Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

Abstract

The (neo-) lacto series glycosphingolipids (nsGSLs) comprise of glycan epitopes that are present as blood group antigens, act as primary receptors for human pathogens and are also increasingly associated with malignant diseases. Beta-1, 3-N-acetyl-glucosaminyl-transferase 5 (B3GNT5) is suggested as the key glycosyltransferase for the biosynthesis of nsGSLs. In this study, we investigated the impact of CRISPR-Cas9 -mediated gene disruption of B3GNT5 (∆B3GNT5) on the expression of glycosphingolipids and N-glycoproteins by utilizing immunostaining and glycomics-based PGC-UHPLC-ESI-QTOF-MS/MS profiling. ∆B3GNT5 cells lost nsGSL expression coinciding with reduction of α2-6 sialylation on N-glycoproteins. In contrast, disruption of B4GALNT1, a glycosyltransferase for ganglio series GSLs did not affect α2-6 sialylation on N-glycoproteins. We further profiled all known α2-6 sialyltransferase-encoding genes and showed that the loss of α2-6 sialylation is due to silencing of ST6GAL1 expression in ∆B3GNT5 cells. These results demonstrate that nsGSLs are part of a complex network affecting N-glycosylation in ovarian cancer cells.

L1 Cell Adhesion Molecule Confers Radioresistance to Ovarian Cancer and Defines a New Cancer Stem Cell Population

“…A detailed description of the CRISPR-Cas9 design, molecular cloning, cell-sorting strategy and characterization of homozygously deleted cells is provided in Supplemental information. Design and experimental strategy have been performed as recently described [19,53].…”

The P1 histo‐blood group antigen is present on human red blood cell glycoproteins

“…The anti‐P1 (clone P3NIL100) has been shown to be specific for P1 and not bind to the P k epitope even if P1 and P k share the terminal Galα1‐4Gal motif . To assess the reagent specificity in our system, RBCs were treated with α‐galactosidases.…”

“…The anti-P1 (clone P3NIL100) has been shown to be specific for P1 and not bind to the P k epitope even if P1 and P k share the terminal Galα1-4Gal motif. 38 To assess the reagent specificity in our system, RBCs were treated with α-galactosidases. The bacterial galactosidase specificity is limited to α1-3Gal, while the green coffee bean galactosidase cleaves terminal galactose (Gal) residues in α-linkage at carbon atom position 3, 4, or 6 in the Gal and is therefore able to destroy the P1 epitope, which has an α1-4-bound Gal at the terminal position.…”

Glycosphingolipid metabolism in cell fate specification

“…4C). An increase in histone acetylation at the GM2S (B4GALNT1) promoter was indeed observed in developing Arab et al, 1997Kovbasnjuk et al, 2005Geyer et al, 2016 SSEA-3 Breast cancer Chang et al, 2008 Globo H Breast cancer Ovarian cancer Thyroid carcinoma Chang et al, 2008Hakomori, 1989Cheng et al, 2016 DSGG Renal cell carcinoma Hepatocellular carcinoma Satoh et al, 1996Wu et al, 2012 SSEA-4 Breast cancer Glioblastoma Aloia et al, 2015Lou et al, 2014 Ganglio-series GSLs GM3 Acute myeloid leukemia (AML) Melanoma Multiple cancer types Wang et al, 2012Guthmann et al, 2004Zheng et al, 2018GM1 Lung cancer Fuentes et al, 1997 Fucosyl-GM1 Hepatocellular carcinoma Small cell lung carcinoma Wu et al, 2012Nilsson et al, 1986 GD3 Melanoma Neuroblastoma Lung cancer Cheresh et al, 1985Cheresh et al, 1986Fuentes et al, 1997 GD2 Melanoma Neuroblastoma Navid et al, 2010 Yang andSondel, 2010 Lacto-series GSLs Lex Breast cancer Colon cancer Gastric cancer Hakomori, 1989Hakomori, 1989Hakomori and Zhang, 1997 Lex-Lex Breast cancer Colon cancer Gastric cancer Hakomori, 1989Hakomori, 1989Hakomori and Zhang, 1997 Lea-Lea Colon cancer Gastric cancer Hakomori, 1989 Hakomori andZhang, 1997 Ley-Lex Pancreatic cancer Multiple cancer types Kim et al, 1988Zheng et al, 2018 SSEA-1 Renal cancer Liebert et al, 1987 Lc3 Acute myeloid leukemia (AML) Ovarian cancer Wang et al, 2012Alam et al, 2017 Acute myeloid leukemia (AML)…”