The neuronal ceroid lipofuscinosis protein CLN5: new insights into cellular maturation, transport, and consequences of mutations

Abstract

Neuronal ceroid lipofuscinoses (NCLs) represent a group of children's inherited neurodegenerative disorders caused by mutations in at least eight different genes. Mutations in the CLN5 gene result in the Finnish variant late infantile NCL characterized by gradual loss of vision, epileptic seizures, and mental deterioration. The CLN5 gene encodes a lysosomal glycoprotein of unidentified function. In this study, we have used both transient and stable expression systems for the characterization of CLN5, focusing on the localization, processing, and intracellular trafficking. We show that CLN5 is proteolytically cleaved, and that the mature polypeptide is transported to the lysosomes. Our data provide the first evidence that soluble CLN5 protein can also undergo mannose-6-phosphate receptor-independent trafficking to the lysosomes. We studied the localization and maturation of the CLN5 carrying the previously uncharacterized vLINCL disease causing mutations in HeLa cells. All analyzed disease mutations disturb the lysosomal trafficking of the mutated CLN5 proteins. The level of lysosomal targeting does not correlate, however, to disease onset, indicating that CLN5 may also function outside lysosomes. This study furthers our understanding of the basic properties of the CLN5 protein, necessary for the characterization of the consequences of disease mutations and for the planning of future therapies for vLINCL.

Adult-onset autosomal recessive ataxia associated with neuronal ceroid lipofuscinosis type 5 gene (CLN5) mutations

“…We evaluated the consequences of the p.Ser312Asn on CLN5 subcellular localization using immunofluorescence microscopy after HEK293 transfection ( Figure 1E-G). Wild-type protein localized to lysosomal vesicles as expected [16], whereas both the CLN5-Ser312Asn and the CLN5-fin (Finnish mutation …”

“…Both bioinformatics analysis and functional studies in HEK293 cells support the pathogenetic role of the mutation. While the wild-type CLN5 colocalize with lysosomes [16], the p.Ser312Asn mutant protein was retained in the ER and did not reach the lysosome, in analogy with p.Tyr392*, a previously well characterized CLN5 protein change causing an infantile-onset NCL in Finnish population. Therefore, our data further sustain the hypothesis that CLN5 mutations, including the one identified, impact the ER-lysosomal trafficking [15,16].…”

“…Human wild-type CLN5-pCMV (a.a. 1-407, CLN5-wt) and CLN5-pCMV carrying the Finnish major disease causing mutation p.Tyr392* (CLN5-fin) have previously been described [16]. The CLN5-Ser312Asn vector was generated by in vitro mutagenesis, using the Quick Change site directed mutagenesis kit (Stratagene, La Jolla, CA, USA).…”

“…Transient transfection with vectors containing CLN5-wt, CLN5-fin or CLN5-Ser312Asn cDNA was performed with Turbofect reagent (Thermo Fisher Scientific, Asheville, NC, USA) using the reported protocol [16] (see supplement).…”

“…We called variants using Genome Analysis Tool Kit (GATK, version 1.4) [12] followed by functional annotation with Annovar [13] and SnpEff [14]. In the second pipeline (P2), we conducted alignment to UCSC hg19 by Short Oligonucleotide Analysis Package (SOAP, version2.21) [15], then used SOAPsnp (version 1.05) [16] to identify single nucleotide variants (SNVs) as well as GATK [12] to detect small insertion-deletions (indels).…”