endoglycosidaseh  (New England Biolabs)


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    O Glycosidase
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    O Glycosidase 10 000 000 units
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    New England Biolabs endoglycosidaseh
    O Glycosidase
    O Glycosidase 10 000 000 units
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    Average 99 stars, based on 1 article reviews
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    endoglycosidaseh - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis"

    Article Title: Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.198655

    The large N-terminal domain of the type I TMP VCAM1 results in a complete block of its membrane integration by mycolactone. (A) VCAM1 translated in the absence or presence of mycolactone (MYC) and treated with EndoglycosidaseH (EndoH). (B) VCAM1 and VCAM1 60 constructs (wild type and S707L/S707L* mutants) used in this study. (C) VCAM1 60 and a version containing an artificial N-glycosylation site (C52N) translated in the absence or presence of mycolactone, without or without EndoH. (D) VCAM1 60 and a variant with a more hydrophobic TMD (VCAM1 60 S707L* ) translated in the absence or presence of mycolactone, without or without subsequent EndoH treatment. Estimated TMD hydrophobicities (kcal/mol) are indicated in D. Graph shows the reduction in the amount of ‘+g’ VCAM1 60 and VCAM1 60 S707L* in the presence of mycolactone, relative to control samples, as described in the legend to Fig. 1 . is also shown in D (graph). The statistical test performed was one-way ANOVA. Error bars show mean±s.d. VCAM1, n =3 VCAM1 60 , n =4; VCAM1 60 S707L* , n =3). P -values are as defined in Fig. 1 legend. (E) Translation of VCAM1, VCAM1 60 and the secretory protein cecropin, possessing a C-terminal opsin tag (CecOPG2), performed with increasing concentrations of CAM741 or an equivalent volume of DMSO (‘−’). (F) VCAM1 60 and VCAM1 60 S707L* translated in the absence or presence of 250 nM CAM741. Other symbols are as defined in Fig. 1 legend.
    Figure Legend Snippet: The large N-terminal domain of the type I TMP VCAM1 results in a complete block of its membrane integration by mycolactone. (A) VCAM1 translated in the absence or presence of mycolactone (MYC) and treated with EndoglycosidaseH (EndoH). (B) VCAM1 and VCAM1 60 constructs (wild type and S707L/S707L* mutants) used in this study. (C) VCAM1 60 and a version containing an artificial N-glycosylation site (C52N) translated in the absence or presence of mycolactone, without or without EndoH. (D) VCAM1 60 and a variant with a more hydrophobic TMD (VCAM1 60 S707L* ) translated in the absence or presence of mycolactone, without or without subsequent EndoH treatment. Estimated TMD hydrophobicities (kcal/mol) are indicated in D. Graph shows the reduction in the amount of ‘+g’ VCAM1 60 and VCAM1 60 S707L* in the presence of mycolactone, relative to control samples, as described in the legend to Fig. 1 . is also shown in D (graph). The statistical test performed was one-way ANOVA. Error bars show mean±s.d. VCAM1, n =3 VCAM1 60 , n =4; VCAM1 60 S707L* , n =3). P -values are as defined in Fig. 1 legend. (E) Translation of VCAM1, VCAM1 60 and the secretory protein cecropin, possessing a C-terminal opsin tag (CecOPG2), performed with increasing concentrations of CAM741 or an equivalent volume of DMSO (‘−’). (F) VCAM1 60 and VCAM1 60 S707L* translated in the absence or presence of 250 nM CAM741. Other symbols are as defined in Fig. 1 legend.

