Glycan moieties associated with proteins and lipids have been implicated in the regulation of numerous biological and physiological events. We recently observed that altering the (neo-) lacto series biosynthesis in glycosphingolipids (GSLs) impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells1. To understand this unexpected interplay between two distinct classes of glycoconjugates, we investigated the role of different glucosylceramide-related GSL series by the deletion of specific glycosyltransferase-encoding genes in ovarian cancer using CRISPR-Cas9.
We identified α1-4-galactosyltransferase (A4GALT), β-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5) and β-1,4-N-acetyl-galactosaminyltransferase 1 (B4GALNT1) as key transferases directly involved in the extension of the lactosylceramide to globo, (neo-) lacto and ganglio-series, respectively.
We successfully deleted these transferases through CRISPR-Cas9 -mediated gene disruption in the ovarian cancer cell line IGROV1. In addition, the UDP-Glucose Ceramide Glucosyltransferase that catalyses the first glycosylation step in the biosynthesis of GSLs was also deleted to identify its impact on protein glycosylation. The membrane glycans from proteins and lipids were analysed using negative mode porous graphitic carbon liquid chromatography mass spectrometry2,3.
We observed massive changes in protein glycosylation in all mutant cells, specifically N-linked glycans showed several terminal glycan epitopes such as sialic acids, LacDiNac and bisecting GlcNAc that were significantly different. For example, there was an increase in bisecting structures in ΔA4GALT and ΔB3GNT5 cells, while in ΔB4GALNT1 cells these structures were decreased. The impairment of α2-6 sialylation in ΔA4GALT and ΔB3GNT5 cells was consistent with previous observations, whereas the overall sialyation was increased in ΔB4GALNT1 cells albeit with diminished α2-6 sialylation.
This is the first ever study of systematically modifying GSL biosynthesis to characterise their effect on protein and lipid glycosylation. GSLs play an important role in moderating cell surface glycosylation and therefore its physiological state, an important factor in ovarian cancer metastasises.