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

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Intracellular trafficking of CLN3, the protein underlying the childhood neurodegenerative disease, Batten disease

Juvenile neuronal ceroid lipofuscinoses (Batten disease) is a progressive neurodegenerative disorder resulting from mutations in the CLN3 gene, which encodes a hydrophobic 438 amino acid protein of unknown function. Prior studies have shown that CLN3 is expressed in multiple tissues, with highest levels in brain and testis. Experiments using cells overexpressing CLN3 indicate that CLN3 is a lysosomal resident protein. However, studies to date have not addressed trafficking of endogenous CLN3. As such, the purpose of the present study was two-fold. First, to develop a culture model to allow evaluation of native CLN3 transport. Second, to utilize available epitope-specific antibodies to determine if CLN3 reaches the plasma membrane en route to the lysosome. Our data using a NCCIT (embryonic testicular carcinoma) cell model coupled with surface biotinylation and antibody trapping demonstrated that at least a proportion of CLN3 trafficks to the lysosome via the cell membrane. Moreover, inhibition of the micro3A subunit of the AP-3 adapter protein complex increased levels of CLN3 at the cell surface.     

Cln3 function is linked to osmoregulation in a Dictyostelium model of Batten disease

Mutations in CLN3 cause a juvenile form of neuronal ceroid lipofuscinosis (NCL), commonly known as Batten disease. Currently, there is no cure for NCL and the mechanisms underlying the disease are not well understood. In the social amoeba Dictyostelium discoideum, the CLN3 homolog, Cln3, localizes predominantly to the contractile vacuole (CV) system. This dynamic organelle functions in osmoregulation, and intriguingly, osmoregulatory defects have been observed in mammalian cell models of CLN3 disease. Therefore, we used Dictyostelium to further study the involvement of CLN3 in this conserved cellular process. First, we assessed the localization of GFP-Cln3 during mitosis and cytokinesis, where CV system function is essential. GFP-Cln3 localized to the CV system during mitosis and cln3^? cells displayed defects in cytokinesis. The recovery of cln3^? cells from hypotonic stress and their progression through multicellular development was delayed and these effects were exaggerated when cells were treated with ammonium chloride. In addition, Cln3-deficiency reduced the viability of cells during hypotonic stress and impaired the integrity of spores. During hypertonic stress, Cln3-deficiency reduced cell viability and inhibited development. We then performed RNA sequencing to gain insight into the molecular pathways underlying the sensitivity of cln3^? cells to osmotic stress. This analysis revealed that cln3-deficiency upregulated the expression of tpp1A, the Dictyostelium homolog of human TPP1/CLN2. We used this information to show a correlated increase in Tpp1 enzymatic activity in cln3^? cells. In total, our study provides new insight in the mechanisms underlying the role of CLN3 in osmoregulation and neurodegeneration.     

Loss of the Batten disease gene CLN3 prevents exit from the TGN of the mannose 6-phosphate receptor

The neuronal ceroid lipofuscinoses (NCLs, Batten disease) are a group of inherited childhood-onset neurodegenerative disorders characterized by the lysosomal accumulation of undigested material within cells. To understand this dysfunction, we analysed trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR), which delivers the digestive enzymes to lysosomes. A common form of NCL is caused by mutations in CLN3, a multipass transmembrane protein of unknown function. We report that ablation of CLN3 causes accumulation of CI-MPR in the trans Golgi network, reflecting a 50% reduction in exit. This CI-MPR trafficking defect is accompanied by a fall in maturation and cellular activity of lysosomal cathepsins. CLN3 is therefore essential for trafficking along the route needed for delivery of lysosomal enzymes, and its loss thereby contributes to and may explain the lysosomal dysfunction underlying Batten disease.     

CLN3P, the Batten disease protein, localizes to membrane lipid rafts (detergent-resistant membranes)

Juvenile neuronal ceroid lipofuscinosis is an inherited pediatric neurodegenerative disorder, which occurs as a result of mutations in the CLN3 gene that is located on chromosome 16p12.1. The encoded protein, CLN3P, is a putative transmembrane protein with no known function. In this study, we demonstrate that CLN3P resides on membrane lipid raft domains (detergent-resistant membranes) and provide important new data towards possible functions of the protein.     

