Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Carcinoembryonic antigen targeted glycomics and glycoproteomics reveals novel features of human glycosylation (64694)

Andreia Almeida 1 2 , Francis Jacob 3 , Kathrin Stavenhagen 4 5 , Kathirvel Alagesan 6 , Michaela Mischak 6 , Manfred Wuhrer 4 7 , Arun Everest-Dass 6 , Celso A. Reis 8 9 10 , Daniel Kolarich 2 6
  1. Institute for Glycomics, Griffith University, Southport, Queensland, Australia
  2. Department of Biomolecular Systems, Max-Planck Institute for Colloids and Interface, Potsdam, Germany
  3. Glyco-Oncology, Ovarian Cancer Research, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland
  4. Division of BioAnalytical Chemistry, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands
  5. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
  6. Institute for Glycomics, Griffith University, Southport, Queensland, Australia
  7. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands
  8. i3S – Institute for Research and Innovation in Health, University of Porto, Porto, Portugal
  9. Institute of Molecular Pathology and Immunology of the University of Porto, University of Porto, Porto, Portugal
  10. Instituto de Ciencias Biomedicas Abel Salazar (ICBAS), University of Porto, Porto, Portugal

Carcinoembryonic Antigen (CEA) is an FDA-approved tumour marker strongly associated with tumour progression and metastasis. With 28 potential sites of N-glycosylation, CEA is densely coated with glycans, however, the diagnostic potential embedded in its glycosylation to improve the selectivity of current cancer diagnostics is under-utilised.

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The glycosylation of CEA purified from four different body sources [human colon cancer (cell line and tissue), tissue from liver metastasis of colon cancer and ascites fluid] was investigated by porous-graphitised-carbon (PGC) LC-ESI-MS/MS glycomics, while CEA glycosylation sites where mapped using glycopeptides obtained by pronase digestion and analysed on a dual LC system combining reverse-phase and PGC chromatography within a single LC-ESI-MS/MS analysis.

 

The analysed CEAs exhibited 278 individual N-glycan structures, which vastly increases our knowledge on CEA N-glycosylation and makes it the most heterogeneously N-glycosylated protein known to date. Despite this huge diversity, CEA N-glycosylation carries body origin-specific glycosylation signatures, including an unusual glyco-epitope containing a b1-4 galactosidase resistant hexose attached to the bisecting GlcNAc. This hitherto unknown glyco-epitope was in particular present on colon derived CEA, while almost absent in any other CEAs analysed. Other distinct glycosylation differences such as N-glycan branching, degree of sialylation and level of bisecting N-glycans were uncovered between the CEA's from different body sources. Antennae fucosylation, such Lex and Leb/y determinants were present in all CEAs, however with significant abundance differences between the different sources. Next to confirming that 27 out of the 28 predicted N-glycosylation sites are glycosylated, we also identified a novel, 29th site of N-glycosylation on Asn76 that is located within a non-canonical 71N-R-Q73 sequence motif in the N-domain.

 

In summary, CEA-specific glycosylation bears a yet unmined potential to improve the specificity of the CEA-marker, but also to understand the role glycosylation plays for its function.