The effects of modified sialic acids on mucus and erythrocytes on influenza A virus HA and NA functions

Karen N. Barnard; Brynn K. Alford-Lawrence; David W. Buchholz; Brian R. Wasik; Justin R. LaClair; Hai Yu; Rebekah Honce; Stefan Ruhl; Petar Pajic; Erin K. Daugherity; Xi Chen; Stacey L. Schultz-Cherry; Hector C. Aguilar; Ajit Varki; Colin R. Parrish

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/800300 doi: bioRxiv preprint acetylneuraminic acid hydroxylase (CMAH) in the cytoplasm of cells, and this enzyme is missing or inactive in some animals, including humans (9, 12) . The addition of O-Ac to the C7 and/or C9 positions is mediated by the sialylate O-acetyltransferase enzyme, Cas1 domain containing 1 (CasD1), which has been suggested to add an O-acetyl group to the C7 position, from which it would migrate to the C8 and C9 position under physiological conditions, allowing the possibility 85 of the addition of another O-acetyl group to C7 (7, 13, 14) . The regulatory processes that control the number or positions of acetyl groups have not been well defined, although distinct differences in expression of 7,9-O-Ac and 9-O-Ac Sia have been reported in mouse and human tissues, chicken embryos, and in some other animals (7) . CasD1 uses acetyl-CoA to modify Sia in the activated CMP-Sia form before it is added to the glycan chain, and likely has a preference 90 for CMP-Neu5Ac as a substrate and is less active on CMP-Neu5Gc (14). The sialate Oacetylesterase (SIAE) enzyme can remove the 7,9-O-and 9-O-Ac modifications, although its activities and roles are not well understood (17) (18) (19) (20) . The 4-O-Ac Sia is produced in some tissues of many animals by a distinct sialate 4-O-acetyltransferase that is also likely expressed in the Golgi compartment; however, the gene for this enzyme has also not yet been identified 95 (21) (22) (23) . The 7, 8 , and/or 9-O-Ac Sia appear to be present at low levels -a few percent or less -in the cell-associated Sia on many cultured cells, but may be present at higher levels (10 to 50%) in Sia on the secreted mucus of various animals and on erythrocyte-associated glycans (15, 24) . However, the expression, distribution, and regulations of these modified Sia are not well 6 pathogens (26, 27) . Many pathogens therefore express proteins that attach to Sia, as well as expressing receptor-modifying enzymes such as sialidases (neuraminidases) that remove the Sia from the underlying glycan. Bacterial adhesins and toxins may recognize Sia on the surface of cells, and many bacteria can also use Sia as a metabolic carbon source after release through 110 the activity of neuraminidases, and uptake into the cell by Sia transporters (3, 28-31). These bacterial interactions with Sia are potentially affected by chemical modifications (30, 32) . Both enveloped and non-enveloped viruses may also bind Sia as primary receptors or co-receptors for cell recognition and infection, although only the enveloped viruses appear to express neuraminidases or sialate O-acetyl esterases, possibly to reduce aggregation of viral particles 115 during budding (5, 33) . For some viruses, Sia modifications are required for infection as viral proteins specifically bind to modified Sia -examples include human coronavirus OC43 and HKU1, and influenza C and D viruses, which all require 9-O-Ac Sia for cell infection (34, 35) .