The yeast model for Batten disease: a role for Btn2p in the trafficking of the Golgi-associated vesicular targeting protein,Yif1p

Btn2p is a novel coiled coil cytosolic protein in Saccharomyces cerevisiae. We report that Btn2p interacts with Yif1p, a component of a protein complex at the Golgi that functions in ER to Golgi transport. Deletion of Btn2p, btn2-delta, results in mis-localiztion of Yif1p to the vacuole. Therefore, Btn2p may have an apparent role in intracellular trafficking of proteins. Btn2p was originally identified as being up-regulated in a btn1-delta strain, which exhibits dysregulation of vacuolar pH, and this up-regulation of Btn2p was presumed to contribute to maintaining a stable vacuolar pH [Pearce et al. Nat. Genet. 22 (1999) 55]. We propose that up-regulation of Btn2p in btn1-delta is an indicator of altered trafficking within the cell, and as btn1-delta serves as a model for the lysosomal storage disorder Batten disease, that altered intracellular trafficking may contribute to some of the cellular pathological hallmarks of this disease.     

A Lysosomal Proteinase, the Late Infantile Neuronal Ceroid Lipofuscinosis Gene (CLN2) Product, Is Essential for Degradation of a Hydrophobic Protein, the Subunit c of ATP Synthase

The specific accumulation of the hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with the late infantile form of neuronal ceroid lipofuscinosis (LINCL) is caused by lysosomal proteolytic dysfunction. The defective gene in LINCL (CLN2 gene) has been identified recently. To elucidate the mechanism of lysosomal storage of subunit c, antibodies against the human CLN2 gene product (Cln2p) were prepared. Immunoblot analysis indicated that Cln2p is a 46-kDa protein in normal control skin fibroblasts and carrier heterozygote cells, whereas it was absent in cells from four patients with LINCL. RT-PCR analysis indicated the presence of mRNA for CLN2 in cells from the four different patients tested, suggesting a low efficiency of translation of mRNA or the production of the unstable translation products in these patient cells. Pulse-chase analysis showed that Cln2p was synthesized as a 67-kDa precursor and processed to a 46-kDa mature protein (t(1/2) = 1 h). Subcellular fractionation analysis indicated that Cln2p is localized with cathepsin B in the high-density lysosomal fractions. Confocal immunomicroscopic analysis also revealed that Cln2p is colocalized with a lysosomal soluble marker, cathepsin D. The immunodepletion of Cln2p from normal fibroblast extracts caused a loss in the degradative capacity of subunit c, but not the beta subunit of ATP synthase, suggesting that the absence of Cln2p provokes the lysosomal accumulation of subunit c.     

Biochemical Characterization of a Lysosomal Protease Deficient in Classical Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) and Development of an Enzyme-Based Assay for Diagnosis and Exclusion of LINCL in HUman Specimens and Animal Models

Classical late-infantile neuronal ceroid lipofuscinosis (LINCL), a progressive and fatal neurodegenerative disease of childhood, results from mutations in a gene (CLN2) that encodes a protein with significant sequence similarity to prokaryotic pepstatin-insensitive acid proteases. We have developed a sensitive protease activity assay that allows biochemical characterization of the CLN2 gene product in various human biological samples, including solid tissues (brain and chorionic villi), blood (buffy coat leukocytes, platelets, granulocytes, and mononuclear cells), and cultured cells (lymphoblasts, fibroblasts, and amniocytes). The enzyme has a pH optimum of 3.5 and is rapidly inactivated at neutral pH. A survey of fibroblasts and lymphoblasts demonstrated that lack of activity was associated with LINCL arising from mutations in the CLN2 gene but not other neuronal ceroid lipofuscinoses (NCLs), including the CLN6 variant LINCL, classical infantile NCL, classical juvenile NCL, and adult NCL (Kufs' disease). A study conducted using blood samples collected from classical LINCL families whose affliction was confirmed by genetic analysis indicates that the assay can distinguish homozygotes, heterozygotes, and normal controls and thus is useful for diagnosis and carrier testing. Analysis of archival specimens indicates that several specimens previously classified as LINCL have enzyme activity and thus disease is unlikely to arise from mutations in CLN2. Conversely, a specimen previously classified as juvenile NCL lacks pepinase activity and is associated with mutations in CLN2. In addition, several animals with NCL-like neurodegenerative symptoms [mutant strains of mice (nclf and mnd), English setter, border collie, and Tibetan terrier dogs, sheep, and cattle] were found to contain enzyme activity and are thus unlikely to represent models for classical LINCL. Subcellular fractionation experiments indicate that the CLN2 protein is located in lysosomes, which is consistent with its acidic pH optimum for activity and the presence of mannose 6-phosphate. Taken together, these findings indicate that LINCL represents a lysosomal storage disorder that is characterized by the absence of a specific protease activity.     

