Abstract
CLN5 is a soluble lysosomal protein with unknown function. Mutations in CLN5 lead to neuronal ceroid lipofuscinosis, a group of inherited neurodegenerative disorders that mainly affect children. CLN5 has eight potential N-glycosylation sites based on the Asn-X-Thr/Ser consensus sequence. Through site-directed mutagenesis of individual asparagine residues to glutamine on each of the N-glycosylation consensus sites, we showed that all eight putative N-glycosylation sites are utilized in vivo. Additionally, localization studies showed that the lack of N-glycosylation on certain sites (N179, N252, N304, or N320) caused CLN5 retention in the endoplasmic reticulum, indicating that glycosylation is important for protein folding. Interestingly, one particular mutant, N401Q, is mislocalized to the Golgi, suggesting that N401 is not important for protein folding but essential for CLN5 trafficking to the lysosome. Finally, we analyzed several patient mutations in which N-glycosylation is affected. The N192S patient mutant is localized to the lysosome, indicating that this mutant has a functional defect in the lysosome. Our results suggest that there are functional differences in various N-glycosylation sites of CLN5 which affect folding, trafficking, and lysosomal function of CLN5.
Highlights
Neuronal ceroid lipofuscinoses (NCLs), known collectively as Batten disease, are a group of progressive neurodegenerative disorders that predominantly affect children
We show that all eight putative N-glycosylation sites of human CLN5 are utilized in vivo
This suggests that the Man-6-P modification on N401 is the major determinant of human CLN5 lysosomal transport via Man-6-P receptors (MPRs)-dependent route
Summary
Neuronal ceroid lipofuscinoses (NCLs), known collectively as Batten disease, are a group of progressive neurodegenerative disorders that predominantly affect children. With different ages of onset and progression, several forms of NCL have been characterized as infantile (INCL), late infantile (LINCL), juvenile (JNCL), and adult NCL (ANCL) [2]. NCL is classified as a lysosomal storage disorder based on the buildup of autofluorescent lipopigments, lipofuscin-like ceroids, in the lysosomes of neurons as well as some other cell types [3,4]. The lipopigments consist of proteins, lipids, and carbohydrates. Even though the proteins underlying NCLs are ubiquitously expressed, neuronal cells are by far the most affected cell type by these dysfunctional NCL proteins [7]
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