Research ArticleMICROBIOLOGY

Influenza binds phosphorylated glycans from human lung

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Science Advances  13 Feb 2019:
Vol. 5, no. 2, eaav2554
DOI: 10.1126/sciadv.aav2554
  • Fig. 1 An HL-SGM was generated and validated by lectin binding.

    (A) To identify endogenous receptors recognized by IAV, we generated a shotgun glycan microarray comprising the N-glycans from the human lung (7, 42, 43). Lungs (provided by LifeLink) were processed following the oxidative release of natural glycans (ORNG) method (17) and labeled with the bifunctional linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB) (40). The labeled N-glycans were printed on glass slides to generate the HL-SGM. RFU, relative fluorescence units; m/z, mass/charge ratio; MS, mass spectrometry; MSn, tandem mass spectrometry; MAGS, Metadata Assisted Glycan Sequencing. (B) Biotinylated lectins were bound to the HL-SGM at the noted concentrations and were detected with cyanine 5–conjugated streptavidin. ConA, concanavalin A; WGA, wheat germ agglutinin; RCA, R. communis agglutinin; AAL, A. aurantia lectin; ECL, E. cristagalli lectin; LEL, L. esculentum lectin.

  • Fig. 2 A range of IAVs all displays binding on the HL-SGM to chart IDs not bound by sialylated glycan binding lectins, SNA, and MAL-I.

    (A) Fluorescently labeled viruses, representative of different subtypes and host species, were bound to the array and display divergent binding profiles. (B) Penn binding to HL-SGM and comparison to SNA and MAL-I. IAV virus Penn was labeled with Alexa Fluor 488 and bound to the HL-SGM. The panels for MAL-I and SNA binding are included to demonstrate the nonoverlap between the Sia-binding lectins (light blue boxes) and the virus binding in the lower fraction numbers (green box).

  • Fig. 3 The Penn strain binds to sialylated glycans and to glycan fractions containing phosphorylated structures.

    (A) NA treatment of HL-SGM and interrogation with SNA (25 μg/ml; detected with cyanine 5–conjugated streptavidin) and Penn reveal that IAV binds to array after removal of Sia as determined by loss of SNA binding. (B) Matrix-assisted laser desorption/ionization–time-of-flight MS (MALDI-TOF-MS) analysis of fractions, R10N13 (chart ID 118; high binding) indicating sialylated N-glycans and R04N23 (chart ID 47; high binding after NA treatment) indicating phosphorylated glycans, and tandem MS (MS/MS) of selected peaks. (C) Neuraminidase treatment of highest binding fraction R10N13 (predicted structures shown to the left; blue square, N-acetylglucosamine; red triangle, fucose; green circle, mannose; yellow circle, galactose; purple diamond, Sia) and binding of SNA (red bars) and MAL-I (blue bars) reveal the presence of both linkage types within the fraction.

  • Fig. 4 A combination of NA and phosphatase treatment of the HL-SGM results in no binding of Penn, while single enzymatic treatments allow for binding to certain glycan fractions.

    Binding inhibition with Fv M6P-1 (100 μg/ml) is also shown.

  • Fig. 5 Different phosphorylated sugars produce no inhibition to Penn on the HL-SGM in hapten competition assays, indicating that charge alone is not mediating virus binding to nonsialylated glycans.

    The IAV-only RFUs are represented in gray, while the IAV and hapten RFUs are in green. In addition, the HL-SGM was treated with neuraminidase A (denoted as NA) to remove Sia before binding experiments, and the IAV-only RFUs are in gray, while the IAV and hapten RFUs are in purple.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav2554/DC1

    Supplementary Methods

    Table S1. IAV strains used in this work.

    Table S2. Raw glycan microarray data (Excel file).

    Fig. S1. Success of the HL-SGM print is further validated by lectin binding after A. ureafaciens NA treatment.

    Fig. S2. HPLC chromatograms overlaid with the HL-SGM binding profile for Penn indicate that glycan specificity is more important for virus binding than abundance of glycan.

    Fig. S3. Conditions for NA treatment of the microarrays are confirmed on a defined N-glycan microarray, and those enzymatic conditions are applied to the HL-SGM for an avian and swine strain, revealing the same Sia-independent binding.

    Fig. S4. MALDI-TOF-MS characterization of additional glycan fractions reveals phosphorylated glycans and sialylated glycans.

    Fig. S5. Presence of sialylated or phosphorylated glycans within the human lung fractions is confirmed by peak shifts in the HPLC profile after enzymatic treatment due to phosphatase or NA sensitivity.

    Fig. S6. Phosphatase conditions for the HL-SGM were optimized on a defined mannose phosphate glycan microarray using binding of Fv M6P-1.

    Fig. S7. Hapten competition studies indicate that binding to sialylated glycans can be inhibited by sialyllactose, but not Fv M6P-1, which binds to the mannose phosphate array while Penn does not.

    Fig. S8. Proteomics of Penn grown in canine kidney cells identifies the canine MPR protein.

  • Supplementary Materials

    The PDF file includes:

    • Supplementary Methods
    • Table S1. IAV strains used in this work.
    • Fig. S1. Success of the HL-SGM print is further validated by lectin binding after A. ureafaciens NA treatment.
    • Fig. S2. HPLC chromatograms overlaid with the HL-SGM binding profile for Penn indicate that glycan specificity is more important for virus binding than abundance of glycan.
    • Fig. S3. Conditions for NA treatment of the microarrays are confirmed on a defined N-glycan microarray, and those enzymatic conditions are applied to the HL-SGM for an avian and swine strain, revealing the same Sia-independent binding.
    • Fig. S4. MALDI-TOF-MS characterization of additional glycan fractions reveals phosphorylated glycans and sialylated glycans.
    • Fig. S5. Presence of sialylated or phosphorylated glycans within the human lung fractions is confirmed by peak shifts in the HPLC profile after enzymatic treatment due to phosphatase or NA sensitivity.
    • Fig. S6. Phosphatase conditions for the HL-SGM were optimized on a defined mannose phosphate glycan microarray using binding of Fv M6P-1.
    • Fig. S7. Hapten competition studies indicate that binding to sialylated glycans can be inhibited by sialyllactose, but not Fv M6P-1, which binds to the mannose phosphate array while Penn does not.
    • Fig. S8. Proteomics of Penn grown in canine kidney cells identifies the canine MPR protein.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Raw glycan microarray data (Excel file).

    Files in this Data Supplement:

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