Understanding the Batten disease associated protein CLN5

Abstract

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative lysosomal storage diseases (LSD) that typically result in loss of vision, epilepsy, loss of motor function and cognitive decline. With a reported incidence of up to 1 in 12,500 births (Rider & Rider, 1988), the NCLs collectively are the most common neurodegenerative disease of childhood. At present all forms result in premature death for affected individuals. Mutations throughout the CLN5 gene are predominantly responsible for a late-infantile variant of NCL. The CLN5 protein is a soluble lysosomal protein of unknown function, with no strong sequence identity to any previously characterised protein.

The aim of this project was to better understand the normal role of the CLN5 protein by investigating its structure and function. To investigate structure, an optimised expression construct of CLN5 was designed for purification and crystallisation studies. HEK293FT cells were modified using lentiviral transduction to overexpress CLN5. CLN5 protein was collected from the media of these cells and purified using nickel affinity chromatography. Crystallisation trials of glycosylated CLN5 were performed, however no crystals were grown. Further analysis with glycosidases PNGase F and Endo H illustrated the importance of glycosylation in the folding, function and continued stability of CLN5.

Complementary to structural studies, sequence profile matching identified that CLN5 shares weak homology with a protease that utilises a cysteine-histidine catalytic dyad. To test for possible protease activity, the predicted catalytic cysteine was mutated via site directed mutagenesis to investigate function. Colocalisation analysis of the mutant compared to WT found that the mutant was still correctly localised to the lysosome, laying the groundwork for future functional analysis.

Combined, this work has advanced our understanding of CLN5 and will inform future structural and functional studies.