O-linked glycosylation is prominent in all domains of life, and O-glycoproteins participate in diverse cellular functions including signaling and cell communication, cell adhesion, immune surveillance and host-pathogen interactions, inflammation, and endocytosis. As a consequence, abnormal O-glycosylation is implicated in a number of human diseases including, cancer, Congenital Disorders of Glycosylation, and autoimmune diseases such as Tn syndrome and IgA nephropathy. In particular, the tumor associated O-glycans, Tn and its derivative sialyl Tn (STn), appear in the majority of cancers and are indicative of metastatic potential and poor prognosis. Tn-glycoproteins are likely to have altered function as a result of their abnormal O-glycosylation, and such outcomes may be important in promoting tumor development and metastasis. To address this, the Tn- glycoproteome of tumors must first be defined. Colorectal cancer (CRC) is the second leading cause of cancer- related deaths in the US, partly due to the lack of early detection methods.
Aim 1 will define the Tn-glycoproteome in human colorectal tumor specimens, using a robust proteomics methodology for identifying Tn-glycopeptides from cell lysates. We expect to identify O-glycoprotein membrane proteins including signaling receptors, adhesion receptors, and signaling ligands that may be useful targets for therapeutic intervention, and soluble Tn-glycoproteins that are potentially valuable biomarkers for early diagnosis. In disease models Tn expression is often a direct result of reduced or loss of T-synthase activity, which appends a galactose residue to the Tn antigen. T-synthase activity requires a specific protein chaperone, termed Cosmc, and expression of both T-synthase and Cosmc are essential for normal O-glycosylation. Reduced expression of Cosmc has been identified as the cause of Tn expression in human and mouse tumor cell lines and in Tn syndrome patients. Although Cosmc has emerged as a key regulator of Tn expression in vivo, a biophysical description of Cosmc/T-synthase interactions is lacking.
Aim 2 will (A) determine mechanisms of Tn expression by characterizing T- synthase and Cosmc interactions using biophysical tools, and (B) identify alternative chaperones using high- throughput screening. Determining the molecular mechanisms of Cosmc/T-synthase interactions will directly suggest other strategies that mimic the in vivo activation pathway, including molecular chaperones. Alternative molecular chaperones such as pharmacological chaperones and antibodies are a new class of valuable therapeutics that could be useful in the treatment of Tn diseases, including cancer, IgA nephropathy, and Tn syndrome.
Tn antigens are molecular markers of disease and indicate poor patient prognosis and high metastatic potential in a number of cancers. This research will characterize Tn antigens in human colorectal cancers, and study the underlying biochemical mechanisms for their expression. These results will directly suggest (1) early cancer detection methods and (2) new therapeutic strategies for cancer treatment (3) new therapeutics for autoimmune inflammatory diseases such as IgA nephropathy and Tn syndrome.
| Hanes, Melinda S; Moremen, Kelley W; Cummings, Richard D (2017) Biochemical characterization of functional domains of the chaperone Cosmc. PLoS One 12:e0180242 |
