Chemical engineering of a single galactosyltranferase, to study its specific role in protein glycosylation and disease.

Summary

Despite broad acknowledgement of the relevance of glycoproteins in biological processes like cell-cell interactions, immune response, and cancer progression, our current understanding of glycoproteins is limited. A highly relevant, highly glycosylated protein class, is the mucin family, which are found to have altered glycosylation in various types of cancer. Mucin-type glycans are O-glycans, initiated by an N-acetylgalactosamine (GalNAc) attached to serine or threonine by polypeptide N-acetyl-α-galactosaminyltranferases (ppGalNAcTs). The proposed research will develop a novel tool to identify proteins specifically glycosylated by ppGalNAcT6, which is overexpressed in most types of breast cancer, To achieve this, I will engineer the active site of ppGalNAcT6 such that it can accommodate an enlarged UDP-GalNAc with an azide-spacer at the 2?-position. The UDP-sugar is designed such that other ppGalNAcTs will not recognize it. Thus, azides are introduced to glycoproteins specifically modified by the ppGalNAcT of interest. Azides can be used for labeling using well-established azide-related chemistry. Ultimately, this system will be translated to cells and a liposomal delivery system will be developed to deliver the UDP-sugar to the cells. Proteomic analyses will be performed to determine the protein range glycosylated by ppGalNAcT6 and obtain broader insight in the selectivity and mode of action of ppGalNAcT6.

Output

Publications for the general public

  • MF Debets(2015): Towards protein substrate identification of single ppGalNAcTs
  • MF Debets(2015): ppGalNAcT protein substrate identification
  • MF Debets(2015): Towards Liposomal delivery of UDP-sugars

Details

Project number

680-50-1310

Main applicant

Dr. M.F. Debets

Affiliated with

Universiteit Leiden, Faculteit der Wiskunde en Natuurwetenschappen, Leiden Institute of Chemistry

Team members

Dr. M.F. Debets

Duration

01/06/2014 to 31/05/2016