    Techniques Used: Blocking Assay, Construct, Variant Assay

    ER integration of the type I TMP CD3δ in the presence of mycolactone is driven by its TMD. (A) Truncated mRNAs coding for CD3δ (top panel) and CD3δ D111L (bottom panel) and lacking stop codons translated in the absence or presence of mycolactone (MYC) without puromycin-mediated release. The nascent chain length of each truncation is shown, as well as the number of residues synthesised C-terminal to the TMD to provide an estimate of its distance from the peptidyl-transferase centre (PTC) of the ribosome. Truncations where all or part of the TMD is likely obscured by the ribosomal exit tunnel (based on Cabrita et al., 2016 ) are indicated by the bracketed area. CD3δ 158 is encompassed by a dashed bracket, since its TMD is likely on the border of having just fully emerged from the ribosomal exit tunnel. Arrowheads indicate maximal glycosylation resulting from the TMD-dependent rescue of integration in the presence of mycolactone. (B) Versions of (i) CD3δ and (ii) CD3δ D111L lacking signal sequences (ΔSS) translated in the absence and presence of mycolactone, without or without subsequent EndoglycosidaseH (EndoH) treatment. (C) Predicted mechanism of type I TMP integration in the absence (i) and presence (ii) of mycolactone. Other symbols are as defined in Fig. 1 legend. ‘+g’, glycosylated; ‘0g’, non-glycosylated; ‘C’, C-terminus; FL, full length; ‘N’, N-terminus.
    Figure Legend Snippet: ER integration of the type I TMP CD3δ in the presence of mycolactone is driven by its TMD. (A) Truncated mRNAs coding for CD3δ (top panel) and CD3δ D111L (bottom panel) and lacking stop codons translated in the absence or presence of mycolactone (MYC) without puromycin-mediated release. The nascent chain length of each truncation is shown, as well as the number of residues synthesised C-terminal to the TMD to provide an estimate of its distance from the peptidyl-transferase centre (PTC) of the ribosome. Truncations where all or part of the TMD is likely obscured by the ribosomal exit tunnel (based on Cabrita et al., 2016 ) are indicated by the bracketed area. CD3δ 158 is encompassed by a dashed bracket, since its TMD is likely on the border of having just fully emerged from the ribosomal exit tunnel. Arrowheads indicate maximal glycosylation resulting from the TMD-dependent rescue of integration in the presence of mycolactone. (B) Versions of (i) CD3δ and (ii) CD3δ D111L lacking signal sequences (ΔSS) translated in the absence and presence of mycolactone, without or without subsequent EndoglycosidaseH (EndoH) treatment. (C) Predicted mechanism of type I TMP integration in the absence (i) and presence (ii) of mycolactone. Other symbols are as defined in Fig. 1 legend. ‘+g’, glycosylated; ‘0g’, non-glycosylated; ‘C’, C-terminus; FL, full length; ‘N’, N-terminus.

    Techniques Used:

    Mycolactone does not interfere with type III TMP integration. (A) Translation of GypC in the absence or presence of mycolactone (MYC), followed by subsequent treatment with EndoglycosidaseH (EndoH). (B) Graph shows change in the amount of glycosylated (+g) GypC and related constructs in the presence of mycolactone, relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. GypC, n =10; others, n =3. Ns, not significant. (C) Estimated TMD hydrophobicities (kcal/mol) of GypC and related constructs. (D) Translation of two variants of GypC with reduced TMD hydrophobicity. (E) GypC truncations lacking stop codons. For crosslinking experiments, truncations contained a single artificially introduced cysteine residue at either position 52 or 84, as denoted by an asterisk. (F) Truncated GypC chains synthesised in the absence or presence of mycolactone without puromycin-mediated release. The glycosylation of nascent chains when still attached to the ribosome (indicated by ‘peptRNA’) was observed. (G) Truncated GypC chains containing a single cysteine residue [either *(52) or *(84)] synthesised in the absence or presence of mycolactone without puromycin-mediated release to generate membrane integration intermediates. Samples were treated with the crosslinking reagent BMH, subjected to extraction with alkaline sodium carbonate, and analysed by SDS-PAGE. Adducts between the nascent chain and Sec61β (xSec61β) or the nascent chain and Sec61α/Sec61α and Sec61β (xSec61α/αβ) are indicated (see also Fig. S3B ). Mycolactone-sensitive adducts are indicated by arrowheads. Other symbols are as defined in Fig. 1 legend. FL, full length.
    Figure Legend Snippet: Mycolactone does not interfere with type III TMP integration. (A) Translation of GypC in the absence or presence of mycolactone (MYC), followed by subsequent treatment with EndoglycosidaseH (EndoH). (B) Graph shows change in the amount of glycosylated (+g) GypC and related constructs in the presence of mycolactone, relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. GypC, n =10; others, n =3. Ns, not significant. (C) Estimated TMD hydrophobicities (kcal/mol) of GypC and related constructs. (D) Translation of two variants of GypC with reduced TMD hydrophobicity. (E) GypC truncations lacking stop codons. For crosslinking experiments, truncations contained a single artificially introduced cysteine residue at either position 52 or 84, as denoted by an asterisk. (F) Truncated GypC chains synthesised in the absence or presence of mycolactone without puromycin-mediated release. The glycosylation of nascent chains when still attached to the ribosome (indicated by ‘peptRNA’) was observed. (G) Truncated GypC chains containing a single cysteine residue [either *(52) or *(84)] synthesised in the absence or presence of mycolactone without puromycin-mediated release to generate membrane integration intermediates. Samples were treated with the crosslinking reagent BMH, subjected to extraction with alkaline sodium carbonate, and analysed by SDS-PAGE. Adducts between the nascent chain and Sec61β (xSec61β) or the nascent chain and Sec61α/Sec61α and Sec61β (xSec61α/αβ) are indicated (see also Fig. S3B ). Mycolactone-sensitive adducts are indicated by arrowheads. Other symbols are as defined in Fig. 1 legend. FL, full length.

