ABSTRACT
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.
Subject(s)
Mutation , Niemann-Pick Diseases/enzymology , Niemann-Pick Diseases/genetics , Sphingomyelin Phosphodiesterase/genetics , Amino Acid Sequence , Animals , Brain/enzymology , Cell Line , Cells, Cultured , Disease Models, Animal , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Sequence Alignment , Sphingomyelin Phosphodiesterase/chemistry , Sphingomyelin Phosphodiesterase/metabolismABSTRACT
BACKGROUND: Intestinal permeability is determined by measuring nonmetabolized sugars. In animals, intestinal permeability is determined in urine, using cumbersome and expensive metabolic cages. We developed an HPLC method for determining concentrations of lactulose (L) and L-rhamnose (R) in blood-drop of rabbits and mice, and we compared these results with the procedure based on sugars excreted in urine. We measured the intestinal permeability induced by a fragment (DeltaG) of the zonula occludens toxin which opens the paracellular pathway. METHODS: The animals received sugar solution and later received the same solution+DeltaG. Five-hour urine collection and timed blood tests were performed after ingestion of sugars. Sugars were measured with HPLC, and the percentage of recovered sugars was expressed as L/R ratio. RESULTS: At 60 min after administration of sugars, the mean L/R ratio for rabbits and mice was 0.026 and 0.052, respectively. At 60 min after administration of sugars+DeltaG, the mean L/R ratio for rabbits and mice was 0.22 and 0.53. The mean L/R ratio in the urine was 0.023 at basal condition and 0.25 after DeltaG ingestion. CONCLUSIONS: Testing small serum samples for sugar permeability is effective for monitoring changes in permeability of the gut in animals. This cheap simple method allows us to measure in vivo the biological activity of other molecules which modulate the paracellular pathway.
