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

Abstract

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 α2,6-linkage (α2,6-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 α2,6-Sia on short N-glycans and α2,3-Sia on larger N-glycans. By contrast, SIAT1 cells predominantly express α2,6-Sia glycans and greatly reduced level of α2,3-Sia glycans. Additionally, they express bisected, sialylated N-glycans that are scant in MDCK cells. The hCK cells exclusively express α2,6-Sia glycans. Unexpectedly, hCK glycoproteins bound robustly to the plant lectin MAL-1, indicating α2,3-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.

Viral envelope proteins fused to multiple distinct fluorescent reporters to probe receptor binding

“…Interestingly, A/HK/1/68 displayed different binding properties, dependent on the localization of the fusion protein, with the N-terminal FP being detrimental for receptor binding. For A/SG/16, fluorescence signal was higher in comparison to the MDCKs, which is potentially related to differences in glycan presentation on these cells [40-42].…”

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

“…Rearrangement of the sialyltransferase expression in hCK cells supported increased replication of many human H3N2 viruses [15]. However, only small quantities of glycans with multiple LacNAc repeating units appeared to be present on both MDCK and hCK cells [24]. Recently, we and others have shown that poly-LacNAc containing N -glycans are critical for the binding of contemporary H3N2 viruses [8, 12].…”

“…To allow the isolation of further evolved contemporary H3N2 viruses, with higher titers and fewer mutations, MDCK cells were further modified to eliminate α2,3-linked SIAs while also overexpressing α2,6-linked SIAs, resulting in "humanized" MDCK (hCK) cells [15]. Analysis of the N-glycans of MDCK, MDCK-SIAT1, and hCK cells indicated a low abundance of glycans with at least three successive LacNAc repeating units terminating in an α2,6-linked SIA [24]. The enzyme beta-1,3-Nacetylglucosaminyltransferase (B3GNT2) is responsible for the addition of Nacetylglucosamine to glycans, while the galactose is transferred to the glycan by the enzyme beta-1,4-galactosyltransferase 1 (B4GALT1).…”

“…Analysis of the N -glycans of MDCK, MDCK-SIAT1, and hCK cells indicated a low abundance of glycans with at least three successive LacNAc repeating units terminating in an α2,6-linked SIA [24]. The enzyme beta-1,3-N-acetylglucosaminyltransferase (B3GNT2) is responsible for the addition of N-acetylglucosamine to glycans, while the galactose is transferred to the glycan by the enzyme beta-1,4-galactosyltransferase 1 (B4GALT1).…”

Factors associated with viral RNA shedding and evaluation of potential viral infectivity at returning to school in influenza outpatients after treatment with baloxavir marboxil and neuraminidase inhibitors during 2013/2014–2019/2020 seasons in Japan: an observational study

“…Clinical samples (100 μL) were inoculated in confluent Madin-Darby canine kidney (MDCK) cells during the 2013/2014 and 2018/2019 seasons and SIAT-MDCK cells in 2019/2020 season propagated in 48-well plates to isolate the influenza viruses. Globally from 2018, isolating influenza A/H3N2 from MDCK cells became difficult; therefore, we changed the cells to MDCK-SIAT 1 that can proliferate A/H3N2 more easily [ 22 , 23 ]. The 48-well plates were then incubated at 34 °C with 5% CO 2 and observed daily for 5 days to detect the specific cytopathic effect (CPE) [ 24 ].…”