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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.

SUBMITTER: Byrd-Leotis L

PROVIDER: S-EPMC9325728 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:H3N2 strains of influenza A virus emerged in humans in 1968 and have continued to circulate, evolving in response to human immune pressure. During this process of "antigenic drift," viruses have progressively lost the ability to agglutinate erythrocytes of various species and to replicate efficiently under the established conditions for amplifying clinical isolates and generating vaccine candidates. We have determined the glycome profiles of chicken and guinea pig erythrocytes to gain insights into reduced agglutination properties displayed by drifted strains and show that both chicken and guinea pig erythrocytes contain complex sialylated N-glycans but that they differ with respect to the extent of branching, core fucosylation, and the abundance of poly-N-acetyllactosamine (PL) [-3Galβ1-4GlcNAcβ1-]n structures. We also examined binding of the H3N2 viruses using three different glycan microarrays: the synthetic Consortium for Functional Glycomics array; the defined N-glycan array designed to reveal contributions to binding based on sialic acid linkage type, branched structures, and core modifications; and the human lung shotgun glycan microarray. The results demonstrate that H3N2 viruses have progressively lost their capacity to bind nearly all canonical sialylated receptors other than a selection of biantennary structures and PL structures with or without sialic acid. Significantly, all viruses displayed robust binding to nonsialylated high-mannose phosphorylated glycans, even as the recognition of sialylated structures is decreased through antigenic drift.IMPORTANCE Influenza subtype H3N2 viruses have circulated in humans for over 50 years, continuing to cause annual epidemics. Such viruses have undergone antigenic drift in response to immune pressure, reducing the protective effects of preexisting immunity to previously circulating H3N2 strains. The changes in hemagglutinin (HA) affiliated with drift have implications for the receptor binding properties of these viruses, affecting virus replication in the culture systems commonly used to generate and amplify vaccine strains. Therefore, the antigenic properties of the vaccines may not directly reflect those of the circulating strains from which they were derived, compromising vaccine efficacy. In order to reproducibly provide effective vaccines, it will be critical to understand the interrelationships between binding, antigenicity, and replication properties in different growth substrates.

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

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