To test whether recovery of the SAs after neuraminidase-mediated desialylation affects the virus attachment, appropriate experiments were designed. Cells grown on coverslips were washed twice with PBS, incubated for 30 min at 37 °C with 500 mU/mL NA. Subsequently cell cultures were washed with PBS and treated with 1 mM cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac, C8271 Sigma-Aldrich, Poznan, Poland) and either α-2,3-sialyltransferase (α2,3-ST) from Pasteurella multocida (S1951, Sigma-Aldrich, Poznan, Poland) or α-2,6-sialyltransferase (α2,6-ST) from Photobacterium damsel (S2076, Sigma-Aldrich, Poznan, Poland) at varying concentrations for 2 h at 37 °C. Following treatment, cells were washed thrice with ice-cold PBS and infected with iodixanol concentrated CRCoV, BCoV orHCoV-OC43. After 2 h at 4 °C, unbound virions were washed off with PBS and cells were fixed with 4% formaldehyde. Activity of neuraminidase and sialyltransferases was verified with α-2,6-SAs specific fluorescein labeled Maackia Amurensis lectin I (Vector Labs) and α-2,3-SAs specific Cy3 labeled Sambucus Nigra lectin (Vector Labs)
Sambucus nigra lectin
Manufactured by Vector Laboratories
Sourced in United States
Sambucus nigra lectin is a protein derived from the European black elderberry plant. It exhibits binding affinity for N-acetylneuraminic acid (sialic acid) and can be used to detect the presence of sialic acid-containing glycoconjugates in various biological samples.
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Lab products found in correlation
Maackia amurensis lectin 2, by Vector Laboratories (3 mentions)
Maackia amurensis lectin 1, by Vector Laboratories (2 mentions)
Peanut agglutinin, by Vector Laboratories (2 mentions)
Biotinylated maackia amurensis lectin 2, by Vector Laboratories (2 mentions)
Parafilm, by Avantor (2 mentions)
Anti hla1, by Abcam (2 mentions)
Anti sm22alpha, by Abcam (2 mentions)
Sequencing grade trypsin, by Promega (2 mentions)
Cmp neu5ac, by Merck Group (1 mentions)
Cd8α 53 6.7, by BD (1 mentions)
Cd3 17a2, by BD (1 mentions)
Tcrγδ gl3, by BioLegend (1 mentions)
Fortessa x20, by BD (1 mentions)
Horseradish peroxidase conjugated streptavidin, by BioLegend (1 mentions)
Anti vegfr2 antibody, by Cell Signaling Technology (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Hydrazide resin, by Bio-Rad (1 mentions)
Orbitrap velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
2d nano lc system, by Thermo Fisher Scientific (1 mentions)
Sodium periodate, by Bio-Rad (1 mentions)
14 protocols using sambucus nigra lectin
1
Quantifying Sialic Acid Recovery After Neuraminidase Treatment
2
Multiparameter Flow Cytometry Analysis of Immune Cell Populations
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Organs were collected from WT, CD45.1, CD45.2, OT-I, or CD8β-deficient mice and single-cell suspensions prepared using standard protocols. Following removal of red blood cells using ACK, nonspecific receptors were blocked with monoclonal antibody (mAb) 2.4G2, before cells (1–5 × 106) were stained with mAb to mouse NKp46 (29A1.4; BioLegend), CD3 (17A2; BD Biosciences), TCRγδ (GL3, BioLegend), TCRαβ (H57-597, BioLegend), CD8α (53-6.7; BD Biosciences), CD8β (H35-17.2; BD Biosciences). Alternatively cells were stained with the lectins peanut agglutinin (Vector Laboratories), sambucus nigra lectin (Vector Laboratories), or maackia amurensis lectin II (Vector Laboratories) before detecting using Streptavidin (BD Biosciences). Cells stained with tetramers were fixed in 2% paraformaldehyde for 15 min and washed twice with FACS buffer (1% FCS/PBS) before being resuspended in FACS buffer. All other FACS combinations were acquired unfixed. For acquisition, events were electronically gated on FSC-A versus FSC-H (singlets), followed by FSC-A and SSC-A (to exclude doublets and debris). Among the remaining population, at least 5000 electronic events of interest were collected using an LSR-II or X- 20 Fortessa (BD Biosciences).
