Cell biology and biochemical studies of ovine batten disease

Abstract

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of fatal inherited neurodegenerative lysosomal storage diseases of humans, which result in severe cortical atrophy, blindness, seizures, and the accumulation of fluorescent lysosome-derived organelles in neurons and most other cells throughout the body. Two naturally occurring of forms of NCL in sheep, CLN5 and CLN6, are used to study human disorders. A mutation in a soluble lysosomal protein (CLN5) causes NCL in Borderdale sheep and the South Hampshire sheep have a defective intracellular endoplasmic reticulum membrane protein (CLN6). The functions of these proteins are still unclear. Generation of highly specific and sensitive antibodies to these proteins would be very useful for biological and functional studies. Studies described in this thesis used ovine CLN5 and CLN6 NCL models to establish the full length coding sequences of the genes and expression of the encoded proteins. Antibodies against ovine CLN5 and CLN6 were generated and used for characterisation studies of both proteins in vitro. The full length ovine CLN5 gene sequence was amplified using a two-step PCR and ligation strategy with internal overhanging primers. Ovine CLN5 exon 1 has an allelic variant, but it was not associated with the CLN5 sheep mutation. Recombinant ovine CLN5 and CLN6 proteins were not expressed in Escherichia coli (E. coli) prokaryotic systems, perhaps because the gene sequences may be toxic to the host. Recombinant ovine CLN5 and CLN6 proteins were expressed successfully in eukaryotic mammalian cells using lentiviral vectors. The adenoviral antibodies against ovine CLN5 and CLN6 specifically recognized ovine CLN5 and CLN6 as shown by immunocytochemistry, Western blotting and mass spectrometry. The rabbit anti-CLN5 antibodies specifically identified the ovine mature CLN5 glycosylated form with a molecular weight of 60 kDa in media and cells and a molecular weight of 35 kDa when deglycosylated by PNGase F. The rabbit anti-CLN6 antibodies specifically detected overexpressed ovine CLN6 at molecular weight of 27 kDa in cells by Western blotting. Mass spectrometry revealed that the CLN5 N-signal sequence is cleaved off when it is mature. The deglycosylation study confirmed that CLN5 is a soluble glycosylated protein, containing high-mannose or complex type glycans at six out of eight predicted N-glycosylation sites. A lysosomal localisation of ovine CLN5 study was confirmed by rabbit anti-CLN5 antibodies labelling of the cells that also expressed the lysosomal associated membrane protein LAMP1, while ER intracellular localisation of ovine CLN6 was revealed by co-staining with rabbit anti-CLN6 antibodies and an ER marker. The CLN5 antibodies also detected endogenous CLN5 expression throughout the normal sheep and human brains. This study also provides evidence for the hypothesis of interactions between CLN5 and CLN6 in cultured and virally transduced neural cell lines using the anti-CLN5 and anti-CLN6 antibodies. Co-expression of CLN6 and CLN5 up-regulated CLN5 expression in CLN5 affected cells. In conclusion, the work described in this thesis elucidates the capability of lentiviral system expression of CLN5 and CLN6 proteins, and proved the reliability of the generated adenoviral CLN5 and CLN6 antibodies.