ACE-2 infected cells (anti-spike positive) in panel A, normalized to siNT. Three technical replicates and four and three biological replicates were done for Vero and HEK cells, respectively. (E) Infectious viral particles (PFU/mL) in supernatants from infected Vero E6 siRNA cells in panel A, normalized to siNT. Bars indicate mean values, error bars represent ± standard deviations (SD), and asterisks indicate significance ( P 

    Article Snippet: Two milliliters of SARS-CoV-2 P4 supernatant containing 2 × 107 PFU/mL was purified using an Amicon Ultra column (molecular size cutoff, 100 kDa; Merck) by centrifugation and washing in PBS at 2,000 × g ; 40 mL purified supernatant was combined with 2,500 U recombinant glycerol-free PNGase F and Glycobuffer 2 (New England Biolabs) by following the manufacturer’s protocol for nondenaturing digestion and incubated at 37°C for 5 h. Parallel controls included incubation of purified virions with or without PNGase F and Glycobuffer, incubation with heat-inactivated (75°C for 10 min) PNGase F, and incubation with PNGase F and Glycobuffer during virus inoculation of host cells for 30 min. After incubation, samples were either directly used for infection or combined with radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors to generate lysates for blotting.

    Techniques: Infection, Synthesized, Modification, Transfection, Staining

    SARS-CoV-2 surface N- glycans are essential for infection. (A) Infective particles in infected Vero E6 supernatants (MOI of 0.001, 48 h) treated with 0, 0.1, 5, and 75 μM NGI-1. (B) Total SARS-CoV-2 RNA in supernatants in panel A. (C) Proportion of infected Vero E6 cells treated with 0, 0.1, 5, and 75 μM NGI-1 and infected with virus from either untreated (DMSO) or 5 μM NGI-1-treated cell supernatants (MOI of 0.05, 24 h); inoculum normalized by total viral RNA. (D) Western blot using anti-spike (green) and anti-M (red) antibodies on lysed native purified virions (C) of mock treated (P-), PNGase F-treated (P+), and heat-inactivated PNGase F-treated (P IN ) cells or supernatant from uninfected cells (Un). Treatment legend (top), apparent molecular mass (left), and band identity (right) are given. Full-length spike (S0), spike aggregates (SA), and glycosylated (g) and unglycosylated (Ug) M protein are shown; asterisks highlight downshifted bands. (E) Spike protein schematic highlighting all N- glycosylation sites and asparagines (N) converted to aspartic acid (boldface red D) after PNGase F deglycosylation as found by mass spectrometry and spike subunits (S1, S2), N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), ands transmembrane domain (TM). (F) Whole-well scans of confluent Vero E6 cells infected with SARS-CoV-2 supernatant (SN) or purified virions analyzed in panels D (MOI of 0.05, 24 h). Anti-spike antibodies, green; scale bar, 5 mm. (G) Representative images of Vero E6 cells in panel F. Anti-spike, green; merged with DAPI counterstain, magenta. Scale bar, 200 μm. (H) Percentage of infected cells in panel F, including purified virions coinoculated with active PNGase F (P CO ) and cells either pretreated (P+ cell ) or not treated (P- cell ) with active PNGase F prior to inoculation with native virions, normalized to SN. Bars indicate mean values, error bars show +SD, and asterisks indicate significance ( P

    Journal: mBio

    Article Title: Inhibition of Protein N-Glycosylation Blocks SARS-CoV-2 Infection

    doi: 10.1128/mbio.03718-21

    Figure Lengend Snippet: SARS-CoV-2 surface N- glycans are essential for infection. (A) Infective particles in infected Vero E6 supernatants (MOI of 0.001, 48 h) treated with 0, 0.1, 5, and 75 μM NGI-1. (B) Total SARS-CoV-2 RNA in supernatants in panel A. (C) Proportion of infected Vero E6 cells treated with 0, 0.1, 5, and 75 μM NGI-1 and infected with virus from either untreated (DMSO) or 5 μM NGI-1-treated cell supernatants (MOI of 0.05, 24 h); inoculum normalized by total viral RNA. (D) Western blot using anti-spike (green) and anti-M (red) antibodies on lysed native purified virions (C) of mock treated (P-), PNGase F-treated (P+), and heat-inactivated PNGase F-treated (P IN ) cells or supernatant from uninfected cells (Un). Treatment legend (top), apparent molecular mass (left), and band identity (right) are given. Full-length spike (S0), spike aggregates (SA), and glycosylated (g) and unglycosylated (Ug) M protein are shown; asterisks highlight downshifted bands. (E) Spike protein schematic highlighting all N- glycosylation sites and asparagines (N) converted to aspartic acid (boldface red D) after PNGase F deglycosylation as found by mass spectrometry and spike subunits (S1, S2), N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), ands transmembrane domain (TM). (F) Whole-well scans of confluent Vero E6 cells infected with SARS-CoV-2 supernatant (SN) or purified virions analyzed in panels D (MOI of 0.05, 24 h). Anti-spike antibodies, green; scale bar, 5 mm. (G) Representative images of Vero E6 cells in panel F. Anti-spike, green; merged with DAPI counterstain, magenta. Scale bar, 200 μm. (H) Percentage of infected cells in panel F, including purified virions coinoculated with active PNGase F (P CO ) and cells either pretreated (P+ cell ) or not treated (P- cell ) with active PNGase F prior to inoculation with native virions, normalized to SN. Bars indicate mean values, error bars show +SD, and asterisks indicate significance ( P

