Expression and lysosomal targeting of CLN7, a major facilitator superfamily transporter associated with variant late-infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCLs) constitute a group of progressive neurodegenerative disorders resulting from mutations in at least eight different genes. Mutations in the most recently identified NCL gene, MFSD8/CLN7, underlie a variant of late-infantile NCL (vLINCL). The MFSD8/CLN7 gene encodes a polytopic protein with unknown function, which shares homology with ion-coupled membrane transporters. In this study, we confirmed the lysosomal localization of the native CLN7 protein. This localization of CLN7 is not impaired by the presence of pathogenic missense mutations or after genetic ablation of the N-glycans. Expression of chimeric and full-length constructs showed that lysosomal targeting of CLN7 is mainly determined by an N-terminal dileucine motif, which specifically binds to the heterotetrameric adaptor AP-1 in vitro. We also show that CLN7 mRNA is more abundant in neurons than astrocytes and microglia, and that it is expressed throughout rat brain, with increased levels in the granular layer of cerebellum and hippocampal pyramidal cells. Interestingly, this cellular and regional distribution is in good agreement with the autofluorescent lysosomal storage and cell loss patterns found in brains from CLN7-defective patients. Overall, these data highlight lysosomes as the primary site of action for CLN7, and suggest that the pathophysiology underpinning CLN7-associated vLINCL is a cell-autonomous process.

An EBV-based mammalian cell expression vector for efficient expression of cloned coding sequences.

The construction of a mammalian cell expression vector using human cytomegalovirus immediate early gene enhancer to initiate transcription of inserted coding sequences is described. The vector also carries Epstein-Barr virus EBNA-1 nuclear antigen gene, ori-P sequences and hygromycin B resistance gene hph from E. coli. The expression capacity of this construct was tested by inserting the chloramphenicol acetyltransferase (CAT) gene into the vector. The EBV-CAT construct was transfected into various cell lines and high levels of CAT activity were obtained in human and monkey cells. In these cells, the vector DNA also replicates as an extrachromosomal element having 1 to 20 copies per cell. In most cases, the vector copy number and the expression level of inserted gene was in positive correlation in different cell clones.

Correction of peripheral lysosomal accumulation in mice with aspartylglucosaminuria by bone marrow transplantation.

OBJECTIVE: Bone marrow transplantation has been shown to alleviate symptoms outside the CNS in many lysosomal storage diseases depending on the type and stage of the disease, but the effect on neurological symptoms is variable or still unclear. Aspartylglucosaminuria (AGU) is a lysosomal storage disease characterized by mental retardation, recurrent infections in childhood, hepatosplenomegaly and coarse facial features. Vacuolized storage lysosomes are found in all tissues of patients and uncleaved enzyme substrate is excreted in the urine. The recently generated AGU mouse model closely mimicks the human disease and serves as a good model to study the efficiency of bone marrow transplantation in this disease. METHODS: Eight-week-old AGU mice were lethally irradiated and transplanted with bone marrow from normal donors. The AGA enzyme activity was measured in the liver and the brain and the degree of correction of tissue pathology was analyzed by light and electron microscopy. Reverse bone marrow transplantation (AGU bone marrow to wild-type mice) was also performed. RESULTS: Six months after transplantation the AGA enzyme activity was 13% of normal in the liver, but only 3% in the brain. Tissue pathology was reversed in the liver and the spleen, but not in the brain and the kidney. The urinary excretion of enzyme substrate was diminished but still detectable. No storage vacuoles were found in the tissues after reverse transplantation, but subtle excretion of uncleaved substrate was detected in the urine. CONCLUSION: Liver and spleen pathology of AGU was corrected by bone marrow transplantation, but there was no effect on lysosomal accumulation in the CNS and in the kidneys.

Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer.