    Techniques Used: Construct, SDS Page

    Mycolactone traps headfirst-inserting type II TMPs in an N-lumenal–C-cytosolic topology. (A) ASGPR H1 and ASGPR H1Δ. Translation of ASGPR H1 (B) and ASGPR H1Δ (C) performed in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Membrane fractions were subjected to extraction with alkaline sodium carbonate prior to analysis. (D) Graph shows the amount of glycosylated (‘+g’) and non-glycosylated (‘0g’) ASGPR H1 and ASGPR H1Δ in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ or ‘0g’ substrate obtained in the presence of mycolactone by the quantity of '+g' or '0g' substrate obtained in the absence of mycolactone and are expressed as percentages. Dashed red line represents the value for comparative material for samples treated with a vehicle control. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. (E) Diagram showing type II TMPs that insert using a hairpin mechanism (i) or a headfirst/inversion mechanism (ii), as well as the headfirst insertion of type III TMPs (iii). Faded steps represent those that are prevented by mycolactone. Dashed arrow shows the predicted route taken by headfirst-inserting type II TMPs when inversion is prevented by mycolactone.
    Figure Legend Snippet: Mycolactone traps headfirst-inserting type II TMPs in an N-lumenal–C-cytosolic topology. (A) ASGPR H1 and ASGPR H1Δ. Translation of ASGPR H1 (B) and ASGPR H1Δ (C) performed in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Membrane fractions were subjected to extraction with alkaline sodium carbonate prior to analysis. (D) Graph shows the amount of glycosylated (‘+g’) and non-glycosylated (‘0g’) ASGPR H1 and ASGPR H1Δ in the presence of mycolactone, relative to control samples. These values were determined by dividing the quantity of ‘+g’ or ‘0g’ substrate obtained in the presence of mycolactone by the quantity of '+g' or '0g' substrate obtained in the absence of mycolactone and are expressed as percentages. Dashed red line represents the value for comparative material for samples treated with a vehicle control. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. (E) Diagram showing type II TMPs that insert using a hairpin mechanism (i) or a headfirst/inversion mechanism (ii), as well as the headfirst insertion of type III TMPs (iii). Faded steps represent those that are prevented by mycolactone. Dashed arrow shows the predicted route taken by headfirst-inserting type II TMPs when inversion is prevented by mycolactone.

    Techniques Used:

