Developing a novel approach to map signals required for GalNAcT2 relocation.

Abstract

Glycosylation is an important post-translational modification as it has multiple biological functions like cell-cell communications and cell-matrix interactions. Upon Src stimulation, polypeptide N-acetylgalactosaminyl transferases (GalNAcTs) are specifically redistributed from golgi to endoplasmic reticulum (ER). This is termed GalNAcT activation (GALA) pathway. As a consequence, there is an increase in Tn antigen synthesis which is a hallmark of cancer tissues. Aberrant glycosylation has been shown to affect cell adhesive properties. Since GALA pathway activation alters surface glycosylation, it might play a role in cancer progression by facilitating metastasis. Hence, it is important to determine the region(s) in GalNAcT2 that is required for the relocation. To achieve this, cloning of GalNAcT2 GFP and GFP FkBP mutants with different deletions or substitutions have been performed. Expressions of these proteins were determined using confocal imaging and western blotting. Results showed that these mutations did not affect GalNAcT2 mutants’ golgi localisation. Furthermore, to minimise the loss of GFP signals through anterograde trafficking of GalNAcT2 GFP FkBP mutant proteins, a new system was developed to trap these relocated proteins in ER upon addition of drug. To conclude, this project has set the foundation for studying the region(s) in GalNAcT2 required for relocation upon GALA pathway activation.