Characterization of common SMPD1 mutations causing types A and B Niemann-Pick disease and generation of mutation-specific mouse models
| Autor: | Peter I. Darroch, Xingxuan He, Iwan Jones, Fourogh Katouzian, Edward H. Schuchman |
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| Rok vydání: | 2008 |
| Předmět: |
Models
Molecular Endocrinology Diabetes and Metabolism Transgene Mutant Molecular Sequence Data Mice Transgenic Biology medicine.disease_cause Biochemistry Article Cell Line Mice Endocrinology Genetics medicine Lysosomal storage disease Animals Humans Amino Acid Sequence Molecular Biology Gene Cells Cultured Mice Knockout Niemann-Pick Diseases Mutation Brain respiratory system medicine.disease Molecular biology Protein Structure Tertiary Blot Mice Inbred C57BL Disease Models Animal Protein Transport Sphingomyelin Phosphodiesterase Acid sphingomyelinase Niemann–Pick disease Sequence Alignment medicine.drug |
| Zdroj: | Molecular genetics and metabolism. 95(3) |
| ISSN: | 1096-7206 |
| Popis: | Herein we describe detailed characterization of four common mutations (L302P, H421Y, R496L and DeltaR608) within the acid sphingomyelinase (ASM) gene causing types A and B Niemann-Pick disease (NPD). In vitro and in situ enzyme assays revealed marked deficiencies of ASM activity in NPD cell lines homoallelic for each mutation, although Western blotting and fluorescent microscopy showed that the mutant ASM polypeptides were expressed at normal levels and trafficked to lysosomes. Co-immunoprecipitation of the polypeptides with the ER chaperone, BiP, confirmed these findings, as did in vitro expression of the mutant cDNAs in reticulocyte lysates. We further developed a computer assisted, three-dimensional model of human ASM based on homologies to known proteins, and used this model to map each NPD mutation in relation to putative substrate binding, hydrolysis and zinc-binding domains. Lastly, we generated transgenic mice expressing the R496L and DeltaR608 mutations on the complete ASM knock-out background (ASMKO), and established breeding colonies for the future evaluation of enzyme enhancement therapies. Analysis of these mice demonstrated that the mutant ASM transgenes were expressed at high levels in the brain, and in the case of the DeltaR608 mutation, produced residual ASM activity that was significantly above the ASMKO background. |
| Databáze: | OpenAIRE |
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