HEK293T cell lines defective for O-linked glycosylation

Abstract

Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE), galactokinase 1 (GALK1), and galactokinase 2 (GALK2) genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc) to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2), O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Human Immunodeficiency Virus and Simian Immunodeficiency Virus Maintain High Levels of Infectivity in the Complete Absence of Mucin-Type O-Glycosylation

“…By generating knockouts (KOs) in the GALE, GALE plus GALK1, and GALE plus GALK2 genes, we eliminated the cell lines' ability to O-glycosylate. Cellular proteins in these HEK293T knockout cell lines were found to be devoid of all O-linked carbohydrates by mass spectrometry (29). Also, gp120 made as the monomeric secreted protein in these knockout cell lines was found to be devoid of O-linked carbohydrate (29).…”

“…NL4-3 viral stocks devoid of all O-glycans. This was accomplished using HEK293T cell lines previously generated by our laboratory that are genetically modified by CRISPR/ Cas9 to knock out enzymes of the Leloir pathway of galactose metabolism (29). These enzymes are needed to generate the UDP-sugar precursors necessary for N-and O-linked glycosylation.…”

“…Cellular proteins in these HEK293T knockout cell lines were found to be devoid of all O-linked carbohydrates by mass spectrometry (29). Also, gp120 made as the monomeric secreted protein in these knockout cell lines was found to be devoid of O-linked carbohydrate (29). To determine the importance of O-glycosylation for NL4-3, virions were packaged in these cell lines to yield virus devoid of all O-linked glycans.…”

“…The GALEϩGALK2 KO cell line is defective in the synthesis of UDP-galactose and UDP-GalNAc as well as the salvage of GalNAc. Both of these cell lines have been well characterized and shown to be completely devoid of O-glycosylation regardless of the medium conditions utilized (29).…”

The human UDP-galactose 4’-epimerase (GALE) is required for cell surface glycome structure and function

“…Finally, our results may have implications for understanding global protein N-glycosylation in both model systems and galactosemic patients. Past studies of mammalian GALE have largely focused on its role in O-glycan biosynthesis (35,(84)(85)(86). Therefore, it is especially noteworthy that we discovered alterations in the N-glycans of several cell surface receptors ( Figs.…”