Endoglycosidase h

Manufactured by New England Biolabs

Sourced in United States

Endoglycosidase H is an enzyme that cleaves the linkage between the first and second N-acetylglucosamine residues of high mannose and some hybrid oligosaccharides from glycoproteins.

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Lab products found in correlation

Pngase f, by New England Biolabs (11 mentions) Peptide n glycosidase f, by New England Biolabs (5 mentions) Phosphor screen, by Fujifilm (4 mentions) Tyloxapol, by Merck Group (3 mentions) Monoq 10 100 gl, by GE Healthcare (3 mentions) Tran35s label, by PerkinElmer (3 mentions) Endo h, by New England Biolabs (3 mentions) Thrombin, by Merck Group (2 mentions) Complete protease inhibitor, by Roche (2 mentions) Beechwood xylan, by Megazyme (2 mentions) Acetone, by Merck Group (2 mentions) Gd3 gd3, by Matreya (2 mentions) Mini protean tetra cell, by Bio-Rad (2 mentions) Talon metal affinity resin, by Takara Bio (2 mentions) 35s methionine cysteine, by PerkinElmer (2 mentions) Protein a agarose, by Thermo Fisher Scientific (2 mentions) Ppiczαa, by Thermo Fisher Scientific (2 mentions) Typhoon fla 9000, by GE Healthcare (2 mentions) α2 3 6 8 9 neuraminidase a, by New England Biolabs (2 mentions) Glycoprotein stain, by Thermo Fisher Scientific (2 mentions)

85 protocols using endoglycosidase h

CD1a-Lipid Interaction Characterization

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Human CD1a/β2m dimers were expressed in human embryonic kidney (HEK) 293S GnTI− cells and purified as previously described^{18 (link)}. The lipid loading was performed at 20C overnight with C18:1 lysylPG (Avanti 840521) or 18:0–18:1 PG (Avanti 840503) solubilized in 0.5%CHAPS/20mM Tris pH 8, 150mM NaCl. Unbound lipid and detergent were separated from the protein by ion exchange chromatography using a MonoQ 10/100 GL column (GE Healthcare). For crystallization the expression tags were removed using thrombin (Sigma-Aldrich) and the protein was de-glycosylated with endoglycosidase H (New England BioLabs). For SPR, the protein was biotinylated using BirA ligase. The stability of lysylPG during lipid loading was assessed by incubating CD1a-endo with 10x molar excess of lysylPG or PG with different buffers (50mM citrate pH 5.5 or 50mM MES pH 5.5 or 20mM Tris pH 8) at 20C overnight. To confirm lysylPG stability once bound to CD1a CD1a-lysylPG was incubated at 20C overnight in 0.1M MMT buffer (MES, DL-Malic acid, Tris) at different pH (5 or 7 or 9). Lipid loading and degradation was assessed by isoelectric focusing where 1 μg of protein was run on a 5–8 PhastGel (Cytiva).

Fish D.N, & Abraham E. (1999). Pharmacokinetics of a Clarithromycin Suspension Administered via Nasogastric Tube to Seriously Ill Patients. Antimicrobial Agents and Chemotherapy, 43(5), 1277-1280.

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Pulse-chase analysis of PD-L1 maturation

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Cells were starved for 20 min in methionine-free, cysteine-free medium, labelled with [³⁵S]methionine/[³⁵S]cysteine) (Perkin-Elmer) for 30 min and then chased in medium containing excess cold methionine and cysteine (Sigma-Aldrich) at 37°C. Samples taken at the indicated time-points were lysed in 1% NP-40/TBS pH 7.6 with Roche complete protease inhibitor and 10mM iodoacetamide (IAA). Post-nuclear supernatants were pre-cleared with a combination of IgG-sepharose and Protein G Dynabeads for 1 to 2 h on a rotator at 4°C, and then incubated (rotating) with a PD-L1-specific antibody (AF156, R & D systems) for 1 h before adding Protein G Dynabeads for a further 2 h at 4°C. After 4 washes in 0.2% lysis buffer, samples were eluted in SDS sample buffer for 10 min at 70°C. Where indicated, eluates were treated with Endoglycosidase H (New England Biolabs) according to the manufacturer’s instructions before separation by SDS-PAGE. Gels were fixed, dried at 80°C for 2 h and processed for autoradiography. Phosphor screens were exposed to the radiolabelled proteins in fixed, dried gels overnight. Screens were scanned using a TYPHOON scanner and bands quantified using ImageJ.

