Article Figures & Data
Figures
- FIG 1
(A) Sialic acids (purple diamonds) terminate glycan chains on glycolipids and glycoproteins as part of the glycocalyx on the surface of cells. They can also terminate glycans on secreted glycoproteins, like mucins, that make up the protective mucosal barrier in gastrointestinal and respiratory tissue. (B) The sialate O-acetyltransferase, CasD1, adds acetyl groups to sialic acid (N-acetylneuraminic acid [Neu5Ac]) at C-7, from which it migrates to the C-9 position (Neu5,9Ac2) under physiological conditions. This can allow for an additional acetyl group to be added by CasD1 to C-7 (Neu5,7,9Ac3). The sialate O-acetylesterase, SIAE, can remove these acetyl modifications, restoring the unmodified Neu5Ac form of sialic acid.
- FIG 2
Surface and internal expression of 9-O-Ac and 7,9-O-Ac on different cell lines. (A) Fluorescent staining of human HEK-293 and A549 and canine MDCK ATCC line (NBL2), MDCK type I, and MDCK type II cells. Cells were probed with HE-Fcs probes derived from BCoV and PToV, which recognize 9-O-Ac and 7,9-O-Ac, respectively. Cells were permeabilized (perm) using Carbo-Free blocking reagent with 0.001% Tween 20, while nonpermeabilized cells (non-perm) received only Carbo-Free block. All cells were imaged at ×60; nuclei were stained with DAPI. Scale bar, 10 μm. (B) Representative flow cytometry graphs showing distribution of positive staining for HEK-293, A549, and MDCK-NBL2 cell lines. BCoV and PToV HE-Fcs probes were used and permeabilization (perm) and nonpermeabilization (non-perm) methods were as in immunofluorescence assay staining.
- FIG 3
Staining of HEK-293, A549, and MDCK-NBL2 cells with PToV HE-Fc for 9-O-Ac and costaining for the Golgi marker GM130. Cells were permeabilized using 0.001% Tween 20 and imaged at ×60 magnification. Scale bar, 10 μm.
- FIG 4
Editing expression of CasD1 in A549, HEK-293, and MDCK-NLB2 cells. (A) Schematic of edits in the CasD1 gene and genotypes of edited cells for HEK-293, A549, and MDCK-NBL2 cells. (B) Phenotype of edited cells by flow cytometry using 9-O-Ac probe (PToV HE-Fc). Cells were permeabilized using 0.001% Tween 20 to determine internal expression, since most modified Sia are retained internally. The graph is representative of three independent experiments. (C) Immunofluorescence microscopy images of the different engineered cells stained with PToV HE-Fc to detect 9-O-Ac. Cells were permeabilized to reveal both surface and internal expression. Cells were imaged at ×60 magnification. Scale bar, 10 μm. (D) Staining of MDCK WT, ΔCasD1, and CasD1-OX cells showing a representative display of 7,9-O-Ac with the BCoV HE-Fc probe. The cells were permeabilized to reveal both surface and internal expression. Cells were imaged at ×60 magnification. Scale bar, 10 μm. (E) qPCR of CasD1 and SIAE expression in HEK-293 WT, ΔCasD1, and CasD1-OX cells. CasD1 still shows mRNA due to a mismatch between qPCR primers and edit sites; mRNA is still present but does not produce functional protein. Expression is indicated relative to the housekeeping gene, GAPDH. Data were analyzed using a t test in Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (F) HPLC data for total Sia collected from cells by mild acid hydrolysis for WT, ΔCasD1, and CasD1-OX cells in HEK-293, A549, and MDCK cells. Data were analyzed by a t test using Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 5
Editing the expression of SIAE in HEK-293 and A549 cells. (A) Schematic of edits in the SIAE gene, which was targeted to remove both isotypes of SIAE. The genotypes of edited cells show frameshifts that lead to stop codons in all cases. (B) Staining of the different engineered cells with PToV HE-Fc to detect 9-O-Ac. Cells were permeabilized using 0.001% Tween 20 to determine surface and internal expression. Cells imaged at ×60. Scale bar, 10 μm. (C) Flow cytometry using PToV HE-Fc showing the relative display of 9-O-Ac. Cells were permeabilized using 0.001% Tween 20 to show both surface and internal expression. The graph is representative of three independent experiments. (D) qPCR of relative SIAE and CasD1 mRNA expression in HEK-293 WT, ΔSIAE, and ΔSIAE+CasD1 cells compared to GAPDH. SIAE qPCR primers overlap with edit site. Data were analyzed by a t test using Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (E) HPLC data for total Sia collected from cells by mild acid hydrolysis for WT, ΔSIAE, and ΔSIAE+CasD1 cells in HEK-293 and A549 cells. Data were analyzed by a t test using Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (F) Growth curve for WT, ΔSIAE, and ΔSIAE+CasD1 cells in HEK-293 and A549 cells. Cells were counted every 24 h. Each experiment was performed in triplicate. Data were analyzed by two-way analysis of variance (ANOVA) using Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 6