    Mycolactone sensitivity is dependent upon which TMD-flanking region is translocated. (A) A chimeric protein containing Ii downstream of a pre-prolactin (PPL) signal sequence (i) and the two topologies it might assume following integration into RMs, depending on whether the region that is translocated is N-terminal (ii) or C-terminal (iii) of the TMD. (B) Translation of PPL-Ii and PPL-Ii G47L Q48L* in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Samples were analysed following immunoprecipitation of Ii. (C) Graph showing the amount of signal-cleaved (‘sc’) or glycosylated (‘+g’) substrate in the presence of mycolactone relative to control samples. These values were determined by dividing the quantity of ‘sc’ or ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘sc’ or ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values and other symbols are as defined in Fig. 1 legend.
    Figure Legend Snippet: Mycolactone sensitivity is dependent upon which TMD-flanking region is translocated. (A) A chimeric protein containing Ii downstream of a pre-prolactin (PPL) signal sequence (i) and the two topologies it might assume following integration into RMs, depending on whether the region that is translocated is N-terminal (ii) or C-terminal (iii) of the TMD. (B) Translation of PPL-Ii and PPL-Ii G47L Q48L* in the absence or presence of mycolactone (MYC), followed by treatment with EndoglycosidaseH (EndoH). Samples were analysed following immunoprecipitation of Ii. (C) Graph showing the amount of signal-cleaved (‘sc’) or glycosylated (‘+g’) substrate in the presence of mycolactone relative to control samples. These values were determined by dividing the quantity of ‘sc’ or ‘+g’ substrate obtained in the presence of mycolactone by the quantity of ‘sc’ or ‘+g’ substrate obtained in the absence of mycolactone and are expressed as percentages. The statistical test performed was two-way ANOVA. Error bars show mean±s.d. ( n =3). P -values and other symbols are as defined in Fig. 1 legend.

    Techniques Used: Sequencing, Immunoprecipitation

    Mycolactone efficiently blocks type II TMP integration. (A) Full-length Ii (wild type and G47L Q48L mutant) and the Ii 125 truncation used in this study. (B) Estimated TMD hydrophobicities (kcal/mol) of Ii and Ii G47L Q48L . (C) Graph shows the reduction in the amount of glycosylated (+g) Ii and related constructs in the presence of mycolactone (MYC), relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. Translation in the absence or presence of mycolactone performed using Ii (D), Ii G47L Q48L (E) and Ii 125 (F), which was followed by treatment with EndoglycosidaseH (EndoH). (G) Ii truncations used in this study. For crosslinking experiments, truncations contained either a native cysteine residue (C28) or one that was artificially introduced [*(50)]. A truncated version of TNFα used for crosslinking analysis (as described in MacKinnon et al., 2014 ) is shown for comparative purposes. Crosslinking was performed on Ii truncations (H) and Ii 125 *(50) (I) and the resulting adducts are labelled as described in the Fig. 4 G legend. Other symbols are as defined in Fig. 1 legend. Puro, puromycin.
    Figure Legend Snippet: Mycolactone efficiently blocks type II TMP integration. (A) Full-length Ii (wild type and G47L Q48L mutant) and the Ii 125 truncation used in this study. (B) Estimated TMD hydrophobicities (kcal/mol) of Ii and Ii G47L Q48L . (C) Graph shows the reduction in the amount of glycosylated (+g) Ii and related constructs in the presence of mycolactone (MYC), relative to control samples as described in the legend to Fig. 1 . The statistical test performed was one-way ANOVA. Error bars show mean±s.d. ( n =3). P -values are as defined in Fig. 1 legend. Translation in the absence or presence of mycolactone performed using Ii (D), Ii G47L Q48L (E) and Ii 125 (F), which was followed by treatment with EndoglycosidaseH (EndoH). (G) Ii truncations used in this study. For crosslinking experiments, truncations contained either a native cysteine residue (C28) or one that was artificially introduced [*(50)]. A truncated version of TNFα used for crosslinking analysis (as described in MacKinnon et al., 2014 ) is shown for comparative purposes. Crosslinking was performed on Ii truncations (H) and Ii 125 *(50) (I) and the resulting adducts are labelled as described in the Fig. 4 G legend. Other symbols are as defined in Fig. 1 legend. Puro, puromycin.

    Techniques Used: Mutagenesis, Construct

    2) Product Images from "Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes"

    Article Title: Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0009071

    Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose N -glycans. (A) Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumoniae in E . coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analyzed by HILIC-UHPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different N -glycans are listed in Table 1 . (B) Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures in Table 1 . The dagger symbol (‡) refers to m/z 1130.55 as [M+2H] 2+ ion; the appearance of the Man 3 GlcNAc 2 -Proc as singly and doubly charged ion in positive mode, reflects on its high relative abundancy (~54%) in this sample. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. GU, glucose homopolymer ladder. [ 22 ].
    Figure Legend Snippet: Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose N -glycans. (A) Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumoniae in E . coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analyzed by HILIC-UHPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different N -glycans are listed in Table 1 . (B) Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures in Table 1 . The dagger symbol (‡) refers to m/z 1130.55 as [M+2H] 2+ ion; the appearance of the Man 3 GlcNAc 2 -Proc as singly and doubly charged ion in positive mode, reflects on its high relative abundancy (~54%) in this sample. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide. GU, glucose homopolymer ladder. [ 22 ].