Burr M.L., Sparbier C.E., Chan Y.C., Williamson J.C., Woods K., Beavis P.A., Lam E.Y., Henderson M.A., Bell C.C., Stolzenburg S., Gilan O., Bloor S., Noori T., Morgens D.W., Bassik M.C., Neeson P.J., Behren A., Darcy P.K., Dawson S.J., Voskoboinik I., Trapani J.A., Cebon J., Lehner P.J, & Dawson M.A. (2017). CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature, 549(7670), 101-105.

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Recombinant Pichia Protein Expression

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EasySelect™ Pichia Expression kit and pPICZα plasmid vectors were acquired from Invitrogen (Waltham, MA, USA). The restriction enzymes ClaI, XbaI, and SacI were purchased from Takara Bio Inc. (Kusatsu, Shiga Prefecture, Japan). Gel extraction and plasmid mini prep kits were both obtained from Macherey–Nagel (Düren, Germany). Beechwood xylan, wheat arabinoxylan, carob galactomannan, konjac glucomannan, barley β-glucan, and xylooligosaccharides (XOS) (DP 2–6) were purchased from Megazyme (Bray, Co. Wicklow, Ireland). Aldotetrauronic (terminal substitution, borohydride reduced) and aldopentauronic acids (internal substitution, borohydride reduced), and β-xylosidase from Bacillus pumilus were a kind gift from Megazyme. Endoglycosidase H was from New England Biolabs (Ipswich, MA, USA).

Katsimpouras C., Dedes G., Thomaidis N.S, & Topakas E. (2019). A novel fungal GH30 xylanase with xylobiohydrolase auxiliary activity. Biotechnology for Biofuels, 12, 120.

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Recombinant Expression and Purification of KIR3DL1

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The extracellular domains of KIR3DL1*001/*005/*015 (residues 1–299) were subcloned into vectors for insect cell and mammalian cell expression. For insect cell expression, the genes were subcloned into a modified baculoviral pFastBac-expression vector (Invitrogen) containing a secretion signal peptide sequence, an N-terminal hexa-histidine tag, and a C-terminal BirA tag (Stifter et al., 2014 (link)). For mammalian expression, the genes were subcloned into the pHLSec expression vector containing a secretion signal peptide sequence and an N-terminal hexa-histidine tag (Aricescu et al., 2006 (link)). Insect cell expression of KIR3DL1 was performed by baculoviral infection of BTI-TN-5B1-4 cells (Hi-5 cells; Invitrogen). Mammalian expression was performed by transient transfection of HEK293S GnTI⁻ cells (Aricescu et al., 2006 (link)). From both cell lines, KIR3DL1 was secreted into the expression media and purified by binding to nickel Sepharose resin followed by size exclusion chromatography (S200 16/60 column; GE Healthcare). The insect cell material was used for single-antigen bead experiments but was not amenable to crystallization. The mammalian cell material was used in x-ray crystallography experiments after overnight incubation with endoglycosidase H (New England Biolabs, Inc.).

Saunders P.M., Pymm P., Pietra G., Hughes V.A., Hitchen C., O’Connor G.M., Loiacono F., Widjaja J., Price D.A., Falco M., Mingari M.C., Moretta L., McVicar D.W., Rossjohn J., Brooks A.G, & Vivian J.P. (2016). Killer cell immunoglobulin-like receptor 3DL1 polymorphism defines distinct hierarchies of HLA class I recognition. The Journal of Experimental Medicine, 213(5), 791-807.