The ability of lysosomal enzymes to be secreted and subsequently captured by adjacent cells provides an excellent basis for investigating different therapy strategies in lysosomal storage disorders. Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminidase (AGA) leading to interruption of the ordered breakdown of glycoproteins in lysosomes. As a consequence of the disturbed glycoprotein catabolism, patients with AGU exhibit severe cell dysfunction especially in the central nervous system (CNS). The uniform phenotype observed in these patients will make effective evaluation of treatment trials feasible in future. Here we have used fibroblasts and lymphoblasts from AGU patients and murine neural cell lines as targets to evaluate in vitro the feasibility of enzyme replacement and gene therapy in the treatment of this disorder. Complete correction of the enzyme deficiency was obtained both with recombinant AGA enzyme purified from CHO-K1 cells and with retrovirus-mediated transfer of the AGA gene. Furthermore, we were able to demonstrate enzyme correction by cell-to-cell interaction of transduced and nontransduced cells.

Monitoring the CNS pathology in aspartylglucosaminuria mice.

Aspartylglucosaminuria (AGU) is a recessively inherited lysosomal storage disorder caused by the deficiency of the aspartylglucosaminidase (AGA) enzyme. The hallmark of AGU is slowly progressing mental retardation but the progression of brain pathology has remained uncharacterized in humans. Here we describe the long-term follow-up of mice carrying a targeted AGU-mutation in both alleles. Immunohistochemistry, histology, electron microscopy, quantitative magnetic resonance imaging (MRI) and behavioral studies were carried out to evaluate the CNS affection of the disease during development. The lysosomal storage vacuoles of the AGA -/- mice were most evident in central brain regions where MRI also revealed signs of brain atrophy similar to that seen in the older human patients. By immunohistochemistry and MRI examinations, a subtle delay of myelination was observed in AGA -/- mice. The life span of the AGA -/- mice was not shortened. Similar to the slow clinical course observed in human patients, the AGA -/- mice have behavioral symptoms that emerge at older age. Thus, the AGU knock-out mice represent an accurate model for AGU, both histopathologically and phenotypically.

Efficient synthesis of influenza virus hemagglutinin in mammalian cells with an extrachromosomal Epstein-Barr virus vector.

The capability of an Epstein-Barr virus hybrid vector (EBV-CMV), containing the cytomegalovirus (CMV) immediate early enhancer and simian virus 40 promoter, to produce large amounts of authentic mammalian proteins was studied. The cDNA of influenza virus hemagglutinin (HA), a cell surface glycoprotein, was inserted into this vector and the EBV-CMV-HA plasmid was transfected into two human and two monkey cell lines. Southern-blot analysis revealed that the EBV-CMV-HA plasmid was maintained in extrachromosomal state and the recombinant cell clones contained on the average three copies (range 1-24) of the transfected DNA. The recombinant HA polypeptides from different cell clones, selected either randomly or by fluorescence-activated cell sorter, were analysed using immunological techniques. Three of the four cell lines expressed recombinant HA on the cell surface in glycosylated form. The highest production levels, 11.5 micrograms/10(6) cells, were obtained in HeLa cells containing only two copies of EBV-CMV-HA DNA per cell. The protein levels correlated with the mRNA levels in Northern-blot analysis. A corresponding vector, containing the same regulatory signals for HA expression, but lacking the EBV sequences, yielded clones with significantly lower expression levels. The results confirm that the extrachromosomal EBV-CMV vector is very useful in the production of apparently authentic mammalian glycoproteins.

Expression of Semliki Forest virus capsid protein from SV40 recombinant virus.

Here, the proteolytic processing of the Semliki Forest virus (SFV) capsid protein was studied in the absence of other viral functions. Two different fragments of the SFV messenger cDNA, coding for capsid protein and 174 and 38 extra amino acids from the envelope proteins, respectively, were cloned in the late region of the SV40 viral DNA. Cells infected with the SV40 recombinant virus stocks were analyzed for the production of SFV capsid mRNA and polypeptide. Immunofluorescence staining of the infected cells indicated that the produced SFV capsid protein accumulated mainly in the nucleus. Polyacrylamide gel electrophoresis of the immunoprecipitated SFV capsid proteins showed that both recombinants yielded a labelled band equivalent in size to the SFV capsid protein. Thus the proteolytic processing takes place even under conditions where the capsid protein is the only virus-specified protein synthesized.

