Oral Presentation 23rd Annual Lorne Proteomics Symposium 2018

Glycolipid biosynthesis modulates protein glycosylation in ovarian cancer cells (#42)

Arun Everest-Dass 1 2 , Shahidul Alam 3 , Yen-Lin Huang 3 , Daniel Kolarich 1 , Francis Jacob 3 4 , Nicolle Packer 1 2 4 , Mark von Itzstein 1 4
  1. Institute for Glycomics , Griffith University, Gold Coast, QLD, Australia
  2. Biomolecular Discovery and Design Research Centre , Macquarie University, Sydney, NSW, Australia
  3. Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel, Basel, Switzerland
  4. #, Equal Authors

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.

  1. Alam, S., M. Anugraham, Y. L. Huang, R. S. Kohler, T. Hettich, K. Winkelbach, Y. Grether, M. N. Lopez, N. Khasbiullina, N. V. Bovin, G. Schlotterbeck and F. Jacob (2017). "Altered (neo-) lacto series glycolipid biosynthesis impairs alpha2-6 sialylation on N-glycoproteins in ovarian cancer cells." Sci Rep 7: 45367
  2. Anugraham, M., F. Jacob, S. Nixdorf, A. V. Everest-Dass, V. Heinzelmann-Schwarz and N. H. Packer (2014). "Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines: glycan structures reflect gene expression and DNA methylation status." Mol Cell Proteomics 13(9): 2213-2232.
  3. Anugraham, M., A. V. Everest-Dass, F. Jacob and N. H. Packer (2015). "A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells." Rapid Commun Mass Spectrom 29(7): 545-561.