3
VEGFR2 Glycosylation Analysis
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Liver tissue lysates were obtained and quantified as described previously. For each immunoprecipitation reaction, 10 μL anti-VEGFR2 antibody (cat# 2479; Cell Signaling Technology) was added to 1 mg total protein lysate and the mixture was incubated for 1.5 hours with rotation at 4°C. A total of 20 μL protein A/G agarose beads (cat# sc-2003; Santa Cruz) were added and rotated overnight at 4°C. The mixture was centrifuged at 2500 rpm for 5 minutes to pellet the beads and washed for 5 times with RIPA buffer. The proteins immunoprecipitated on beads were denatured by boiling for 10 minutes and further resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. For lectin blotting, the membrane was blocked and blotted with 5 μg/mL biotinylated maackia amurensis lectin II (cat# B-1265; Vector Laboratories) or 10 μg/mL sambucus nigra lectin (cat# B-1305-2; Vector Laboratories) in blocking buffer (5% BSA in Tris-buffered saline with Tween 20) at 4°C overnight. After four 5-minute washes, the membrane was incubated in horseradish peroxidase–conjugated streptavidin (cat# 405210; BioLegend) and visualized by chemiluminescence detection using the Tanon 4600SF Imaging System.
4
Epicardial Cell Transplantation in Chicken Embryos
5
Glycoprotein Enrichment and Analysis
6
Sialic Acid Levels in CLL Leukocytes
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Leukocytes from PB of CLL patients were isolated using an ammonium chloride-based red blood lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, and 0.2 mM ethylenediaminetetraacetic acid [EDTA] tetrasodium salt, all from Merck; Rahway, NJ, USA). To determine the levels of α2-3 and α2-6 linked sialic acids, leukocytes (2x106 per tube) were incubated for 15 minutes (min) at room temperature (RT) with gentle rocking with Maackia amurensis lectin II and Sambucus nigra lectin (1:20000; Vector Laboratories; Newark, CA, USA), which preferentially bind the α2-3 and α2-6 linked sialic acids, respectively. After incubation, cells were washed, stained with APC-conjugated streptavidin beads (1:400; BD Bioscience, Franklin Lakes, NJ, USA), FITC-conjugated anti-CD5 and PE-conjugated anti-CD19 antibodies (both from Immunological Science, Rome, Italy), AlexaFluor 647-conjugated cutaneous lymphocyte antigen antibody (clone Heca452; BD Bioscience) and incubated for 30 min at RT with gentle rocking. After incubation, cells were washed, resuspended in 500 µL staining buffer supplemented with 7-aminoactinomycin D (7-AAD, 1:80; Immunological Science) to exclude dead cells. Cells were acquired on a BD FACS Canto I (BD Biosciences). Data were analyzed using the Infinicyt software v 2.0.5.b.007 (Cytognos; Salamanca, Spain).
7
Epicardial Cell Transplantation in Chicken Embryos
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Chicken (Gallus gallus domesticus) eggs (Winter Egg Farm, Cambridge, UK) were incubated in a digital cabinet incubator (OVA Easy 380, Brinsea) at 38 degrees. At Hamburger Hamilton developmental stage 19 (HH19), the eggshell was fenestrated, the window covered with parafilm (VWR) and eggs were placed horizontally in the incubator. At HH35, epicardial cells were transplanted in PSC with matrigel (1:2 dilution) onto the chorionic chicken vasculature. HESCs were fully differentiated to epicardial cells before administration into the chicken embryos. The eggs were then returned in the incubator until stage HH40. The matrigel plugs were harvested and fixed in 4% paraformaldehyde before being stained with anti-HLA1 (abcam) anti-SM22alpha (Abcam) and sambucus nigra lectin (Vector laboratories).
8
Lectin Histochemistry of Glycans
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Tissue sections were microwaved at 95°C in 10 mM citrate buffer pH 6.0 for 10 minutes, blocked with 0.05% bovine serum albumin at RT for 10 minutes, and incubated with 1:100 biotinylated Sambucus nigra lectin (SNA), Maackia amurensis lectin I (MAAI), or Maackia amurensis lectin II (MAAII) (Vector Laboratories) at RT for 1 hour followed by 1:100 streptavidin, alkaline phosphatase (Vector Laboratories) at RT for 45 minutes. The sections were developed with Vector Red Substrate Kit, Alkaline Phosphatase (AP) (Vector Laboratories) at RT for 5–15 minutes.
