N‑Glycolylneuraminic acid is recognized by the hemagglutinin (HA) of certain influenza A viruses that have acquired Neu5Gc‑binding ability. [1]

Expression of Neu5Gc on human MCF7 cells (CMAH‑MCF7 cells) significantly reduced the infectivity of Neu5Gc‑binding IAVs. Relative infectivity in CMAH‑MCF7 cells compared to parental MCF7 cells: A/Memphis/102/1972 (H3N2) showed 36.3%; A/equine/Fontainebleau/1/1979 (H3N8) showed 16.8%; A/equine/Tennessee/5/1986 (H3N8) showed 14.0%. In contrast, A/Memphis/1/1971 (H3N2), which does not bind Neu5Gc, showed similar infectivity in both cell lines. [1]
TCID50 values of Neu5Gc‑binding IAVs were significantly reduced in CMAH‑MCF7 cells compared to MCF7 cells. For A/Aichi/2/1968 (H3N2): log10 TCID50/ml was 9.56±0.51 in MCF7 vs 9.22±0.48 in CMAH‑MCF7 (ratio 0.46). For A/Memphis/102/1972: 8.83±0.44 vs 8.08±0.36 (ratio 0.18). For A/equine/Miami/1963 (H3N8): 9.83±0.29 vs 8.89±0.19 (ratio 0.11). For A/equine/Fontainebleau/1/1979: 7.39±0.35 vs 6.06±0.42 (ratio 0.05). For A/equine/Tennessee/5/1986: 8.61±0.10 vs 7.78±0.19 (ratio 0.15). For A/equine/Ibaraki/1/2007, TCID50 could not be measured in CMAH‑MCF7 cells due to low infectivity. A/Memphis/1/1971 (non‑binding) showed no difference (ratio 1.00). [1]
Treatment of human A549 cells with the Neu5Gc precursor N‑glycolylmannosamine (ManNGc, 0.5 or 2 mM for 3 days) induced Neu5Gc expression on the cell surface and decreased infectivity of A/Memphis/102/1972 and A/equine/Fontainebleau/1/1979, whereas treatment with ManNAc (5 mM) had no effect. [1]
A HA mutant IAV (T155Y, derived from A/Memphis/1/1971 with Thr155→Tyr substitution) that acquired Neu5Gcα2,6Gal binding ability showed 27.9% relative infectivity in CMAH‑MCF7 cells compared to MCF7 cells, while the wild‑type HA (WT) without Neu5Gc binding showed no difference. TCID50 of T155Y was significantly reduced in CMAH‑MCF7 cells (log10 TCID50/ml: 7.67±0.29 in MCF7 vs 6.58±0.52 in CMAH‑MCF7; ratio 0.08), whereas WT showed no difference (ratio 1.00). [1]
Confocal microscopy after virus adsorption (5.0×10⁵ TCID50) at 4°C and incubation at 37°C for 10 min showed that WT entered both MCF7 and CMAH‑MCF7 cells, while T155Y was mainly observed on the plasma membrane of CMAH‑MCF7 cells, indicating that Neu5Gc expression arrests virus entry at the internalization stage. [1]

HPLC analysis of sialic acid species: Cells (50 μl suspension) were mixed with 50 mM H₂SO₄ (75 μl) and 2 μM N‑propylneuraminic acid (25 μl) as internal control. After hydrolysis at 80°C for 12 h, the mixture was filtered by centrifugation using 3K molecular weight cut‑off filters at 15,100×g for 90 min. The flow‑through (60 μl) was mixed with DMB reagent (60 μl) containing 7 mM 1,2‑diamino‑4,5‑methylenedioxybenzene, 1 M β‑mercaptoethanol, and 18 mM sodium hydrosulfite in water. After incubation at 60°C for 2.5 h in the dark (derivatization), the mixture was cooled on ice. A 100‑μl aliquot was injected into an HPLC system with a C18 column (4.6×150 mm, 5 μm) at 40°C, flow rate 1.2 ml/min of methanol:water = 25:75 (v/v). Fluorescence was measured at excitation 373 nm and emission 448 nm. Standard curves were established using 0.1, 1, and 10 μM Neu5Ac and Neu5Gc. Neu5Gc content in CMAH‑MCF7 cells was 1.71±0.28 nmol/mg protein, Neu5Ac 1.54±0.70 nmol/mg protein (Neu5Gc/Neu5Ac ratio 1.11). In MCF7 cells, Neu5Gc was barely detectable (0.15±0.03 nmol/mg protein) and Neu5Ac was 1.90±0.27 nmol/mg protein (ratio 0.08). [1]