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis,Autophagy, and CLN3 Protein Function

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) Ca²⁺ pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca²⁺-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca²⁺ chelation. Interrogation of intracellular Ca²⁺ handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca²⁺ pools and in store-operated Ca²⁺ uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca²⁺ handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening.     

Jid1 is a J-protein functioning in the mitochondrial matrix, unable to directly participate in endoplasmic reticulum associated protein degradation

J-proteins are a class of molecular chaperones that serve to stimulate the activity of Hsp70s and are often located to recruit Hsp70 to a particular cellular function. Protein degradation associated with the endoplasmic reticulum (ERAD) is one such cellular process that requires Hsp70 on both faces of the endoplasmic reticulum. At least five J-proteins, including Jid1 (DnaJ protein Involved in ER-associated Degradation), have been implicated in controlling ERAD. Here we show that Jid1 is confined within the mitochondrial matrix - Jid1 has the same topology as the J-proteins Pam18 and Mdj2, which stimulate mitochondrial Hsp70 to drive protein import into the mitochondrial matrix. The location of Jid1 within mitochondria makes it unavailable to participate directly in the regulation of ERAD.     

A mutation which disrupts the hydrophobic core of the signal peptide of bilirubin UDP-glucuronosyltransferase, an endoplasmic reticulum membrane protein, causes Crigler-Najjar type IIs

Crigler-Najjar (CN) disease is caused by a deficiency of the hepatic enzyme, bilirubin UDP-glucuronosyltransferase (B-UGT). We have found two CN type II patients, who were homozygous for a leucine to arginine transition at position 15 of B-UGT1. This mutation is expected to disrupt the hydrophobic core of the signal peptide of B-UGT1. Wild type and mutant B-UGT cDNAs were transfected in COS cells. Mutant and wild type mRNA were formed in equal amounts. The mutant protein was expressed with 0.5% efficiency, as compared to wild type. Mutant and wild type mRNAs were translated in vitro. Wild type transferase is processed by microsomes, no processing of the mutant protein was observed.     

3D3/lyric: a novel transmembrane protein of the endoplasmic reticulum and nuclear envelope, which is also present in the nucleolus

We previously performed a gene-trap screen in mouse cells with particular focus on clones in which the trapped protein-reporter fusions localise to compartments of the nucleus. Here we describe one such gene-trap line in which the fusion protein showed a unique, patchy distribution at the nuclear periphery. We have cloned the endogenous mouse and human cDNAs encoding the protein trapped in the F9/3D3 cell line. The predicted proteins (64 kDa) encoded by this novel gene are highly conserved and similar to an unpublished rat protein in sequence databases called p80 or lyric. The amino acid sequence of 3D3/lyric indicates that it may be a type-1b membrane protein with a single transmembrane domain (TMD). Antibodies against the endogenous protein recognise multiple isoforms, consistent with multiple 3D3/lyric mRNAs detected by Northern blot analysis. Subcellular fractionation and immunostaining show that 3D3/lyric is located not only principally in the endoplasmic reticulum (ER), but also in the nuclear envelope (NE), which is contiguous with this compartment. Furthermore, 3D3/lyric is also found in the nucleolus and is therefore a rare example of a protein that suggests a possible connection between this compartment and the endoplasmic reticulum.     

Mutant HFE H63D protein is associated with prolonged endoplasmic reticulum stress and increased neuronal vulnerability

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.     

