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N glycanase

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N-glycanase is an enzyme that cleaves the bond between the asparagine residue and N-linked glycans in glycoproteins. It is commonly used in the study and analysis of protein glycosylation patterns.

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Lab products found in correlation

Pi plc, by Merck Group (2 mentions) Acquity uplc, by Thermo Fisher Scientific (2 mentions) Ltq ot, by Thermo Fisher Scientific (2 mentions) Lys c protease, by Thermo Fisher Scientific (2 mentions) O glycanase, by New England Biolabs (1 mentions) Aminolink plus coupling gel, by Thermo Fisher Scientific (1 mentions) Endoglycosidase h, by New England Biolabs (1 mentions) Endo h, by New England Biolabs (1 mentions)

6 protocols using n glycanase

1

Characterization of LAMP1 Glycosylation

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Whole cerebellum protein lysate was extracted, as described above, from age-matched 7-week wild type (Npc1+/+) and mutant (Npc1-/-) mice. N-glycanase, an asparagine amidase that cleaves the Asn-GlcNac bond of N-glycans, and Endoglycosidase H (Endo H), a glycosidase that cleaves within the chitobiose core of complex mannose and some hybrid oligosaccharides (New England Biolabs, Inc., Ipswich, MA, USA), were used to characterize the glycosylation complexity of LAMP1. Cerebellum protein lysates were treated with and without N-glycanase or Endo H in duplicates, according to the kit protocol (New England Biolabs). The treated lysates were analyzed by Western blot for LAMP1.
Cawley N.X., Sojka C., Cougnoux A., Lyons A.T., Nicoli E., Wassif C.A, & Porter F.D. (2020). Abnormal LAMP1 glycosylation may play a role in Niemann-Pick disease, type C pathology. PLoS ONE, 15(1), e0227829.
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2

Deglycosylation and Hydrolase Binding Assay of OMC45

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OMC45 polypeptide was treated with N-glycanase and O-glycanase separately following the manufacturer’s instructions. Both N-glycanase and O-glycanase were purchased from New England Biolabs., MA. After enzymatic digestion, deglycosylated OMC45 polypeptide was analzed by Western blot stained with anti-OMC45.
For the hydrolase binding experiments, the homogenous fraction of OMC45 polypeptide was first treated with N-glycanase (to remove all N-linked oligosaccharide moieties) and the resulting OMC45 polypeptide was treated with O-glycanase (an endoenzyme known to cleave O-linked OS chains). Both incubations were done overnight at 37°C according to the New England Biolabs’ instructions. After enzymatic digestion, deglycosylated OMC45 polypeptide and native OMC45 polypeptide were conjugated to AminoLink Plus coupling gel (Pierce Chemical Co.) separately at pH 10.0 according to the manufacturer’s instructions. As a control, same units of N-glycanase and O-glycanase were conjugated to AminoLink Plus coupling gel. A centrifugation assay was performed to determine the binding efficacy of hydrolases to the deglysosylated OMC45 polypeptide following the method of Nagdas et al. [13 (link)].
Nagdas S.K., Smith L., Mcnamara A., Hernandez–Encarnacion L, & Medina-Ortiz I. (2015). Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases. Molecular and cellular biochemistry, 410(0), 11-23.
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3

Sperm Plasma Membrane N-glycosidase Removal

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The cauda sperm plasma membrane was treated with N-glycanase (New England Biolabs, Ipswich, MA, USA), an endoenzyme that cleaves all N-linked oligosaccharide chains from glycoproteins for 24 hours at 37°C [22 (link)]. For phosphatidylinositol-specific phospholipase C (PIPLC) treatment, cauda sperm plasma membranes were washed in phosphate-buffered saline (PBS) and then incubated in PBS containing 2.0 U/mL PIPLC (Sigma-Aldrich) at 22°C for 30 minutes [23 (link)].
Nagdas S.K., McLean E.L., Richardson L.P, & Raychoudhury S. (2014). Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa. Biochemistry Research International, 2014, 573293.
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4