Immunofluorescence staining of Neu5Gc: Cells (5×10³/well) grown on glass slides were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature, washed, and incubated with chicken anti‑Neu5Gc antibody (recognizes Neu5Gcα2,3Gal but not Neu5Acα2,3Gal) on ice for 1 h, followed by FITC‑conjugated rabbit anti‑chicken IgY on ice for 1 h. Nuclei were stained with DAPI. Sialidase treatment (2500 mU/ml Salmonella typhimurium sialidase at 37°C for 1 h before fixation) abolished staining. Neu5Gc was detected in CMAH‑MCF7 cells but not in MCF7 cells. [1]
Flow cytometry for Neu5Gc: Cells (2×10⁵/well) were harvested with trypsin, fixed with 2% PFA‑PBS for 10 min, incubated with chicken anti‑Neu5Gc antibody for 30 min at room temperature, then with FITC‑conjugated anti‑chicken IgY for 30 min. At least 1×10⁴ cells analyzed. Mean fluorescence intensity (MFI) ratio (relative to secondary antibody only) was 3.57 for CMAH‑MCF7 cells and 1.57 for MCF7 cells. [1]
Flow cytometry for SAα2,6Gal and SAα2,3Gal linkages: Fixed cells were incubated with FITC‑conjugated SNA lectin (for SAα2,6Gal) or FITC‑conjugated MAL‑I lectin (for SAα2,3Gal) for 30 min at room temperature. MFI of SAα2,6Gal linkage was increased in CMAH‑MCF7 cells compared to MCF7 cells, while SAα2,3Gal MFI was similar. [1]
Viral infectivity (TCID50) assay: Cells (2.5×10⁴/well in 96‑well plate) were infected with 10‑fold serial dilutions of virus in serum‑free medium at 37°C for 14‑20 h, fixed with methanol for 30 sec, incubated with mouse anti‑IAV nucleoprotein (NP) monoclonal antibody for 30 min, then with HRP‑conjugated goat anti‑mouse IgG+M for 30 min, and developed with H₂O₂, N,N‑diethyl‑p‑phenylenediamine sulfate, and 4‑chloro‑1‑naphthol. TCID50 was calculated by counting infection‑positive wells. [1]
Virus infection assay for cell counting: Cells (1×10⁵/well in 24‑well plate) were infected with virus suspension (9.39×10³ to 1.27×10⁵ TCID50 for MCF7 cells) in 250 μl SFM at 37°C for 30 min, washed, cultured in SFM for 20‑22 h, fixed with methanol, and stained with anti‑NP antibody as above. Infected cells were counted under a microscope. [1]
ManNGc treatment of A549 cells: A549 cells were treated with 10 mU/ml Arthrobacter ureafaciens sialidase for 1 h at 37°C, then cultured for 3 days in SFM containing ManNAc (5 mM) or ManNGc (0.5 or 2 mM). Control cells received DMSO vehicle. Neu5Gc expression was confirmed by flow cytometry with anti‑Neu5Gc antibody. Infectivity of A/Memphis/102/72 and A/equine/Fontainebleau/79 (5‑6×10³ TCID50) was measured after 30 min infection and 10 h incubation. [1]
Confocal microscopy for virus entry: MCF7 and CMAH‑MCF7 cells (5.0×10⁴/well on 8‑well coverslips) were adsorbed with WT or T155Y virus (5.0×10⁵ TCID50) in cold DMEM on ice for 1.5 h, washed, incubated at 37°C for 10 min, cooled on ice for 5 min, fixed with 4% PFA for 15 min, permeabilized with 0.05% Triton X‑100 for 15 min. Cells were stained with mouse anti‑NP antibody and biotinylated wheat germ agglutinin (WGA) at 4°C for 1 h, followed by TRITC‑conjugated anti‑mouse IgG, Cy5‑conjugated streptavidin, and DAPI. Images were taken with a confocal microscope. [1]

[1]. N-glycolylneuraminic acid on human epithelial cells prevents entry of influenza A viruses that possess N-glycolylneuraminic acid binding ability. J Virol. 2014 Aug;88(15):8445-56.

N-Glucoyl-β-neuraminic acid is an N-acylneuraminic acid in which the acyl substituent on the nitrogen atom is a glycoyl group, and the anomeric carbon atom is in the β configuration. It is a mammalian metabolite and antigen. GcNeu has been reported and data are available for detection in the Mexican axolotl (Ambystoma mexicanum). See also: N-Glucoyl-neuraminic acid (note moved to).

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N‑Glycolylneuraminic acid (Neu5Gc) is a sialic acid species present in many non‑human mammals but not synthesized in humans due to a 92‑bp deletion in the CMAH gene causing a frameshift. However, humans can metabolically incorporate Neu5Gc from dietary sources (red meat, milk). In this study, Neu5Gc expressed on human epithelial cells (CMAH‑MCF7 cells or ManNGc‑treated A549 cells) acted as a decoy receptor for influenza A viruses that have acquired Neu5Gc‑binding ability, suppressing infection by arresting virus entry at the internalization stage. This decoy function may protect against transmission of Neu5Gc‑binding IAVs. The study used human IAVs (A/Aichi/2/1968, A/Memphis/1/1971, A/Memphis/102/1972) and equine IAVs (A/equine/Miami/1963, A/equine/Fontainebleau/1/1979, A/equine/Tennessee/5/1986, A/equine/Ibaraki/1/2007), as well as HA mutant viruses (WT and T155Y) generated on the A/WSN/1933 backbone. Neu5Gc expression did not affect infectivity of IAV lacking Neu5Gc binding. [1]

C11H19NO10

325.26926

325.1

1113-83-3

123802

White to off-white solid powder

1.9±0.1 g/cm3

826.1±75.0 °C at 760 mmHg

188-190°C

453.4±37.1 °C

0.0±0.6 mmHg at 25°C

1.651

-2.96

8

10

6

22

418

6

C1[C@@H]([C@H]([C@@H](O[C@@]1(C(=O)O)O)[C@@H]([C@@H](CO)O)O)NC(=O)CO)O

FDJKUWYYUZCUJX-AJKRCSPLSA-N

InChI=1S/C11H19NO10/c13-2-5(16)8(18)9-7(12-6(17)3-14)4(15)1-11(21,22-9)10(19)20/h4-5,7-9,13-16,18,21H,1-3H2,(H,12,17)(H,19,20)/t4-,5+,7+,8+,9+,11-/m0/s1

(2S,4S,5R,6R)-2,4-dihydroxy-5-[(2-hydroxyacetyl)amino]-6-[(1R,2R)-1,2,3-trihydroxypropyl]oxane-2-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~83.33 mg/mL (~256.19 mM)

Solubility in Formulation 1: 10 mg/mL (30.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)