Effect of substrate availability and O-GlcNAse inhibition on hyaluronan synthesis and intracellular trafficking of HAS3 in MV3 melanoma cells
Year: 2014
Type: dissertation
Abstract: Hyaluronan (HA) is a unique glycosaminoglycan present in the extracellular matrix of many vertebrates.HA is synthesized by a group of enzymes found in the plasma membrane called HA synthases (HAS1-3).The synthesized HA can perform a variety of functions by binding to cell surface receptors, affecting cell proliferation, adhesion, migration and invasion.HAS uses UDP-Glucuronic acid (UDP-GlcUA) and UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) to synthesize HA.Increased content of HA is seen in many cancers but its exact role and mechanism behind the tumor formation is still not clear.High expression of different HASs is also reported in many tumors.The aim of our work is to study the effect of substrate availability on intracellular trafficking of HAS3 and to investigate its role in controlling HA synthesis.For this purpose we have used chemical agents (4-methylumbelliferone and Mannose), which can alter the levels of UDP-sugars.We have also explored the possible effect on HAS3 endocytosis and recycling endocytosis.Other HA dependent functions like cell proliferation and migration are also studied.Dendra2-HAS3 is a photo convertible protein constructed to study the traffic of HAS3.MV3 metastatic melanoma cell lines with transient or stable transfection were used in this study.Live cell imaging with confocal microscope were employed for the trafficking experiments.Bio-Image XD, a new imaging software was used to analyze the trafficking data.The major findings in our experiments demonstrated that alteration of UDP-sugars (by 4-MU, Mannose) resulted in significant reduction of HAS3 trafficking from Golgi to Plasma membrane, which in turn reduced the HA synthesis.In a similar way the endocytosis of Dendra2-HAS3 was increased and significant reduction of HAS3 recycling endosomes were also noticed.Other HA dependent biological functions like proliferation and migration were also affected by the same chemical agents.Mannose significantly reduced the cell proliferation rate and slightly increased the migration, whereas 4-MU had only a mild effect on proliferation and migration.On the other hand, Thiamet G which can prevent the removal of GlcNAc modification from serine and threonine residues in O-GlcNAc modified proteins including HAS3 had no impact in these experiments, with the exception of reducing HAS3 recycling vesicles.From the above results it can be concluded that UDP-sugar substrates of HA have a direct connection with HAS3 traffic, which plays a key role in controlling the HA synthesis, endocytosis and recycling of HAS3.
Author: Avinash Rahul Bathina
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: green
Funder: Itä-Suomen Yliopisto
OpenAlex ID: W3097590924