Endoplasmic reticulum associated glucose-6-phosphatase activity is developmentally regulated and enriched in microsomes of endo/mesoderm in sea urchins

Summary Glucose-6-phosphatase (G-6-Pase) activity was analyzed during early embryogenesis of the sea urchinS. purpuratus. This activity is detected in very low levels in unfertilized eggs and early embryos but is present at high levels in preparations of endoplasmic reticulum (microsomes) from gastrula stage embryos. The approximately eight-fold increase in the relative activity of G-6-Pase associated with the ER occurs abruptly during a 12 h interval at gastrulation, and thereafter remains at a level comparable to that found in mammalian liver microsomes. The enzyme activity associated with the ER of gastrula stage embryos was completely eliminated from the microsomal pellet when cell lysates were first treated with non-ionic detergent. Analysis of germlayer tissues from late stage pluteus embryos revealed that G-6-Pase activity was more highly enriched in microsomes of endo/mesoderm tissues as compared to microsomes from ectoderm. The increase in ER associated G-6-Pase activity during embryonic development and its enriched activity in the ER of endo/mesoderm, as well as the observation that the signal recognition particle becomes associated with the ER at gastrulation (Le Blanc and Infante 1989), opens the question that this cellular organelle may be differentiating during embryogenesis in sea urchins.     

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.     

NMDA-induced retinal injury is mediated by an endoplasmic reticulum stress-related protein, CHOP/GADD153

We investigated the role of an endoplasmic reticulum stress-associated protein, CHOP/GADD153, after NMDA-induced mouse retinal damage. After injection of NMDA into the vitreous, TUNEL-positive cells were detected in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 6 h after NMDA injection, and these gradually increased in number up to 24 h. Analysis by real-time RT-PCR revealed that CHOP mRNA was induced by about 3-fold, at 2 h after NMDA injection. Immunoreactivity for the CHOP protein was intense in cells of the GCL following NMDA treatment. Immunoblot analysis showed that NMDA injection increased the expression of CHOP protein in the retina. Compared with wild-type mice, CHOP/ mice were more resistant to NMDA-induced retinal cell death as determined by TUNEL assay. At 7 days after NMDA treatment, the thickness of the inner plexiform layer and INL were larger in CHOP/ mice than in wild-type mice. The number of residual cells in the GCL following NMDA treatment was significantly higher in CHOP/ mice than in wild-type mice. In conclusion, CHOP is induced in mouse retina by NMDA treatment, and CHOP/ mice are more resistant to NMDA-induced retinal damage, suggesting that CHOP plays an important role in NMDA-induced retinal cell death.     

VIPL,a VIP36-like membrane protein with a putative function in the export of glycoproteins from the endoplasmic reticulum

Subsets of glycoproteins are thought to require lectin-like membrane receptors for efficient export out of the endoplasmic reticulum (ER). To identify new members related to two previously characterized intracellular lectins ERGIC-53/p58 and VIP36, we carried out an extensive database search using the conserved carbohydrate recognition domain (CRD) as a search string. A gene, more closely related to VIP36 than to ERGIC-53/p58, and hence called VIPL (VIP36-Like), was identified. VIPL has been conserved through evolution from zebra fish to man. The 2.4-kb VIPL mRNA was widely expressed to varying levels in different tissues. Using an antiserum prepared against the CRD, the 32-kDa VIPL protein was detected in various cell lines. The single N-linked glycan of VIPL remained endoglycosidase H-sensitive during a 2-h pulse-chase, even when the protein was overexpressed or mutated to allow export to the plasma membrane. VIPL localized primarily to the ER and partly to the Golgi complex. Like VIP36, the cytoplasmic tail of VIPL terminates in the sequence KRFY, a motif characteristic for proteins recycling between the ER and ERGIC/cis-Golgi. Mutating the retrograde transport signal KR to AA resulted in transport of VIPL to the cell surface. Finally, knock-down of VIPL mRNA using siRNA significantly slowed down the secretion of two glycoproteins (M(R) 35 and 250 kDa) to the medium, suggesting that VIPL may also function as an ER export receptor.