    Techniques Used: Labeling, Incubation, Hydrophilic Interaction Liquid Chromatography

    3) Product Images from "Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)"

    Article Title: Calnexin-Assisted Biogenesis of the Neuronal Glycine Transporter 2 (GlyT2)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063230

    GlyT2 expression after a pulse-chased in culture cells. COS7 cells transfected with GlyT2 cDNA in pCDNA3 were pulse-labeled for 15 min with [ 35 S]methionine/cysteine and chased for the times indicated in the conditions given in Material and Methods. The cells were then surface biotinylated, lysed and the protein lysate was either immunoprecipitated with GlyT2 antibody (total transporter, A) or bound to streptavidin-agarose and sequentially immunoprecipitated with GlyT2 antibody (biotinylated fraction, B). Proteins extracted from the beads were resolved in SDS-PAGE. (A) Kinetics of GlyT2 expression. (B) Kinetics of GlyT2 plasma membrane expression in the same cells as in A. Lower panels: (A) densitometry of the 100 kDa and 75 kDa bands in the fluorograms. (B) Biotinylated bands are represented as a percentage of each of the lanes labeled in A. Bars represent the S.E.M. (n = 3). (C) Cells were treated overnight with the vehicle alone (DMSO) or with 10 µg/ml tunicamycin, and then pulse-chased as described above. (D, E) Immunoprecipitates were treated overnight with the vehicle alone (endoglycosidase buffer, -) or with the indicated endoglycosidase (+) in denaturing conditions, and then resolved by SDS-PAGE as described in the Materials and Methods. The transporter proteins (100 kDa, 75 kDa and 60 kDa) are indicated with arrowheads.
    Figure Legend Snippet: GlyT2 expression after a pulse-chased in culture cells. COS7 cells transfected with GlyT2 cDNA in pCDNA3 were pulse-labeled for 15 min with [ 35 S]methionine/cysteine and chased for the times indicated in the conditions given in Material and Methods. The cells were then surface biotinylated, lysed and the protein lysate was either immunoprecipitated with GlyT2 antibody (total transporter, A) or bound to streptavidin-agarose and sequentially immunoprecipitated with GlyT2 antibody (biotinylated fraction, B). Proteins extracted from the beads were resolved in SDS-PAGE. (A) Kinetics of GlyT2 expression. (B) Kinetics of GlyT2 plasma membrane expression in the same cells as in A. Lower panels: (A) densitometry of the 100 kDa and 75 kDa bands in the fluorograms. (B) Biotinylated bands are represented as a percentage of each of the lanes labeled in A. Bars represent the S.E.M. (n = 3). (C) Cells were treated overnight with the vehicle alone (DMSO) or with 10 µg/ml tunicamycin, and then pulse-chased as described above. (D, E) Immunoprecipitates were treated overnight with the vehicle alone (endoglycosidase buffer, -) or with the indicated endoglycosidase (+) in denaturing conditions, and then resolved by SDS-PAGE as described in the Materials and Methods. The transporter proteins (100 kDa, 75 kDa and 60 kDa) are indicated with arrowheads.

    Techniques Used: Expressing, Transfection, Labeling, Immunoprecipitation, SDS Page

    4) Product Images from "Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes"

    Article Title: Tsetse salivary glycoproteins are modified with paucimannosidic N-glycans, are recognised by C-type lectins and bind to trypanosomes

