Juvenile neuronal ceroid lipofuscinosis (JNCL) is due to mutations in the CLN3 gene. We previously determined that CLN3 protein harbors a highly conserved motif, VYFAE, necessary for its impact on cel Show more
Juvenile neuronal ceroid lipofuscinosis (JNCL) is due to mutations in the CLN3 gene. We previously determined that CLN3 protein harbors a highly conserved motif, VYFAE, necessary for its impact on cell growth and apoptosis. Using molecular modeling we demonstrated that this motif is embedded in a stretch of amino acids that is homologous to and structurally compatible with a galactosylceramide (GalCer) binding domain. This domain is present in the V3 loop of the HIV-1 gp120 envelope protein, beta-amyloid protein, and the infectious form of prionic protein, and defines a binding site for lipid rafts. We determined the subcellular localization of CLN3 in different cell systems including human neurons, primary rat hippocampal neurons, normal human fibroblasts, and JNCL fibroblasts homozygous for the 1.02 kb deletion in genomic DNA. Wild-type CLN3 protein was present within Golgi, lipid rafts in the plasma membrane, and early recycling endosomes, but not late endosomes/lysosomes. Wild-type CLN3 internalized from the plasma membrane to the Golgi via Rab4- and Rab11-positive recycling endosomes. Wild-type CLN3 co-localized with GalCer in the Golgi and in lipid rafts at the plasma membrane in normal cells. Neither mutant CLN3 protein nor GalCer were found at the plasma membrane in JNCL fibroblasts. Mutant CLN3p was retained within the Golgi and partially mis-localized to lysosomes, failing to reach recycling endosomes, plasma membrane, or lipid rafts. These studies identify a novel CLN3 domain that may dictate localization and function of CLN3. Show less
Juvenile Batten disease (JNCL) is an autosomal recessive disease that results from mutations in the CLN3 gene. The wild-type CLN3 gene coding sequence has 15 exons, and the translated protein consists Show more
Juvenile Batten disease (JNCL) is an autosomal recessive disease that results from mutations in the CLN3 gene. The wild-type CLN3 gene coding sequence has 15 exons, and the translated protein consists of 438 amino acids. The most commonly observed mutation is a 1.02 kb deletion in the genomic DNA. This deletion results in a truncated protein due to the loss of amino acids 154-438, and the introduction of 28 novel amino acids at the c-terminus. We demonstrate that, compared to normal controls, CLN3-deficient immortalization of lymphoblasts homozygous for this deletion grow at a slower rate, and show increased sensitivity to etoposide-induced apoptosis, supporting the notion that CLN3 may negatively regulate apoptosis. Using immortalized JNCL lymphoblast cell lines as a model system, we assess the effects of specific CLN3 mutations on cell growth rates and protection from etoposide-induced apoptosis. Protection from etoposide-induced apoptosis occurs and the cell growth rate is restored following transfection of JNCL lymphoblasts with mutant CLN3 cDNA that includes exons 11 or 13. We show that deletion of the glycosylation sites 71NQSH74 and 310NTSL313, and also mutations within the highly conserved amino acid stretches 184WSSGTGGAGLLG195, 291VYFAE295 and 330VFASRSSL337, result in slowed growth and susceptibility to apoptosis. Show less