Sperm Plasma Membrane N-glycosidase Removal

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The cauda sperm plasma membrane was treated with N-glycanase (New England Biolabs, Ipswich, MA, USA), an endoenzyme that cleaves all N-linked oligosaccharide chains from glycoproteins for 24 hours at 37°C [22 (link)]. For phosphatidylinositol-specific phospholipase C (PIPLC) treatment, cauda sperm plasma membranes were washed in phosphate-buffered saline (PBS) and then incubated in PBS containing 2.0 U/mL PIPLC (Sigma-Aldrich) at 22°C for 30 minutes [23 (link)].
Nagdas S.K., McLean E.L., Richardson L.P, & Raychoudhury S. (2014). Identification and Characterization of TEX101 in Bovine Epididymal Spermatozoa. Biochemistry Research International, 2014, 573293.
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5

Mosaic Nanoparticle Composition Analysis

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Label-free quantitation was performed by peptide mass spectrometry to determine the relative abundance of each HA present in the mosaic nanoparticle samples. Each mosaic nanoparticle, either before or after SEC purification, along with a standard mixture of each purified HA-I53_dn5B fusion protein at equimolar concentrations (1:1:1:1), was denatured and reduced using guanidine hydrochloride and DTT. Samples were then alkylated with iodoacetamide, deglycosylated with N-glycanase (New England Biolabs), and digested overnight with LysC protease (ThermoFisher scientific). LC-MS was performed using a Waters Acquity UPLC coupled to a Thermo LTQ-OT using data-dependent acquisition. Peptides were resolved over a Waters CSH C18 1.7 μm, 2.1 × 100 mm column with a linear gradient from 3% to 40% B over 30 minutes (A: 0.1% formic acid; B: acetonitrile with 0.1% formic acid). Peptides were identified from MS/MS data using Protein Prospector using a score cutoff of 15 (http://prospector.ucsf.edu/). Due to the high sequence identity between the HA constructs, only four peptides unique to each specific HA were observed that could be used for label-free quantitation. The integrated peak areas for these peptides relative to the areas from an equimolar mixture of each HA were used to estimate the total abundance of each HA within the mosaic nanoparticle samples (Supplementary Table 3).
Boyoglu-Barnum S., Ellis D., Gillespie R.A., Hutchinson G.B., Park Y.J., Moin S.M., Acton O.J., Ravichandran R., Murphy M., Pettie D., Matheson N., Carter L., Creanga A., Watson M.J., Kephart S., Ataca S., Vaile J.R., Ueda G., Crank M.C., Stewart L., Lee K.K., Guttman M., Baker D., Mascola J.R., Veesler D., Graham B.S., King N.P, & Kanekiyo M. (2021). Quadrivalent influenza nanoparticle vaccines induce broad protection. Nature, 592(7855), 623-628.
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6

Mosaic Nanoparticle Composition Analysis

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Label-free quantitation was performed by peptide mass spectrometry to determine the relative abundance of each HA present in the mosaic nanoparticle samples. Each mosaic nanoparticle, either before or after SEC purification, along with a standard mixture of each purified HA-I53_dn5B fusion protein at equimolar concentrations (1:1:1:1), was denatured and reduced using guanidine hydrochloride and DTT. Samples were then alkylated with iodoacetamide, deglycosylated with N-glycanase (New England Biolabs), and digested overnight with LysC protease (ThermoFisher scientific). LC-MS was performed using a Waters Acquity UPLC coupled to a Thermo LTQ-OT using data-dependent acquisition. Peptides were resolved over a Waters CSH C18 1.7 μm, 2.1 × 100 mm column with a linear gradient from 3% to 40% B over 30 minutes (A: 0.1% formic acid; B: acetonitrile with 0.1% formic acid). Peptides were identified from MS/MS data using Protein Prospector using a score cutoff of 15 (http://prospector.ucsf.edu/). Due to the high sequence identity between the HA constructs, only four peptides unique to each specific HA were observed that could be used for label-free quantitation. The integrated peak areas for these peptides relative to the areas from an equimolar mixture of each HA were used to estimate the total abundance of each HA within the mosaic nanoparticle samples (Supplementary Table 3).
Boyoglu-Barnum S., Ellis D., Gillespie R.A., Hutchinson G.B., Park Y.J., Moin S.M., Acton O.J., Ravichandran R., Murphy M., Pettie D., Matheson N., Carter L., Creanga A., Watson M.J., Kephart S., Ataca S., Vaile J.R., Ueda G., Crank M.C., Stewart L., Lee K.K., Guttman M., Baker D., Mascola J.R., Veesler D., Graham B.S., King N.P, & Kanekiyo M. (2021). Quadrivalent influenza nanoparticle vaccines induce broad protection. Nature, 592(7855), 623-628.
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