title: 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 approximately 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.
authors.affiliation: Department of Biochemistry, Children's Hospital, University of Hamburg, D-20246 Hamburg, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Heine
authors.forename: Bettina
authors.initials: B
authors.lastname: Koch
authors.forename: Stephan
authors.initials: S
authors.lastname: Storch
authors.forename: Alfried
authors.initials: A
authors.lastname: Kohlschütter
authors.forename: David N
authors.initials: DN
authors.lastname: Palmer
authors.forename: Thomas
authors.initials: T
authors.lastname: Braulke
mesh_terms: D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002402:Cathepsin D / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002553:Cerebroside-Sulfatase / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011233:Precipitin Tests; D017510:Protein Folding; D012756:Sheep; D014162:Transfection
chemical_list: C447500:CLN6 protein, human; C486722:Cln6 protein, mouse; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D000939:Epitopes; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D002553:Cerebroside-Sulfatase; D002402:Cathepsin D