Functional consequences of amyloidosis mutation for gelsolin polypeptide -- analysis of gelsolin-actin interaction and gelsolin processing in gelsolin knock-out fibroblasts.

Gelsolin, an actin-modulating protein, derived from a single gene exists in intracellular and secreted forms. A point mutation at position 187 of both forms of gelsolin causes familial amyloidosis of the Finnish type (FAF). Here, we expressed both isoforms of the wild-type and FAF mutant gelsolin in mouse embryonic gelsolin-null fibroblasts. We demonstrate that the FAF mutation does not interfere with the normal actin-modulating function of intracellular gelsolin, and that aberrant processing of secreted FAF gelsolin to FAF amyloid precursor takes place in the gelsolin-negative background. These results suggest that, in patients with FAF, symptoms are caused by the accumulation in their tissues of amyloid derived from plasma gelsolin and are not due to functional differences in cytoplasmic gelsolin.

CLN-1 and CLN-5, genes for infantile and variant late infantile neuronal ceroid lipofuscinoses, are expressed in the embryonic human brain.

Mutations in the CLN-1 and CLN-5 genes underlie the infantile, and Finnish variant of the late-infantile, neuronal ceroid lipofuscinoses, respectively. These disorders are characterized by a massive neuronal death early in childhood. We have studied mRNA and protein expression of CLN-1 and CLN-5 in embryonic human brains. The spatial and temporal distributions of CLN-1 and CLN-5 were similar in the embryonic human brain. Both genes are expressed at the beginning of cortical neurogenesis, and this expression increases as cortical development proceeds. In the developing cortical plate, expression is found in postmitotic migrating neuroblasts and neuroblasts that have completed migration. Expression was intense also in cells of the thalamus as well as in the future Purkinje cell layer of the cerebellum. These findings indicate that expression of CLN-1 and CLN-5 may be significant for development of a wide range of maturating neurons.

Molecular mechanisms of mutations in factor XIII A-subunit deficiency: in vitro expression in COS-cells demonstrates intracellular degradation of the mutant proteins.

Factor XIII deficiency is an autosomal recessive bleeding disorder that is largely caused by various mutations in FXIII A-subunit gene. Characteristically, the patients lack both A-subunit activity and antigen in the circulation. Here we have analysed the consequences of four missense mutations (Met242-->Thr, Arg252-->Ile, Arg326-->Gln, Leu498 to Pro) and one stop mutation (Arg661-->Stop) in the FXIII A-subunit gene by expression in COS-cells. After transient transfection each mutant cDNA expressed mRNA at an equal level to the wild type FXIII. However, the mutant polypeptides accumulated in the cells in significantly reduced quantities and demonstrated only very low enzymatic activity. Analysis of immunoprecipitated metabolically labelled polypeptides demonstrated remarkable instability and intracellular degradation of all mutant FXIII proteins. These results verify the deleterious nature of the individual amino acid changes and confirm that protein instability and susceptibility to proteolysis are consequences of the mutations, as predicted from the three-dimensional model of crystallised FXIII A-subunit.

Structure of the rat catechol-O-methyltransferase gene: separate promoters are used to produce mRNAs for soluble and membrane-bound forms of the enzyme.

The enzyme catechol-O-methyltransferase (COMT) catalyzes the inactivation of catechol-containing molecules by methylation. The cDNAs for the rat and human COMT have recently been cloned and recombinant proteins expressed in prokaryotic and eukaryotic cells. We describe here the structure of the rat COMT gene and its 5'-flanking sequences. The gene spans at least 13 kb and is composed of 5 exons, the first one noncoding. The two ATG codons for the initiation of translation of the membrane-bound (MB-COMT) and soluble (S-COMT) forms of the enzyme reside in the second exon. The gene expresses two mRNA species of 1.6 kb and 1.9 kb that have different tissue distributions. The expression of the transcripts is regulated by at least two promoters, P1 and P2. The P1 promoter expresses the shorter transcript in a tissue-specific manner and is located between the ATG codons in the coding region of the longer transcript. The P2 promoter is constitutive and responsible for the expression of the longer transcript. The shorter 1.6-kb mRNA (S-mRNA) produces only the S-COMT polypeptide, whereas the longer 1.9-kb mRNA (MB-mRNA) is able to direct synthesis of both forms of the COMT enzyme.

