sialidase  (New England Biolabs)

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: In some assays, cells were treated with sialidase (200 U/mL Arthrobacter ureafaciens α2–3,6,8,9-Neuraminidase, New England BioLabs) for 1 hr at 37°C, and washed using HEPES buffer prior to the binding assay.

Techniques: Binding Assay, Expressing, Generated

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: In some assays, cells were treated with sialidase (200 U/mL Arthrobacter ureafaciens α2–3,6,8,9-Neuraminidase, New England BioLabs) for 1 hr at 37°C, and washed using HEPES buffer prior to the binding assay.

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

Journal: eLife

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

doi: 10.7554/eLife.61552

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

Article Snippet: In some assays, cells were treated with sialidase (200 U/mL Arthrobacter ureafaciens α2–3,6,8,9-Neuraminidase, New England BioLabs) for 1 hr at 37°C, and washed using HEPES buffer prior to the binding assay.

Techniques: Binding Assay, Plasmid Preparation, Flow Cytometry

Journal: mAbs

Article Title: Evaluation of a glycoengineered monoclonal antibody via LC-MS analysis in combination with multiple enzymatic digestion

doi: 10.1080/19420862.2015.1113361

Figure Lengend Snippet: HPLC-FLD profile of procainamide labeled N -glycans from rituximab and the glycoengineered rituximab with α2,6 linked sialic acid. A). HPLC profile of rituximab N -glycans with major species shown. B). The glycan profile from the glycoengineered rituximab prepared by Endo S digestion. The major glycan is G2FS2 with α2,6 linked sialic acid. C). The glycan profile from the glycoengineered rituximab after α2-3 Neuraminidase S treatment. D). The glycan profile from the glycoengineered rituximab after α2-3,6,8,9 Neuraminidase A treatment. E). The glycan profile from the glycoengineered rituximab prepared by Endo S2 digestion.

Article Snippet: Trypsin-ultra (P8101S), PNGase F (P0708S), α2-3 Neuraminidase S (P0743S), α2-3,6,8,9 Neuraminidase A (P0722S), Remove-iT Endo S (P0741S) are products from New England Biolabs.

Techniques: High Performance Liquid Chromatography, Labeling