Mucin-type protein O-glycosylation (henceforth called O-glycosylation) is a ubiquitous and essential post translational modification of higher organisms. Most proteins passing through the secretory pathway are decorated with a wide range of mucin-type O-glycans which serve diverse biological functions. Hence, many biological processes and disease states are linked to normal or abnormal O-glycosylation including coronary artery disease, the regulation of kidney function, organogenisis, embryonic development, multiple cancers and fertility. Importantly, the loss of single O-glycan initiating and elongating transferases is developmentally lethal in the fly and mouse, respectively. Presently it is not well understood how these transferases chose their specific targets and what features of their substrates modulate their activities. Such an understanding, at the molecular level, is necessary for deducing the biological roles of O-glycosylation and for predicting sites of O- glycosylation. By understanding all of the factors involved in substrate selection new avenues will open for the development of novel and selective strategies to treat diseases of aberrant O-glycosylation including cancers. Furthermore, the ability to predict transferase specific sites of O-glycosylation will be invaluable for the interpretation of O-glycoproteomics data and for identifying the targets of glycosyltransferases linked to disease from genome wide association studies (GWAS). The planned research will focus on the large family of GalNAc-Ts (T1-T20) that initiate O-glycosylation and the core transferases (C1GALT1, B3GNT6, GCNT1 and ST6GalNAc-1 & 2) that perform the first step(s) of O-glycan elongation.
This research aims to characterize the unique peptide and glycopeptide substrate specificities of the GalNAc-Ts as well as to identify additional substrate features such as clustered charges and prior glycosylation that may control O- glycosylation. Our major working hypothesis is that O-glycan site selection and specific elongation are modulated by the properties of the peptide with one component being the charge distribution of residues flanking the acceptor site. This work will provide an unprecedented understanding of GalNAc-T substrate selection, achieved by correlating our specificity and kinetics data with the crystal structures of substrates bound or modeled onto the GalNAc-Ts and the core elongating transferases. Additional studies will involve characterizing the role of prior Thr versus Ser O-glycosylation, the glycosylation of Tyr residues and the further refinement of our web based isoform specific O-glycosylation prediction tool ISOGlyP. Together, these basic studies will greatly advance our understanding of the properties of these transferases and how they chose their targets and ultimately the mechanisms of their biological role and function in disease with an eventual goal to develop useful therapeutics for the treatment of diseases of aberrant O-glycosylation.
Protein glycosylation (i.e. the addition sugars to proteins) of the so-called mucin-type is an essential modification of higher organisms and is linked to many diseases in man. The results of this work will advance our understanding of the properties of the enzymes that perform this glycosylation and how they chose their targets at the molecular level. The results of these studies are fundamental for understanding the roles of these enzymes in disease and for the future development of novel highly specific therapeutics for use against diseases of O-glycosylation, including metabolic, inflammatory, cardiovascular and neoplastic diseases.
| de Las Rivas, Matilde; Paul Daniel, Earnest James; Coelho, Helena et al. (2018) Structural and Mechanistic Insights into the Catalytic-Domain-Mediated Short-Range Glycosylation Preferences of GalNAc-T4. ACS Cent Sci 4:1274-1290 |
| Sletmoen, Marit; Gerken, Thomas A; Stokke, Bjørn T et al. (2018) Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions. Glycobiology 28:437-442 |
| de Las Rivas, Matilde; Lira-Navarrete, Erandi; Daniel, Earnest James Paul et al. (2017) The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences. Nat Commun 8:1959 |
