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

• Published in: The Journal of biological chemistry 2004-05, Vol.279 (21), p.22347-22352
• Main Authors: Heine, Claudia, Koch, Bettina, Storch, Stephan, Kohlschütter, Alfried, Palmer, David N, Braulke, Thomas
• Format: Article
• Language: English
• Subjects: Animals; Biotinylation; Blotting, Western; Cathepsin D - pharmacology; Cell Line; Cell Membrane - metabolism; Cerebroside-Sulfatase - chemistry; Cerebroside-Sulfatase - metabolism; Cloning, Molecular; Cricetinae; Cross-Linking Reagents - pharmacology; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Endocytosis; Endoplasmic Reticulum - metabolism; Epitopes - chemistry; Fibroblasts - metabolism; Glycosylation; Golgi Apparatus - metabolism; Humans; Immunoblotting; Ligands; Lysosomes - metabolism; Membrane Proteins - chemistry; Membrane Proteins - physiology; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Mutation; Peptides - chemistry; Precipitin Tests; Protein Folding; Sheep; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M400643200

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 ∼27-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.