Defective Endoplasmic Reticulum-resident Membrane Protein CLN6 Affects Lysosomal Degradation of Endocytosed Arylsulfatase A

Abstract

Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a ϳ27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material.

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

“…The full topology of CLN6 is not known but the accessibility to glycosylation of a neo-N -glycosylation site at residues 151 Á153 of CLN6 suggests a luminal orientation for the predicted second loop (Heine et al 2004). In overexpressing cells CLN6 can form dimers (Heine et al 2004).…”

“…The full topology of CLN6 is not known but the accessibility to glycosylation of a neo-N -glycosylation site at residues 151 Á153 of CLN6 suggests a luminal orientation for the predicted second loop (Heine et al 2004). In overexpressing cells CLN6 can form dimers (Heine et al 2004). Analysis of fibroblast cells from CLN6 patients or animal models of the disease have documented that the transport, sorting and processing of newly synthesized lysosomal protease cathepsin D is not affected by defective CLN6.…”

“…Overexpressed CLN3 was found in the late endosomal/lysosomal compartment, at the plasma membrane and in neuronal cells in synaptosomes and endosomes (Järvelä et al 1998, Kyttälä et al 2004) and has been reported to play a role in arginine transport (Kim et al 2003). CLN6 and CLN8 are localized in the endoplasmic reticulum (ER) and CLN8 traffics to the ER-Golgi intermediate compartment (ER-GIC) (Lonka et al 2000, Heine et al 2004, Mole et al 2004. The function and interacting cytosolic and/or luminal proteins of both CLN6 and CLN8 are unknown.…”

btn1, theSchizosaccharomyces pombehomologue of the human Batten disease geneCLN3, regulates vacuole homeostasis

“…The NCLs are inherited in an autosomal recessive manner and six human NCL genes have now been identified (International Batten Disease Consortium, 1995;Gao et al, 2002;Ranta et al, 1999;Savukoski et al, 1998;Sleat et al, 1997;Vesa et al, 1995;Vines et al, 1999;Wheeler et al, 2002). Despite a common cellular phenotype of disturbed lysosomal function, not all of the proteins causing NCL are located in this organelle (Heine et al, 2004;Isosomppi et al, 2002;Järvelä et al, 1999;Järvelä et al, 1998;Lonka et al, 2000;Lonka et al, 2004;Mole et al, 2004;Ranta et al, 2004).…”

Myoclonic Epilepsy in Lysosomal Storage Disorders

“…It is localized in the ER and in neuronal cells it is additionally found along neural extension in subdomains of a tubular ER network. It contains a N-terminal cytoplasmic domain, seven putative transmembrane domains and a C-terminal luminal domain (Heine et al, 2004;Mole et al, 2004). The main storage component in NCL6 cells is the subunit c of the mitochondrial ATP Synthase (Elleder et al., 2006).…”