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.172007

    Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose glycans. [A] Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumonia in E. coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analysed by HILIC-(U)HPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different glycans are listed in Table 1 . [B] Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures shown in Table 1 . Asterisk (*) refers to m/z 1130.55 as [M+2H] 2+ ion. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.
    Figure Legend Snippet: Tsetse fly salivary glycoproteins are composed mainly of paucimannose and oligomannose glycans. [A] Profile of salivary N -glycans from teneral (young, unfed) flies, before and after digestion with exoglycosidases. Aliquots of the total PNGase F-released 2-AB-labeled N -glycan pool were either undigested (i) or incubated with a range of exoglycosidases (ii-iv). (i) Undig, before digestion; (ii) GUH, Streptococcus pneumonia in E. coli β-N-acetylglucosaminidase; (iii) JBM, Jack bean α-Mannosidase; (iv) bkF, Bovine kidney α-fucosidase. Following digestion, the products were analysed by HILIC-(U)HPLC. Peaks labelled A correspond to the product of complete digestion with JBM; those labelled with an asterisk refer to buffer contaminants. The percent areas and structures of the different glycans are listed in Table 1 . [B] Positive-ion ESI-MS spectrum of procainamide-labelled N -glycans from teneral tsetse fly saliva. Numbers refer to the structures shown in Table 1 . Asterisk (*) refers to m/z 1130.55 as [M+2H] 2+ ion. Green circle, mannose; blue square, N -Acetylglucosamine; red triangle, fucose; Proc, procainamide.

    Techniques Used: Labeling, Incubation, Hydrophilic Interaction Liquid Chromatography

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    other:

    Article Title: Two Modes of Regulation of the Fatty Acid Elongase ELOVL6 by the 3-Ketoacyl-CoA Reductase KAR in the Fatty Acid Elongation Cycle
    Article Snippet: Deglycosylation of Proteins Endoglycosidase H (Endo H) and peptide: N -glycosidase F (PNGase F) were purchased from New England Biolabs (Beverly, MA).

    Article Title: p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition
    Article Snippet: These cell lysates were used for deglycosylation reactions with the enzyme Endoglycosidase H for 1 hr at 37°C as per the manufacturer’s recommendations (New England Biolabs, Ipswich, MA).

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    New England Biolabs enzyme endoglycosidase h
    Different forms of Nrf1. HEK-293 cells stably expressing wild-type Nrf1 3×Flag were treated with MG132 and/or cotransin, an inhibitor of protein insertion into the Sec61 translocation channel ( Garrison et al., 2005 ) as indicated (lanes 1 to 4) and total cell lysates were prepared. Lanes 5 and 6 contain full-length Nrf1 3×Flag and Nrf1(104-742) 3×Flag that were translated in vitro (IVT) in rabbit reticulocyte lysate in the absence of membranes. The HEK293 cell lysates and IVT reactions were examined by SDS-PAGE followed by immunoblotting with anti-Flag antibody. Different Nrf1 species are shown (ungly–unglycosylated; unmod–unmodified). Nrf1 p120 (species ‘a’) was converted to species ‘c’, which comigrated with the primary translation product (species ‘d’) upon expression in cells treated with cotransin, suggesting that the slow mobility of p120 arose from modifications (e.g., N-linked glycosylation) that occurred within the endoplasmic reticulum. Retrotranslocation and processing of p120 (species ‘a’) yielded p110 (species ‘b’). Species ‘b’ was not sensitive to <t>endoglycosidase</t> H ( Figure 4E ), suggesting that it was deglycosylated upon retrotranslocation into the cytosol. Nevertheless, species ‘b’ migrated considerably more slowly than the primary translation product for Nrf1(104-742) 3×Flag (species ‘e’), indicating that p110 must carry additional modifications that remain uncharacterized. Please note that deglycosylation by cytosolic enzymes converts the Asn at the site of glycosylation to Asp, which could influence migration on SDS-PAGE. DOI: http://dx.doi.org/10.7554/eLife.01856.007
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    Different forms of Nrf1. HEK-293 cells stably expressing wild-type Nrf1 3×Flag were treated with MG132 and/or cotransin, an inhibitor of protein insertion into the Sec61 translocation channel ( Garrison et al., 2005 ) as indicated (lanes 1 to 4) and total cell lysates were prepared. Lanes 5 and 6 contain full-length Nrf1 3×Flag and Nrf1(104-742) 3×Flag that were translated in vitro (IVT) in rabbit reticulocyte lysate in the absence of membranes. The HEK293 cell lysates and IVT reactions were examined by SDS-PAGE followed by immunoblotting with anti-Flag antibody. Different Nrf1 species are shown (ungly–unglycosylated; unmod–unmodified). Nrf1 p120 (species ‘a’) was converted to species ‘c’, which comigrated with the primary translation product (species ‘d’) upon expression in cells treated with cotransin, suggesting that the slow mobility of p120 arose from modifications (e.g., N-linked glycosylation) that occurred within the endoplasmic reticulum. Retrotranslocation and processing of p120 (species ‘a’) yielded p110 (species ‘b’). Species ‘b’ was not sensitive to endoglycosidase H ( Figure 4E ), suggesting that it was deglycosylated upon retrotranslocation into the cytosol. Nevertheless, species ‘b’ migrated considerably more slowly than the primary translation product for Nrf1(104-742) 3×Flag (species ‘e’), indicating that p110 must carry additional modifications that remain uncharacterized. Please note that deglycosylation by cytosolic enzymes converts the Asn at the site of glycosylation to Asp, which could influence migration on SDS-PAGE. DOI: http://dx.doi.org/10.7554/eLife.01856.007

