Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Barnard, Karen N.; Wasik, Brian R.; LaClair, Justin R.; Buchholz, David W.; Weichert, Wendy S.; Alford-Lawrence, Brynn K.; Aguilar, Hector C.; Parrish, Colin R.

The expression levels of those modified Sia variants were also quantified by HPLC analysis using DMB (1,2-diamino-4,5-methylenedioxybenzene) labeling and fluorescence detection (39) . The cells were treated at 80ËšC for 3 h in the presence of 2 M acetic acid, and therefore the data likely represent the total Sia present in the cells. Even though some cell lines showed strong staining by the HE-Fc probes, the levels of 9-O-Ac were 1 to 2% of the total collected Sia from all cells, while the levels of 7,9-O-Ac were below the level of detection in all cell lines ( Fig. 2 and Table 1 ; see also Fig. S1 in the supplemental material). This includes cells from other species, including cat, mouse, horse, and swine. Unique among the cells tested, A549 cells are secretory and express significant levels of mucin (including MUC1 and MUC5B) (40) . Although mucin proteins are not the only secreted protein found in mucus, we chose to focus on them due to their high levels of glycosylation (80% of weight) and proposed importance as a barrier to pathogens (41) (42) (43) . We found that A549 cells were able to secrete MUC5B into conditioned media (see Fig. S2A in the supplemental material), although we found that the amounts secreted were variable between collections. Analysis of conditioned media from A549 cells showed that approximately 2% of Sia was 9-O-Ac on secreted proteins with no 7,9-O-Ac detected, indicating that, at least in these cells, secreted proteins were not enriched for O-acetylated Sia (Fig. S2B) . However, it would be worth examining proteins secreted by primary cells from humans or that are present in different tissues, including mucins, to determine whether the secreted proteins from A549 are representative of respiratory mucus.