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Endoglycosidase-Based Glycoprotein Analysis

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Five hundred microlitres of pelleted cells was thawed on ice, mixed with 100 μl of 1% digitonin detergent and left to incubate for 45 minutes at 4°C. Detergent–cell mixtures were then centrifuged at 3,000g for 10 min at 4 °C. Supernatant was transferred into fresh Eppendorf tubes and centrifuged at 186,000g for 20 min at 4 °C. High-speed supernatant was then transferred into fresh Eppendorf tubes for a second time. Each sample was divided into four tubes; the first two tubes as the control and the other two tubes mixed with 0.5 μl (250 U) of endoglycosidase H (NEB P0702S) or PNGase F (NEB P0704S), respectively. Samples were left nutating overnight at 4 °C at optimum pH. The following day, all samples were centrifuged at 186,000g for 20 min at 4 °C, mixed with 5× sample loading buffer to a final concentration of 1× and run on a 4–20% SDS–PAGE gel with protein standard. Unstained gel was analysed using a fluorescent gel imager to detect GFP.

Choi W., Clemente N., Sun W., Du J, & Lü W. (2019). The structures and gating mechanism of human calcium homeostasis modulator 2. Nature, 576(7785), 163-167.

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Yeast Protein Extraction for Western Blot

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Yeast protein extracts were prepared for western blotting as previously described47 (link). Samples resolved by SDS-PAGE were transferred to nitrocellulose membranes and blotted using the following antibodies: monoclonal mouse anti-HA (1:2000 dilution, ThermoFisher, 32–6700) and HRP-conjugated horse anti-mouse IgG (1:2000 dilution, Cell Signaling, 7076S). Western blots were developed with Supersignal West Pico Chemiluminescent Substrate (Pierce). To monitor the acquisition of N-linked glycosylation, whole cell protein extracts were digested with 1 U of Endoglycosidase H (New England Biolabs) overnight at 37 °C. Samples were resolved by SDS-PAGE and used for western blotting, as described above.

Medus M.L., Gomez G.E., Zacchi L.F., Couto P.M., Labriola C.A., Labanda M.S., Bielsa R.C., Clérico E.M., Schulz B.L, & Caramelo J.J. (2017). N-glycosylation Triggers a Dual Selection Pressure in Eukaryotic Secretory Proteins. Scientific Reports, 7, 8788.

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Antibody Reagents and Cell Lines

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We purchased mannosidase II rabbit polyclonal antibodies (Affinity Bioreagents, Golden, CO), Rab43 mouse monoclonal antibodies (Abnova, Taipei City, Taiwan), Rab43 rabbit polyclonal antibodies (Atlas Antibodies, Stockholm, Sweden), VAMP4 rabbit polyclonal antibodies (Sigma-Aldrich, St. Louis, MO), and protein disulfide isomerase mouse monoclonal antibodies (ThermoFisher, Waltham, MA). V5 antibodies and the various Alexa Fluor–conjugated secondary antibodies were obtained from Invitrogen (Waltham, MA), and secondary antibodies and actin mouse monoclonal antibodies were obtained from LI-COR. Endoglycosidase H was purchased from New England Biolabs (Ipswich, MA). The polyclonal VSV (Mire et al., 2010 (link)), AE1 (Adair-Kirk et al., 1999 (link)), and I1 anti-G monoclonal antibodies (Lefrancois and Lyles, 1982 (link)) have been described. COS7, AD293, and PH5CH8 cells (Ikeda et al., 1998 (link)) were grown in DMEM containing 10% fetal calf serum.

Cox J.V., Kansal R, & Whitt M.A. (2016). Rab43 regulates the sorting of a subset of membrane protein cargo through the medial Golgi. Molecular Biology of the Cell, 27(11), 1834-1844.