    Journal: eLife

    Article Title: p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition

    doi: 10.7554/eLife.01856

    Figure Lengend Snippet: Different forms of Nrf1. HEK-293 cells stably expressing wild-type Nrf1 3×Flag were treated with MG132 and/or cotransin, an inhibitor of protein insertion into the Sec61 translocation channel ( Garrison et al., 2005 ) as indicated (lanes 1 to 4) and total cell lysates were prepared. Lanes 5 and 6 contain full-length Nrf1 3×Flag and Nrf1(104-742) 3×Flag that were translated in vitro (IVT) in rabbit reticulocyte lysate in the absence of membranes. The HEK293 cell lysates and IVT reactions were examined by SDS-PAGE followed by immunoblotting with anti-Flag antibody. Different Nrf1 species are shown (ungly–unglycosylated; unmod–unmodified). Nrf1 p120 (species ‘a’) was converted to species ‘c’, which comigrated with the primary translation product (species ‘d’) upon expression in cells treated with cotransin, suggesting that the slow mobility of p120 arose from modifications (e.g., N-linked glycosylation) that occurred within the endoplasmic reticulum. Retrotranslocation and processing of p120 (species ‘a’) yielded p110 (species ‘b’). Species ‘b’ was not sensitive to endoglycosidase H ( Figure 4E ), suggesting that it was deglycosylated upon retrotranslocation into the cytosol. Nevertheless, species ‘b’ migrated considerably more slowly than the primary translation product for Nrf1(104-742) 3×Flag (species ‘e’), indicating that p110 must carry additional modifications that remain uncharacterized. Please note that deglycosylation by cytosolic enzymes converts the Asn at the site of glycosylation to Asp, which could influence migration on SDS-PAGE. DOI: http://dx.doi.org/10.7554/eLife.01856.007

    Article Snippet: These cell lysates were used for deglycosylation reactions with the enzyme Endoglycosidase H for 1 hr at 37°C as per the manufacturer’s recommendations (New England Biolabs, Ipswich, MA).

    Techniques: Stable Transfection, Expressing, Translocation Assay, In Vitro, SDS Page, Migration

    Effects of US3 on class I and class II synthesis and maturation. His16 cells were left uninfected (UN; A to C) or were infected with AdtetUS3 and Adtet-trans at 100 and 20 or 200 and 40 (A and C) or 150 and 30 PFU/cell (B), respectively, for 18 h. Infected cells were labeled with [ 35 S]methionine-cysteine in a pulse-chase format. DR-α and DR-β (A), Ii (B), and MHC-I HC (C) were immunoprecipitated from cell extracts with MAb DA6.147, HB10A, and PIN.1, and rabbit anti-HC serum, respectively. (D) His16 cells were infected with Adtet-trans alone at 120 PFU/cell, AdtetUS2 and Adtet-trans at 100 and 20 PFU/cell, respectively, or AdtetUS3 and Adtet-trans at 100 and 20 PFU/cell, respectively. The cells were labeled for 12 min, the label was chased for 120 min, and then DM-α and DM-β were immunoprecipitated with MAbs 5C1 and MaP.DMB/C, respectively. For endo H treatment, precipitated proteins were divided in half and treated with endo H (+) or not treated (−). The proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. r, endo H-resistant species of DM; s, endo H-susceptible species of DM.

