The role of N-glycosylation in folding, trafficking, and functionality of lysosomal protein CLN5.
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.
Molecular, Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas, United States of America.
Akshay
A
Moharir
Sun H
SH
Peck
Theodore
T
Budden
Stella Y
SY
Lee
D011992:Endosomes; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017510:Protein Folding; D055550:Protein Stability; D021381:Protein Transport; D013347:Subcellular Fractions / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
C113332:CLN5 protein, human; D051907:Lysosomal Membrane Proteins; D008565:Membrane Proteins; D050505:Mutant Proteins; D020696:rab5 GTP-Binding Proteins