    Article Snippet: Two milliliters of SARS-CoV-2 P4 supernatant containing 2 × 107 PFU/mL was purified using an Amicon Ultra column (molecular size cutoff, 100 kDa; Merck) by centrifugation and washing in PBS at 2,000 × g ; 40 mL purified supernatant was combined with 2,500 U recombinant glycerol-free PNGase F and Glycobuffer 2 (New England Biolabs) by following the manufacturer’s protocol for nondenaturing digestion and incubated at 37°C for 5 h. Parallel controls included incubation of purified virions with or without PNGase F and Glycobuffer, incubation with heat-inactivated (75°C for 10 min) PNGase F, and incubation with PNGase F and Glycobuffer during virus inoculation of host cells for 30 min. After incubation, samples were either directly used for infection or combined with radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors to generate lysates for blotting.

    Techniques: Infection, Western Blot, Purification, Mass Spectrometry, Binding Assay

    The alternative 90 kDa protein product exhibits a different glycosylation profile. ( A ) Representative western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 1250 U or 2500 U Endo H. ( B ) Representative western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 500 U PNGase F. ( C ). Western blot of  wtSorcs1  and  mutSorcs1  INS1 cells after de-glycosylation treatment with 1250 U Endo H and 500 U PNGase F. ( D ) Representative western blot of subcellular fractionation by sucrose gradient of  wtSorcs1  and  mutSorcs1 -expressing INS1 cells. ( E ) Densitometry analysis of combined bands in subcellular fractionation immunoblots in  wtSorcs1  and  mutSorcs1  INS1 cells. ( F ) Densitometry analysis of 180, 130 and 90 kDa  mutSorcs1  INS1 cell bands in subcellular fractionation immunoblots. *p 

    Journal: Scientific Reports

    Article Title: Type 2 diabetes-associated single nucleotide polymorphism in Sorcs1 gene results in alternative processing of the Sorcs1 protein in INS1 β-cells

    doi: 10.1038/s41598-019-55873-6

    Figure Lengend Snippet: The alternative 90 kDa protein product exhibits a different glycosylation profile. ( A ) Representative western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 1250 U or 2500 U Endo H. ( B ) Representative western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 500 U PNGase F. ( C ). Western blot of wtSorcs1 and mutSorcs1 INS1 cells after de-glycosylation treatment with 1250 U Endo H and 500 U PNGase F. ( D ) Representative western blot of subcellular fractionation by sucrose gradient of wtSorcs1 and mutSorcs1 -expressing INS1 cells. ( E ) Densitometry analysis of combined bands in subcellular fractionation immunoblots in wtSorcs1 and mutSorcs1 INS1 cells. ( F ) Densitometry analysis of 180, 130 and 90 kDa mutSorcs1 INS1 cell bands in subcellular fractionation immunoblots. *p 

    Article Snippet: For glycan cleavage analysis by western blot, WCL were treated with either 1250 U or 2500 U Endo H (NEB, P0702S) or 500 U PNGase F (NEB, P0709S) under denaturing conditions as per manufacturer’s protocols, after overnight induction of Sorcs1 expression prior to analysis by SDS-PAGE.

    Techniques: Western Blot, Fractionation, Expressing

    Sialylated vaginal epithelial N -linked glycans are depleted in BV. ( A ) Schematic for 2-amino benzamide (2AB) profiling of N- linked glycans by high performance anion exchange chromatography (HPAEC). Glycans were released using PNGase-F from protein extracts derived from VECs pools and fluorescently labelled with 2-AB prior to analysis by HPAEC. ( B ) HPAEC profiles of 2-AB labeled N -glycans derived from (i) protein standards, with well-known glycan structures, Rnase B and Bovine Fetuin, (ii) 2 pools of No BV VECs (n=10 specimens/pool), (ii) 2 pools of BV VECs (n=10 specimens/pool). ( C ) HPAEC profiles of N -glycans derived from (i) Rnase B and Bovine Fetuin, and (ii) pools of No BV VECs (same VEC pools as used in B , n=10 specimens/pool) pretreated with commercially available sialidase (+ A.u. sialidase, dotted line) or with buffer alone (- A.u . sialidase, solid line). Structures of different types of N -glycans (high mannose, mono- and di-sialylated) are depicted following the NCBI Symbol Nomenclature for Glycans (yellow circle, galactose; green circle, mannose; blue square, N -acetylglucosamine; purple diamond, sialic acid). A.u. sialidase = sialidase from Arthrobacter ureafaciens. A total of N=40 specimens were used to generate these data. VEC pools generated from these specimens were also used for studies reported in Fig. 2C, 2D and Fig. 4 . See methods for pooling rationale.

