Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies

The Madin-Darby canine kidney (MDCK) cell line is an in vitro model for influenza A virus (IAV) infection and propagation. MDCK-SIAT1 (SIAT1) and humanized MDCK (hCK) cell lines are engineered MDCK cells that express N-glycans with elevated levels of sialic acid (Sia) in alpha 2,6-linkage (alpha 2,6-Sia) that are recognized by many human IAVs. To characterize the N-glycan structures in these cells and the potential changes compared to the parental MDCK cell line resulting from engineering, we analyzed the N-glycans from these cells at different passages, using both mass spectrometry and specific lectin and antibody binding. We observed significant differences between the three cell lines in overall complex N-glycans and terminal galactose modifications. MDCK cells express core fucosylated, bisected complex-type N-glycans at all passage stages, in addition to expressing alpha 2,6-Sia on short N-glycans and alpha 2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express alpha 2,6-Sia glycans and greatly reduced level of alpha 2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express alpha 2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating alpha 2,3-Sia glycans, but such binding was not Sia-dependent and closely mirrored that of an antibody that recognizes glycans with terminal 3-O-sulfate galactose (3-O-SGal). The 3-O-SGal epitope is highly expressed in N-glycans on multiple hCK glycoproteins. These results indicate vastly different N-glycomes between MDCK cells and the engineered clones that could relate to IAV infectivity.

Automated summary

The MDCK cell line is a useful in vitro model for studying influenza A virus infection, and the engineered cell lines SIAT1 and hCK, which express higher levels of sialic acid, are recognized by many human IAVs. Analysis of the N-glycan structures in these cells revealed significant differences compared to the parental MDCK cell line, including different types of glycans and modifications. These differences may impact the infectivity of IAVs.