Infection of WT, ΔCasD1, and CasD1-OX cells with IAV, IBV, ICV, and IDV. (A) HEK-293 WT, ΔCasD1, CasD1-OX cells were inoculated at an MOI of 0.1 with IAV strains pH1N1 (A/California/04/2009) and Victoria H3N2 (A/Victoria/361/2011). The cells were fixed at 24 h, and the infected cells per field were counted. Each experiment was performed in triplicate. Data analyzed by two-way ANOVA using Prism software. (B) MDCK WT, ΔCasD1, and CasD1-OX cells were inoculated at a high MOI with IAV strains pH1N1 (A/California/04/2009) and Victoria H3N2 (A/Victoria/361/2011, and IBV strains B/Memphis/1/2018 and B/Colorado/06/2017 for 48 h and then imaged at ×10 magnification. Scale bar, 100 μm. Representative images of three independent experiments are shown. (C) MDCK WT, ΔCasD1, and CasD1-OX cells were inoculated at a high MOI with ICV strains C/Ann Arbor/1/50, C/Taylor/1233/1947, and C/Victoria/1/2011 and IDV strains D/bovine/MS/C000020N/2014 and D/swine/OK/1334/2011 for 48 h and then imaged at ×10 magnification. Scale bar, 100 μm. Representative images of three independent experiments are shown. (D) Quantification of the relative infected cell counts for panels B and C, as determined through image analysis with ImageJ. Data were analyzed by two-way ANOVA using Prism software. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
- FIG 7
Effects of 7,9-O- and 9-O-Ac on IAV HA binding and NA cleavage. (A) Surface staining for α2,6-linked Sia and α2,3-linked Sia on mouse erythrocytes via flow cytometry and a table showing total Sia analysis of mouse erythrocytes using HPLC. Data for each Sia variant are given as the percentages of total Sia, averaged across three independent samples. (B) Hemagglutination of human H1N1 and H3N2 IAV strains on untreated or esterase-treated mouse erythrocytes. The data are averaged across three independent experiments, with data analyzed by two-way ANOVA using Prism software. (C) Profiles of total Sia freed by either chemical treatment, N1 VLPs, N2 VLPs, or commercial NeuA sialidase as determined by HPLC. The data are averaged across two independent experiments.
- FIG 8
Summary of proposed 7,9-O- and 9-O-acetyl sialic acid production and trafficking in cells. (Step 1) Sia (purple diamond) is added to the growing glycan chain in the Golgi by sialyltransferases using CMP-Neu5Ac or CMP-Neu5Gc substrates, which are synthesized in the nucleus by the addition of cytodine monophosphate (CMP) to Neu5Ac or Neu5Gc and are specifically imported into the Golgi. (Step 2) CMP-Neu5Ac or CMP-Neu5Gc Sia are modified by CasD1, adding one or two acetyl groups to form 9-O-Ac or 7,9-O-Ac, respectively (red circles), before being added to glycan chains. The majority of glycoproteins with these modifications are retained in the Golgi compartment (large arrow) of many cells, including the HEK-293 and A549 cells examined here, whereas only some (mostly 9-O-Ac) are transported to the cell surface (small arrow). (Step 3) Surface-displayed O-acetyl Sia can interact with pathogens, cell receptors, or lectins. For example, ICV uses 9-O-Ac as its receptor. Secreted forms of SIAE may also remove the O-acetyl modifications, altering these lectin-ligand interactions. (Step 4) When glycoproteins are recycled from the cell surface, the lysosomal form of SIAE (LSE) can remove O-acetyl modifications from Sia. Free Sia are exported to the cytosol, where the cytosolic form of SIAE (CSE) can also remove any remaining O-acetyl modifications. Unmodified Sia can then be “activated” in the nucleus by the addition of CMP and transported to the Golgi compartment for addition to new glycan chains.
Tables
- TABLE 1
Total sialic acids were collected from different cell lines via mild acid hydrolysis and analyzed using HPLCa
↵a Percentages of the total sialic acid collected are presented. Representative chromatograms for standard, HEK-293, A549, and MDCK-NBL2 are shown in Fig. S1 in the supplemental material. ND, not detected; *, primary swine nasal (SiNEC) and tracheal (SiTEC) epithelial cells (courtesy of Stacey Schultz-Cherry).
FIG S1
HPLC chromatograms showing wild-type HEK-293 cells, A549 cells, MDCK-NLB2 cells, and bovine mucin standard of O-acetyl modifications. Total sialic acids were collected from cell lines and standards via mild acid hydrolysis. Neu5Gc on cells is likely derived from the fetal bovine serum used in the growth media, which is taken up by cells and displayed on the cell surface. Humans and canines do not have a functional CMAH gene to synthesize Neu5Gc endogenously. Download FIG S1, TIF file, 0.8 MB.
Copyright © 2019 Barnard et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
A549 conditioned medium was analyzed for the presence of secreted mucins and total sialic acid content. (A) Conditioned media from A549 was concentrated using a 30-kDa filter column and then titrated on a western blot for Muc5B expression compared to a purified human Muc5B (a gift from Stefan Ruhl, University of Buffalo). (B) Representative chromatogram of total sialic acid collected from A549 conditioned media using HPLC analysis. The percentage of different sialic acid forms found in total sialic acid collected is summarized in the table. Download FIG S2, TIF file, 0.4 MB.
Copyright © 2019 Barnard et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
MDCK-NBL2 cells were costained for the presence of 9-O-Ac using PToV HE-Fc (green) and canine podoplanin (red) using an anti-podoplanin antibody (courtesy of Yukinari Kato, Tohoku University). Cells were imaged at ×40 and ×60 magnifications, as indicated. Scale bars, 10 μm. Download FIG S3, TIF file, 1.6 MB.
Copyright © 2019 Barnard et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
