A method to assess the lysosomal residence of proteins in cultured cells.

Analytical subcellular fractionation is playing an increasingly important role in proteomic studies to identify and validate components of cellular organelles. For lysosomes, definitive studies in this area have been restricted to rodent tissues due to technical constraints. Our goal was to design a quantitative assay that would allow clear demonstration of lysosomal localization in cultured human cells. We found that culturing HepG2 (human hepatocellular carcinoma) cells in progesterone-containing medium elicited an extensive shift in the buoyant density of lysosomes as measured by isopycnic centrifugation in sucrose density gradients. The density of other organelles remained essentially unchanged; thus, this shift represents a specific test for lysosomal localization. Progesterone treatment of a variety of other cultured cells also elicited a shift in lysosome density. This approach should represent a valuable tool for identification and validation of both luminal and membrane lysosomal proteins.

Aminoglycoside-mediated suppression of nonsense mutations in late infantile neuronal ceroid lipofuscinosis.

The ability of aminoglycoside antibiotics to promote readthrough of eukaryotic stop codons has attracted interest in these drugs as potential therapeutic agents in human disorders caused by nonsense mutations. One disease for which such a therapeutic strategy may be viable is classical late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative disorder with currently no effective treatment. Premature stop codon mutations in the gene CLN2 encoding the lysosomal tripeptidyl-peptidase 1 (TPP-I) are associated with disease in approximately half of children diagnosed with LINCL. The aim of this study was to examine the ability of the aminoglycoside gentamicin to restore TPP-I activity in LINCL cell lines. In one patient-derived cell line that was compound heterozygous for a commonly seen nonsense mutation, Arg208Stop and a different rare nonsense mutation, approximately 7% of normal levels of TPP-I were maximally restored with gentamicin treatment. In other cell lines from patients that were compound heterozygous for Arg208Stop and a splice junction mutation, approximately 0.5% of maximal activity was restored. These results suggest that pharmacological suppression of nonsense mutations by aminoglycosides or functionally similar pharmaceuticals may have therapeutic potential in LINCL.

Congenital ovine neuronal ceroid lipofuscinosis--a cathepsin D deficiency with increased levels of the inactive enzyme.

We recently showed that a form of neuronal ceroid lipofuscinosis (NCL) in white Swedish landrace sheep is caused by a missense mutation in the cathepsin D gene resulting in complete inactivation of the enzyme. Despite the lack of cathepsin D activity, the brains of the cathepsin D deficient sheep showed strongly increased staining for cathepsin D in immunohistochemistry. By Western blotting, a 5-10 fold increase in the level of cathepsin D was confirmed. These results indicate that the missense mutation in congenital NCL sheep results in the synthesis of an inactive yet stable cathepsin D.

Niemann-Pick disease type C: spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

Expression and analysis of CLN2 variants in CHO cells: Q100R represents a polymorphism, and G389E and R447H represent loss-of-function mutations.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal hereditary childhood disease. The gene underlying LINCL, CLN2, encodes a lysosomal enzyme, tripeptidyl peptidase I (TPP-I), deficiency in which leads to lysosomal accumulation of autofluorescent materials accompanied by severe neuronal atrophy. Mutational analysis was conducted to characterize different CLN2 alleles. Two probands of Romany origin were found to be homozygous for an allele that encoded a protein with two changes, designated Q100R+G389E CLN2. To distinguish potential polymorphisms from mutations, a recombinant expression system was used to investigate individual constructs. Elevated levels of TPP-I activity in CHO cells expressing Q100R CLN2 and background activity in CHO cells expressing G389E CLN2 clearly defines G389E as a pathogenic mutation and indicates that Q100R is a polymorphism. Association of the R447H mutation with a delayed onset form of LINCL in two separate families raised the question of whether R447H CLN2 retains residual activity. However, CHO cells expressing R447H CLN2 had TPP-I activity comparable to that of neo transfected cells, indicating that any residual activity was below the level of detection in this experimental system. Hum Mutat 18:165, 2001.

Production and characterization of recombinant human CLN2 protein for enzyme-replacement therapy in late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is a fatal recessive childhood disease caused by mutations in the CLN2 gene, which encodes the lysosomal enzyme tripeptidyl peptidase I. As a step towards understanding the protein and developing therapeutics for the disease, we have produced and characterized recombinant human CLN2 (ceroid lipofuscinosis, neuronal 2) protein from Chinese-hamster ovary cells engineered to secrete high levels of the enzyme. The protein was secreted as an inactive soluble proenzyme of approximately 65 kDa that appears as a monomer by gel filtration. Upon acidification, the protein is processed to mature form and acquires activity. The enzyme is efficiently delivered to the lysosomes of LINCL fibroblasts by mannose 6-phosphate-receptor-mediated endocytosis (EC(50) approximately 2 nM), where it remains active for long periods of time (t(1/2) approximately 12 days). In addition, the enzyme is taken up by rat cerebellar granule neurons by mannose 6-phosphate-dependent and -independent mechanisms. Treatment of LINCL fibroblasts with recombinant CLN2 protein restores normal enzyme activity and ameliorates accumulation of the major storage protein, mitochondrial ATP synthase subunit c.

The human CLN2 protein/tripeptidyl-peptidase I is a serine protease that autoactivates at acidic pH.