Mayer’s hematoxylin was used to counterstain the nuclei in all tissue sections for 1 minute. The sections were blued with Scott’s tap water, air dried, and mounted with Permount (Fisher Scientific).
Mayer’s hematoxylin was used to counterstain the nuclei in all tissue sections for 1 minute. The sections were blued with Scott’s tap water, air dried, and mounted with Permount (Fisher Scientific).
9
Recombinant Human MR Glycosylation Analysis
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Human recombinant full-length MR with His-tag, expressed in mouse myeloma cell line NS0, was purchased from R&D systems, anti-human IgG Alexa-Fluor 488-conjugated from Jackson ImmunoResearch, and anti–penta-His Alexa-Fluor 488 conjugate from Qiagen. Biotinylated Concanavalin A, R. communis agglutinin-I, Maackia amurensis lectin-I, Sambucus nigra lectin, fluorescein Concanavalin A, fluorescein Wheat germ agglutinin, and Man-BSA were purchased from Vector Laboratories; neuraminidase and Protein A-agarose beads from Roche; O-glycosidase, PNGase F, α-galactosidase, β1,3-galactosidase, and β1,4-galactosidase from New England Biolabs; sequencing grade trypsin from Promega; Glucitol-polyacrylamide (PAA), Man3-PAA, and Glucitol-PAA biotin from Lectinity; GlcNAc and GlcNAc-BSA from Carbosynth. All other chemicals were purchased from Sigma-Aldrich if not indicated differently.
10
Characterization of Lectin Binding Patterns
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Maackia amurensis lectin II (MAL II) was purchased from Sigma. Sambucus nigra lectin (SNA), Peanut Agglutinin (PNA) and Wheat Germ Agglutinin (WGA) was bought from Vector Laboratories. Arg-Gly-Asp-D-Phe-Lys (cyclo-RGDfK) modified with 4-carboxy-butyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide was synthesized by Prof. Dr. S. Burov, Saint-Petersburg, Russia and further characterized by us [31 (link)].
Neuraminidase from Vibrio cholerae was obtained from GIBCO laboratories. Neuraminidase hydrolyzes terminal N- or O-acylneuraminic acids which are α2,6-, α2,3-, or α2,8-linked (rate: α2,6 > α2,3 > α2,8) to oligosaccharides, polysaccharides, mucopolysaccharides, glycoproteins, and glycolipids. One unit is the enzyme activity that hydrolyzes 1 μmol N-acetyl-neuraminosyl-D-lactose within 1 min at 37°C under the following incubation conditions: 10 mM N-acetyl-neuraminosyl-D-lactose, 50 mM sodium acetate, 4 mM calcium chloride, bovine serum albumin, 100 μg/ml, pH 5.5. Specific activity of neuraminidase (Vibrio cholera) is 1 μmol N-acetylneuraminic acid per min is split off from human acid α1-glycoprotein (10 mg/ml incubation mixture) by 1 U neuraminidase.
Neuraminidase from Vibrio cholerae was obtained from GIBCO laboratories. Neuraminidase hydrolyzes terminal N- or O-acylneuraminic acids which are α2,6-, α2,3-, or α2,8-linked (rate: α2,6 > α2,3 > α2,8) to oligosaccharides, polysaccharides, mucopolysaccharides, glycoproteins, and glycolipids. One unit is the enzyme activity that hydrolyzes 1 μmol N-acetyl-neuraminosyl-D-lactose within 1 min at 37°C under the following incubation conditions: 10 mM N-acetyl-neuraminosyl-D-lactose, 50 mM sodium acetate, 4 mM calcium chloride, bovine serum albumin, 100 μg/ml, pH 5.5. Specific activity of neuraminidase (Vibrio cholera) is 1 μmol N-acetylneuraminic acid per min is split off from human acid α1-glycoprotein (10 mg/ml incubation mixture) by 1 U neuraminidase.
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