1.Cloning of human lysosomal acid beta-glucosidase gene and its expression in COS7 cells.
Yanli ZHANG ; Dan XU ; Ziyu WANG ; Li MENG ; Feng WANG
Chinese Journal of Biotechnology 2009;25(2):263-267
In this study, we amplified human lysosomal acid beta-glucosidase (GlcCerase) gene by RT-PCR from human placenta, and analyzed the sequence of the PCR product cloned in pMD-19T. The gene identity was 99% comparable to that of the reported human GlcCerase cDNA sequence in GenBank. The GlcCerase gene digested with Xho I was subcloned into eukaryotic express vector pEGFP-C1 to generate recombinant expression vector pEGFP-GlcCerase. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pEGFP-GlcCerase into COS7 cells by liposome. GlcCerase mRNA was expressed and the activity of GlcCerase was also detected in COS7 cells. This study would lay a foundation for the function of GlcCerase and its production by transgenic bioreactor.
Animals
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COS Cells
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Cercopithecus aethiops
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Cloning, Molecular
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Genetic Vectors
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genetics
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Glucosylceramidase
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genetics
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metabolism
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Green Fluorescent Proteins
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genetics
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Humans
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Lysosomes
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enzymology
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Recombination, Genetic
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Transfection
2.Loss of glucocerebrosidase 1 activity causes lysosomal dysfunction and alpha-synuclein aggregation.
Eun Jin BAE ; Na Young YANG ; Cheolsoon LEE ; He Jin LEE ; Seokjoong KIM ; Sergio Pablo SARDI ; Seung Jae LEE
Experimental & Molecular Medicine 2015;47(3):e153-
Lysosomal dysfunction is a common pathological feature of neurodegenerative diseases. GTP-binding protein type A1 (GBA1) encodes beta-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase. Homozygous mutations in GBA1 cause Gaucher disease, the most common lysosomal storage disease, while heterozygous mutations are strong risk factors for Parkinson's disease. However, whether loss of GCase 1 activity is sufficient for lysosomal dysfunction has not been clearly determined. Here, we generated human neuroblastoma cell lines with nonsense mutations in the GBA1 gene using zinc-finger nucleases. Depending on the site of mutation, GCase 1 activity was lost or maintained. The cell line with GCase 1 deficiency showed indications of lysosomal dysfunction, such as accumulation of lysosomal substrates, reduced dextran degradation and accumulation of enlarged vacuolar structures. In contrast, the cell line with C-terminal truncation of GCase 1 but with intact GCase 1 activity showed normal lysosomal function. When alpha-synuclein was overexpressed, accumulation and secretion of insoluble aggregates increased in cells with GCase 1 deficiency but did not change in mutant cells with normal GCase 1 activity. These results demonstrate that loss of GCase 1 activity is sufficient to cause lysosomal dysfunction and accumulation of alpha-synuclein aggregates.
Cell Line
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Enzyme Activation/genetics
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Gene Knockout Techniques
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Gene Order
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Genetic Loci
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Glucosylceramidase/genetics/*metabolism
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Humans
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Lysosomes/*metabolism
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Mutation
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*Protein Aggregation, Pathological/genetics
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Protein Binding
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Zinc Fingers
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alpha-Synuclein/chemistry/*metabolism
3.Preclinical research of a new therapy for Gaucher's disease with F213I mutation.
Chinese Journal of Medical Genetics 2003;20(5):381-384
OBJECTIVETo design and make trial of a new therapy for Gaucher disease.
METHODSA substrate analogue of beta-Glc (glucocerebroside analogue, GCA) was used as a molecular chaperon. Normal and mutant skin fibroblasts were cultured with or without GCA. The activity of beta-Glc was assayed by fluorescent enzymologic techniques. The amount of beta-Glc was determined using Western blot. The beta -Glc was localized by double cell stain experiment. The degradation of glucocerebroside was assessed by thin layer chromatography (TLC) experiment using 14C-Serine.
RESULTSIt was found that GCA could enhance the activity and amount of beta-Glc with F213I mutation. It also promoted the beta-Glc with F213I mutation to the lysosome and accelerated the degradation of glucocerebroside.
CONCLUSIONThe low molecular compound analogous to beta-Glc substrate (GCA ) may be a new therapeutic strategy for Gaucher's disease with F213I mutation.
Amino Acid Substitution ; Blotting, Western ; Cells, Cultured ; Enzyme Induction ; drug effects ; Gaucher Disease ; drug therapy ; enzymology ; genetics ; Glucosylceramidase ; biosynthesis ; genetics ; metabolism ; Glucosylceramides ; metabolism ; pharmacology ; Humans ; Immunohistochemistry ; Mutation ; Substrate Specificity
4.Upregulation of Proinflammatory Cytokines in the Fetal Brain of the Gaucher Mouse.
Young Bin HONG ; Eun Young KIM ; Sung Chul JUNG
Journal of Korean Medical Science 2006;21(4):733-738
Gaucher disease is caused by a deficiency of glucocerebrosidase. Patients with Gaucher disease are divided into three major phenotypes: chronic nonneuronopathic, acute neuronopathic, and chronic neuronopathic, based on symptoms of the nervous system, the severity of symptoms, and the age of disease onset. The characteristics of patients with acute neuronopathic- and chronic neuronopathic-type Gaucher disease include oculomotor abnormalities, bulbar signs, limb rigidity, seizures and occasional choreoathetoid movements, and neuronal loss. However, the mechanisms leading to the neurodegeneration of this disorder remain unknown. To investigate brain dysfunction in Gaucher disease, we studied the possible role of inflammation in neurodegeneration during development of Gaucher disease in a mouse model. Elevated levels of the proinflammatory cytokines, IL-1alpha, IL-1beta, IL-6, and TNF-alpha, were detected in the fetal brains of Gaucher mice. Moreover, the levels of secreted nitric oxide and reactive oxygen species in the brains of Gaucher mice were higher than in wild-type mice. Thus, accumulated glucocerebroside or glucosylsphingosine, caused by glucocerebrosidase deficiency, may mediate brain inflammation in the Gaucher mouse via the elevation of proinflammatory cytokines, nitric oxide, and reactive oxygen species.
Up-Regulation/genetics
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Tumor Necrosis Factor-alpha/genetics/secretion
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Reverse Transcriptase Polymerase Chain Reaction
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Reactive Oxygen Species/metabolism
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RNA, Messenger/genetics/metabolism
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Nitric Oxide/metabolism
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Microglia/cytology/metabolism
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Mice, Knockout
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Mice, Inbred ICR
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Mice, Inbred C57BL
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Mice
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Interleukin-6/genetics/secretion
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Interleukin-1/genetics/secretion
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Inflammation/immunology
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Glucosylceramidase/genetics
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Gaucher Disease/*genetics/metabolism/pathology
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Cytokines/*genetics/immunology/secretion
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Cells, Cultured
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Brain/embryology/*metabolism/pathology
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Animals