    Journal: medRxiv

    Article Title: Resident microbes shape the vaginal epithelial glycan landscape

    doi: 10.1101/2022.02.23.22271417

    Figure Lengend Snippet: Sialylated vaginal epithelial N -linked glycans are depleted in BV. ( A ) Schematic for 2-amino benzamide (2AB) profiling of N- linked glycans by high performance anion exchange chromatography (HPAEC). Glycans were released using PNGase-F from protein extracts derived from VECs pools and fluorescently labelled with 2-AB prior to analysis by HPAEC. ( B ) HPAEC profiles of 2-AB labeled N -glycans derived from (i) protein standards, with well-known glycan structures, Rnase B and Bovine Fetuin, (ii) 2 pools of No BV VECs (n=10 specimens/pool), (ii) 2 pools of BV VECs (n=10 specimens/pool). ( C ) HPAEC profiles of N -glycans derived from (i) Rnase B and Bovine Fetuin, and (ii) pools of No BV VECs (same VEC pools as used in B , n=10 specimens/pool) pretreated with commercially available sialidase (+ A.u. sialidase, dotted line) or with buffer alone (- A.u . sialidase, solid line). Structures of different types of N -glycans (high mannose, mono- and di-sialylated) are depicted following the NCBI Symbol Nomenclature for Glycans (yellow circle, galactose; green circle, mannose; blue square, N -acetylglucosamine; purple diamond, sialic acid). A.u. sialidase = sialidase from Arthrobacter ureafaciens. A total of N=40 specimens were used to generate these data. VEC pools generated from these specimens were also used for studies reported in Fig. 2C, 2D and Fig. 4 . See methods for pooling rationale.

    Article Snippet: Isolation and purification of N -glycans N -linked glycans were isolated from glycoproteins in the crude cell lysate by overnight incubation with glycerol free recombinant PNGase-F (P0709S, NEB) at 37 °C.

    Techniques: Chromatography, Derivative Assay, Labeling, Generated

    MALDI-TOF–MS of  Brugia malayi  adult worm (mixed sex)  N -linked glycans.  PNGase F-released glycans from  B. malayi  glycoproteins were labeled with 2-AA and analyzed using MALDI-TOF–MS in negative-ion reflectron mode. Monoisotopic masses of measured signals are indicated, and proposed glycan structures for ions with signal-to-noise ratios superior to 4 and intensities above 5000 are depicted using the Consortium for Functional Glycomics (CFG) nomenclature (see  symbol key inset ). Compositions and structures were deduced using a panel of glycan sequencing techniques in combination with MALDI-TOF–MS/MS fragmentation and information from the literature. 2-AA, 2-aminobenzoic acid; MS, mass spectrometry; MS/MS, tandem MS; PNGase F, peptide: N -glycosidase F.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Mass Spectrometric and Glycan Microarray–Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens

    doi: 10.1016/j.mcpro.2022.100201

    Figure Lengend Snippet: MALDI-TOF–MS of Brugia malayi adult worm (mixed sex) N -linked glycans. PNGase F-released glycans from B. malayi glycoproteins were labeled with 2-AA and analyzed using MALDI-TOF–MS in negative-ion reflectron mode. Monoisotopic masses of measured signals are indicated, and proposed glycan structures for ions with signal-to-noise ratios superior to 4 and intensities above 5000 are depicted using the Consortium for Functional Glycomics (CFG) nomenclature (see symbol key inset ). Compositions and structures were deduced using a panel of glycan sequencing techniques in combination with MALDI-TOF–MS/MS fragmentation and information from the literature. 2-AA, 2-aminobenzoic acid; MS, mass spectrometry; MS/MS, tandem MS; PNGase F, peptide: N -glycosidase F.

    Article Snippet: N-linked glycans were released using 3500 units of peptide:N-glycosidase F (PNGase F) (catalog no.: P0709; NEB) and incubated at 37 °C for 24 h.

    Techniques: Labeling, Functional Assay, Sequencing, Tandem Mass Spectroscopy, Mass Spectrometry