Defective intracellular transport of CLN3 is the molecular basis of Batten disease (JNCL)
Batten disease [juvenile-onset neuronal ceroid lipofuscinosis (JNCL)], the most common progressive encephalopathy of childhood, is caused by mutations in a novel lysosomal membrane protein (CLN3) with unknown function. In this study, we have confirmed the lysosomal localization of the CLN3 protein by immunoelectron microscopy by co-localizing it with soluble and membrane-associated lysosomal proteins. We have analysed the intracellular processing and localization of two mutants, 461-677del, which is present in 85% of CLN3 alleles and causes the classical JNCL, and E295K [corrected], which is a rare missense mutation associated with an atypical form of JNCL. Pulse-chase labelling and immunoprecipitation of the two mutant proteins in COS-1-cells indicated that 461-677del is synthesized as an approximately 24 kDa truncated polypeptide, whereas the maturation of E295K [corrected] resembles that of the wild-type CLN3 polypeptide. Transient expression of the two mutants in BHK cells showed that 461-677del is retained in the endoplasmic reticulum, whereas E295K [corrected] was capable of reaching the lysosomal compartment. The CLN3 polypeptides were expressed further in mouse primary neurons where the wild-type CLN3 protein was localized both in the cell soma and in neuronal extensions, whereas the 461-677del mutant was arrested in the cell soma. Interestingly, co-localization of the wild-type CLN3 and E295K [corrected] proteins with a synaptic vesicle marker indicates that the CLN3 protein might participate in synaptic vesicle transport/transmission. The data presented here provide clear evidence for a cellular distinction between classical and atypical forms of Batten disease both in neural and non-neural cells.
Järvelä
I
I
National Public Health Institute, Laboratory of Human Molecular Genetics, Helsinki, Finland. irma.jarvela@ktl.fi
Lehtovirta
M
M
Tikkanen
R
R
Kyttälä
A
A
Jalanko
A
A
D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D019556:COS Cells; D002460:Cell Line; D005057:Eukaryotic Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D006801:Humans; D008562:Membrane Glycoproteins; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron; D018832:Molecular Chaperones; D009154:Mutation; D009472:Neuronal Ceroid-Lipofuscinoses / Q000235:genetics*; D009474:Neurons / Q000378:metabolism; D011247:Pregnancy; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion; D013687:Telencephalon / Q000166:cytology; D014162:Transfection
C105199:CLN3 protein, human; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D011506:Proteins; D011993:Recombinant Fusion Proteins