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Enzymatic Preparation and Modification of Glycoproteins

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Yeast nitrogen base (YNB) was obtained from Sunrise Biosciences, and Bacto™ Tryptone and Yeast Extract was purchased from Becton Dickinson and Company (Franklin Lakes, NJ). Antifoam 204 was obtained from Sigma-Aldrich (St. Louis, MO). Certified ACS grade crystalline sucrose was purchased from Fisher Scientific (Pittsburg, PA). Boc-triglycine was supplied by Bachem Americas, Inc. (Torrance, CA). The enzymes PNGase F, Sortase, and B. thetaiotaomicron α-1,2-mannosidase (BT3990, B.t. α-1,2-mannosidase) were produced inhouse.^{52 (link)–55 (link)} Endoglycosidase H and restriction enzymes were obtained from New England Biolabs (Ipswich, MA). Protein G resin was produced by coupling protein G (recombinantly expressed in E. coli)^{56 (link)} with Sepharose^® CL-4B (Sigma-Aldrich, St Louis, MO) using divinyl sulfone as a coupling reagent.⁵⁷ General chemicals were purchased from Sigma-Aldrich and Fisher Scientific unless otherwise noted.

Okbazghi S.Z., More A.S., White D.R., Duan S., Shah I.S., Joshi S.B., Middaugh C.R., Volkin D.B, & Tolbert T.J. (2016). Production, characterization, and biological evaluation of well-defined IgG1 Fc glycoforms as a model system for biosimilarity analysis. Journal of pharmaceutical sciences, 105(2), 559-574.

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Purification and Loading of CD1a Glycoprotein

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The glycoprotein CD1a was expressed in a mammalian expression system and purified as previously described^{9 (link)}. Following an endoglycosidase H (New England BioLabs) treatment, the purified CD1a was first loaded with the ganglioside GD₃ (GD₃) (Matreya LLC) that was dissolved in a solution containing 0.5% tyloxapol (Sigma) and 10 mM Tris buffer at pH 8.0. CD1a was first incubated with GD₃ overnight at room temperature at a molar ratio of 1:15. The CD1a sample loaded with GD₃ was further purified using ion exchange chromatography (MonoQ 10/100 GL-GE Healthcare). Urushiol C15:2 (Chemos) was dissolved in a solution containing 10 mM Tris buffer at pH 8.0 / 0.5% tyloxapol / 50% acetone (Sigma). The GD₃–CD1a sample was then incubated overnight with urushiol at a 1:15 molar ratio and at room temperature in order to achieve the GD₃ displacement by urushiol. A subsequent purification step involving ion exchange chromatography (MonoQ 10/100 GL) was performed to remove the excess of urushiol C15:2, GD₃ –CD1a, and detergent.

Kim J.H., Hu Y., Yongqing T., Kim J., Hughes V.A., Nours J.L., Marquez E.A., Purcell A.W., Wan Q., Sugita M., Rossjohn J, & Winau F. (2016). CD1a on Langerhans cells controls inflammatory skin diseases. Nature immunology, 17(10), 1159-1166.

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Enzymatic Removal and Quantification of Yeast Cell Wall Glycans

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The procedure was performed as described previously.42 (link) Briefly, to remove N-linked glycans from the cell wall, yeast-like cells were incubated for 20 hours at 37°C with 25 U endoglycosidase H (New England Biolabs; Ipswich, MA, USA), whereas removal of O-linked glycans was carried out by resuspending cells in 1 N NaOH and gently shaking for 18 hours at room temperature. Glycan release was confirmed by HPAEC-PAD as described previously.43 (link) In both cases, the cells were pelleted by centrifuging, and the supernatants collected, lyophilized, and used to determine the sugar content with the phenol-sulfuric acid method.44

Lozoya-Pérez N.E., Casas-Flores S., de Almeida J.R., Martínez-Álvarez J.A., López-Ramírez L.A., Jannuzzi G.P., Trujillo-Esquivel E., Estrada-Mata E., Almeida S.R., Franco B., Lopes-Bezerra L.M, & Mora-Montes H.M. (2018). Silencing of OCH1 unveils the role of Sporothrix schenckii N-linked glycans during the host–fungus interaction. Infection and Drug Resistance, 12, 67-85.

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