tetano
Editor, Senior Moderator
ci Rep
. 2022 Jul 26;12(1):12757.
doi: 10.1038/s41598-022-16605-5.
Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies
Lauren Byrd-Leotis[SUP] #[/SUP][SUP] 1 2 3 [/SUP], Nan Jia[SUP] #[/SUP][SUP] 2 [/SUP], Yasuyuki Matsumoto[SUP] 2 [/SUP], Dongli Lu[SUP] 2 [/SUP], Yoshihiro Kawaoka[SUP] 4 [/SUP], David A Steinhauer[SUP] 1 3 [/SUP], Richard D Cummings[SUP] 5 6 [/SUP]
Affiliations
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.
. 2022 Jul 26;12(1):12757.
doi: 10.1038/s41598-022-16605-5.
Sialylated and sulfated N-Glycans in MDCK and engineered MDCK cells for influenza virus studies
Lauren Byrd-Leotis[SUP] #[/SUP][SUP] 1 2 3 [/SUP], Nan Jia[SUP] #[/SUP][SUP] 2 [/SUP], Yasuyuki Matsumoto[SUP] 2 [/SUP], Dongli Lu[SUP] 2 [/SUP], Yoshihiro Kawaoka[SUP] 4 [/SUP], David A Steinhauer[SUP] 1 3 [/SUP], Richard D Cummings[SUP] 5 6 [/SUP]
Affiliations
- PMID: 35882911
- DOI: 10.1038/s41598-022-16605-5
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.