Cloning and characterization of human placental catechol-O-methyltransferase cDNA.

Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. All four positive clones isolated contained an open reading frame, which potentially coded for a 24.4-kD polypeptide, presumably corresponding to the cytoplasmic form of the COMT (S-COMT). In addition to the S-COMT sequences, two of the clones carried extensions in the 5' end, which potentially coded for a 50-amino-acid peptide extending the S-COMT reading frame. This sequence contained a stretch of signal sequence-like hydrophobic amino acids in its amino terminus. The deduced human COMT polypeptide had 80% similarity with the previously characterized rat COMT. Expression of one of the cDNA clones in human K-562 cells resulted in cell clones with 3- to 10-fold increased COMT activity. Cell-free translation of transcripts synthesized in vitro from one of the short cDNAs yielded a 26-kD product, similar in size to human S-COMT. Translation of transcripts from one of the long cDNAs gave 30-kD and 26-kD polypeptides, suggesting translation initiation from two different AUG initiation codons. The 30-kD protein, but not the 25-kD protein, associated with microsomal membranes in translation lysates. A potential polyadenylation signal AATTAA was detected in the 3' ends of two of the clones 265 nucleotides downstream from the COMT translation termination codon. RNA blotting on human placental RNA revealed a 1.5-kb-long COMT-specific transcript. DNA analysis suggested that human, as well as rat, canine and monkey cells have one gene for COMT.

Dissection of the molecular consequences of a double mutation causing a human lysosomal disease.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency in which leads to human storage disease aspartylglucosaminuria (AGU). AGUFin is the most common AGU mutation in the world and is found in 98% of AGU alleles in Finland, where the population displays enrichment of the disease allele. The AGUFin allele actually contains a double mutation, both individual mutations resulting in amino acid substitutions: Arg-161-->Gln and Cys-163-->Ser. The separate consequences of these two amino acid substitutions for the intracellular processing of the AGA polypeptides were analyzed using a stable expression of mutant polypeptides in Chinese hamster ovary (CHO) cells. The synthesized polypeptides were monitored by metabolic labeling, followed by immunoprecipitation, immunofluorescence, and immunoelectron microscopy. The Arg-161-->Gln substitution did not affect the intracellular processing or transport of AGA and the fully active enzyme was correctly targeted to lysosomes. The Cys-163-->Ser substitution prevented the early proteolytic cleavage required for the activation of the precursor AGA polypeptide and the inactive enzyme was accumulated in the endoplasmic reticulum (ER). The precursors of the translation products of the AGUFin double mutant and the Cys-163-->Ser mutant were also observed in the culture medium. When cells expressing the normal AGA or AGUFin double mutation were treated with DTT to prevent the formation of disulfide bonds, both normal and mutated AGA polypeptides remained in the inactive precursor form and were not secreted into the medium. These results indicate that correct initial folding is essential for the proteolytic activation of AGA.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental expression of palmitoyl protein thioesterase in normal mice.