The CLN2 gene mutated in the fatal hereditary neurodegenerative disease late infantile neuronal ceroid lipofuscinosis encodes a lysosomal protease with tripeptidyl-peptidase I activity. To understand the enzymological properties of the protein, we purified and characterized C-terminal hexahistidine-tagged human CLN2p/tripeptidyl-peptidase I produced from insect cells transfected with a baculovirus vector. The N terminus of the secreted 66-kDa protein corresponds to residue 20 of the primary CLN2 gene translation product, indicating removal of a 19-residue signal peptide. The purified protein is enzymatically inactive; however, upon acidification, it is proteolytically processed and concomitantly acquires enzymatic activity. The N terminus of the final 46-kDa processed form (Leu196) corresponds to that of mature CLN2p/tripeptidyl-peptidase I purified from human brain. The activity of the mature enzyme is irreversibly inhibited by the serine esterase inhibitor diisopropyl fluorophosphate, which specifically and stoichiometrically reacts with CLN2p/tripeptidyl-peptidase I at Ser475, demonstrating that this residue represents the active site nucleophile. Expression of wild type and mutant proteins in CHO cells indicate that Ser475, Asp360, Asp517, but not His236 are essential for activity. These data indicate that the CLN2 gene product is synthesized as an inactive proenzyme that is autocatalytically converted to an active serine protease.

Identification of HE1 as the second gene of Niemann-Pick C disease.

Niemann-Pick type C2 disease (NP-C2) is a fatal hereditary disorder of unknown etiology characterized by defective egress of cholesterol from lysosomes. Here we show that the disease is caused by a deficiency in HE1, a ubiquitously expressed lysosomal protein identified previously as a cholesterol-binding protein. HE1 was undetectable in fibroblasts from NP-C2 patients but present in fibroblasts from unaffected controls and NP-C1 patients. Mutations in the HE1 gene, which maps to chromosome 14q24.3, were found in NP-C2 patients but not in controls. Treatment of NP-C2 fibroblasts with exogenous recombinant HE1 protein ameliorated lysosomal accumulation of low density lipoprotein-derived cholesterol.

A mutation in the ovine cathepsin D gene causes a congenital lysosomal storage disease with profound neurodegeneration.

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of neurodegenerative storage diseases characterized by progressive psychomotor retardation, blindness and premature death. Pathologically, there is accumulation of autofluorescent material in lysosome-derived organelles in a variety of cell types, but neurons in the central nervous system appear to be selectively affected and undergo progressive death. In this report we show that a novel form of NCL, congenital ovine NCL, is caused by a deficiency in the lysosomal aspartyl proteinase cathepsin D. A single nucleotide mutation in the cathepsin D gene results in conversion of an active site aspartate to asparagine, leading to production of an enzymatically inactive but stable protein. This results in severe cerebrocortical atrophy and early death, providing strong evidence for an important role of cathepsin D in neuronal development and/or homeostasis.

Prenatal testing for late infantile neuronal ceroid lipofuscinosis.

Classic late infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative disease in which autofluorescent "curvilinear" storage bodies accumulate in tissues from affected patients. Recently, the LINCL gene (CLN2) has been found to code for a pepstatin-insensitive lysosomal protease whose activity is deficient in LINCL specimens. We report the first 2 cases of successful prenatal testing for LINCL by using DNA and enzyme-based methods on amniocytes, and describe a new private mutation in one of the families analyzed. These approaches allow definitive prenatal diagnosis and represent a significant improvement over previous pathological methods.

Subcellular localization of mannose 6-phosphate glycoproteins in rat brain.

The intracellular transport of soluble lysosomal enzymes relies on the post-translational modification of N-linked oligosaccharides to generate mannose 6-phosphate (Man 6-P) residues. In most cell types the Man 6-P signal is rapidly removed after targeting of the precursor proteins from the Golgi to lysosomes via interactions with Man 6-phosphate receptors. However, in brain, the steady state proportion of lysosomal enzymes containing Man 6-P is considerably higher than in other tissues. As a first step toward understanding the mechanism and biological significance of this observation, we analyzed the subcellular localization of the rat brain Man 6-P glycoproteins by combining biochemical and morphological approaches. The brain Man 6-P glycoproteins are predominantly localized in neuronal lysosomes with no evidence for a steady state localization in nonlysosomal or prelysosomal compartments. This contrasts with the clear endosome-like localization of the low steady state proportion of mannose-6-phosphorylated lysosomal enzymes in liver. It therefore seems likely that the observed high percentage of phosphorylated species in brain is a consequence of the accumulation of lysosomal enzymes in a neuronal lysosome that does not fully dephosphorylate the Man 6-P moieties.

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.

Mutational analysis of the defective protease in classic late-infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder.

The late-infantile form of neuronal ceroid lipofuscinosis (LINCL) is a progressive and ultimately fatal neurodegenerative disease of childhood. The defective gene in this hereditary disorder, CLN2, encodes a recently identified lysosomal pepstatin-insensitive acid protease. To better understand the molecular pathology of LINCL, we conducted a genetic survey of CLN2 in 74 LINCL families. In 14 patients, CLN2 protease activities were normal and no mutations were identified, suggesting other forms of NCL. Both pathogenic alleles were identified in 57 of the other 60 LINCL families studied. In total, 24 mutations were associated with LINCL, comprising six splice-junction mutations, 11 missense mutations, 3 nonsense mutations, 3 small deletions, and 1 single-nucleotide insertion. Two mutations were particularly common: an intronic G-->C transversion in the invariant AG of a 3' splice junction, found in 38 of 115 alleles, and a C-->T transition in 32 of 115 alleles, which prematurely terminates translation at amino acid 208 of 563. An Arg-->His substitution was identified, which was associated with a late age at onset and protracted clinical phenotype, in a number of other patients originally diagnosed with juvenile NCL.

Specific alterations in levels of mannose 6-phosphorylated glycoproteins in different neuronal ceroid lipofuscinoses.

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.