    Journal: Journal of Virology

    Article Title: Inhibition of HLA-DR Assembly, Transport, and Loading by Human Cytomegalovirus Glycoprotein US3: a Novel Mechanism for Evading Major Histocompatibility Complex Class II Antigen Presentation

    doi: 10.1128/JVI.76.21.10929-10941.2002

    Figure Lengend Snippet: Effects of US3 on class I and class II synthesis and maturation. His16 cells were left uninfected (UN; A to C) or were infected with AdtetUS3 and Adtet-trans at 100 and 20 or 200 and 40 (A and C) or 150 and 30 PFU/cell (B), respectively, for 18 h. Infected cells were labeled with [ 35 S]methionine-cysteine in a pulse-chase format. DR-α and DR-β (A), Ii (B), and MHC-I HC (C) were immunoprecipitated from cell extracts with MAb DA6.147, HB10A, and PIN.1, and rabbit anti-HC serum, respectively. (D) His16 cells were infected with Adtet-trans alone at 120 PFU/cell, AdtetUS2 and Adtet-trans at 100 and 20 PFU/cell, respectively, or AdtetUS3 and Adtet-trans at 100 and 20 PFU/cell, respectively. The cells were labeled for 12 min, the label was chased for 120 min, and then DM-α and DM-β were immunoprecipitated with MAbs 5C1 and MaP.DMB/C, respectively. For endo H treatment, precipitated proteins were divided in half and treated with endo H (+) or not treated (−). The proteins were subjected to SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. r, endo H-resistant species of DM; s, endo H-susceptible species of DM.

    Article Snippet: Endoglycosidase H (endo H) analyses were carried out with enzyme preparations and protocols supplied by New England Biolabs (Boston, Mass.).

    Techniques: Infection, Labeling, Pulse Chase, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Effects of glycanase treatment of domestic cat and dog TfRs and cells on CPV and FPV infectivity. (A) Cleavage site (arrow) of endoglycosidase H (endo H), located between the chitobiose core of GlcNAc 2 residues in high-mannose and hybrid N-glycans. The

    Journal: Journal of Virology

    Article Title: Single Mutations in the VP2 300 Loop Region of the Three-Fold Spike of the Carnivore Parvovirus Capsid Can Determine Host Range

    doi: 10.1128/JVI.02636-15

    Figure Lengend Snippet: Effects of glycanase treatment of domestic cat and dog TfRs and cells on CPV and FPV infectivity. (A) Cleavage site (arrow) of endoglycosidase H (endo H), located between the chitobiose core of GlcNAc 2 residues in high-mannose and hybrid N-glycans. The

    Article Snippet: To test if endoglycosidase H (endo H) treatment would specifically remove glycans from the dog and cat TfRs, we incubated 13.5 μg of FPLC-purified, denatured TfR with 4,000 U of endo Hf (New England BioLabs, Ipswich, MA) overnight according to the manufacturer's protocols and then analyzed the electrophoretic mobility by SDS-PAGE.

    Techniques: Infection

    SDS-PAGE gels of the IMAC-purified Tt PPO. Samples were run under either denaturing (A) or native (B) conditions. Lane M, prestained protein marker; lanes 1A and 1B, untreated Tt PPO; lanes 2A and 2B, endoglycosidase H-treated Tt PPO.

    Journal: Applied and Environmental Microbiology

    Article Title: Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

    doi: 10.1128/AEM.01628-18

    Figure Lengend Snippet: SDS-PAGE gels of the IMAC-purified Tt PPO. Samples were run under either denaturing (A) or native (B) conditions. Lane M, prestained protein marker; lanes 1A and 1B, untreated Tt PPO; lanes 2A and 2B, endoglycosidase H-treated Tt PPO.

    Article Snippet: In order to investigate potential N -glycosylation on the recombinant protein, endoglycosidase treatment was performed by Endo H enzyme (NEB, USA) under native conditions, according to the manufacturer's manual.

    Techniques: SDS Page, Purification, Marker