Deficiency in palmitoyl protein thioesterase (PPT) results in the rapid death of neocortical neurons in human. Very little is known about the developmental and cell-specific expression of this lysosomal enzyme. Here we show that PPT is expressed as a major 2.65 kb and a minor 1.85 kb transcript in the mouse brain. Transcript levels gradually increase between postnatal days 10 and 30. In situ hybridization analysis revealed that PPT transcripts are found widely but not homogeneously in the brain. The most intense signal was detected in the cerebral cortex (layers II, IV-V), hippocampal CA1-CA3 pyramidal cells, dentate gyrus granule cells and the hypothalamus. Immunostaining of PPT was localized in the cell soma, axons and dendrites, especially in the pyramidal and granular cells of the hippocampus, correlating well, both spatially and temporally, with the immunoreactivity of a presynaptic vesicle membrane protein, synaptophysin. In whole embryos, at embryonic day 8, the PPT mRNA expression was most apparent throughout the neuroepithelium, and from day 9 onwards it was seen in all tissues. The expression pattern of PPT suggests its general significance for the brain cells and reflects the response to maturation and growth of the neural networks. Strong PPT immunoreactivity in the axons and dentrites would imply that PPT may not be exclusively a lysosomal enzyme. A notable correlation with synaptophysin would suggest that PPT may have a role in the function of the synaptic machinery.

Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector.

Human tissue-type plasminogen activator (t-PA) cDNA inserted into an Epstein-Barr virus (EBV) derived expression vector was transfected into human HeLa, 293, K-562 and hamster CHO-K1 cells and the expression of t-PA was studied. The best t-PA producing cell clones were found among CHO-K1 cells (up to 11 micrograms d-1 per 10(6) cells). However, HeLa and 293 cells were most efficiently transfected, e.g. about 70% of the selected cell clones were t-PA positive. The vector DNA copy numbers correlated with the mRNA levels and the protein levels for all cell lines analysed, with the exception for the K-562 cell line, where the production of t-PA was very low. The results obtained indicated that the highest expression levels were achieved in low density cultures.

The repeated regions of Semliki Forest virus defective-inferfering RNA interferes with the encapsidation process of the standard virus.

Two different defective interfering RNAs of Semliki Forest virus have been cloned and sequenced previously. These molecules have repeated sequence blocks between unique terminal regions. The late gene region of SV40 virus has been replaced with the repeating unit detected in both defective-inferfering (DI) RNAs, and by complementation with a tsA mutant of SV40 a mixed stock of recombinant and helper virus was obtained. Upon infection of monkey kidney cells the recombinant expressed the repeated part of the DI RNA (svDI301 RNA). Superinfection of these cells with standard Semliki Forest virus showed that (i) the synthesis of SFV genomic RNA is marginally if at all affected by the svDI301 RNA, (ii) the svDI301 RNA is not replicated by SFV-RNA-dependent RNA polymerase, and (iii) packaging efficiency of the standard SFV genome RNA into virions is clearly decreased in the presence of svDI301 RNA. These results suggest that the terminal regions of the DI RNA molecule are required for efficient replication while the central repeated elements are involved in encapsidation.

Deletion of the C-terminal end of aspartylglucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement.

Aspartylglucosaminuria (AGU) is an inborn error of glycoprotein catabolism and represents the only known human deficiency of an amidase, aspartylglucosaminidase (AGA, EC 3.5.1.26). We report here a detailed characterization of a unique 2 kb deletion of the AGA gene in a North American AGU patient. To facilitate the characterization of the deletion, genomic lamda clones spanning the 3' flanking region of human AGA were isolated and sequenced. The breakpoint of the deletion was determined from the patient's DNA by sequencing the genomic region containing the novel junction. The rearrangement involved a nonhomologous recombination with only 2 bp of homology at the deletion breakpoint. The deletion's 5' breakpoint was located in the last intron of AGA, thus abolishing the normal C-terminal exon. This is in contrast to our previous findings indicating that the deletion in the AGA gene would contain only the complete 3' untranslated region and leave the coding region intact (1). The unique feature of this deletion is a triplication of 19 thymidine nucleotides of an inverted Alu repeat, which is located at the deletion 3' breakpoint. The analysis of the patient's AGA cDNA revealed an open reading frame containing a novel C-terminal exon, coding for a 64 amino acid sequence, which has no homology to the normal exon 9 of AGA. This new exon has a functional splice acceptor site at its 5' end, a stop codon, and a polyadenylation signal at the 3' end. Expression of the mutant AGA cDNA in COS cells showed that mutant mRNA is synthesized in equal amounts compared with normal.(ABSTRACT TRUNCATED AT 250 WORDS)