RESUMEN
Achondroplasia, the most common genetic form of human dwarfism, results from a point mutation (G380R) in the gene for fibroblast growth factor receptor 3 (FGFR-3). Heterozygotes for the mutation share disproportionate, proximal shortening of the limbs, mid-face hypoplasia and relative macrocephaly due to a failure in endochondral ossification. Here we have generated transgenic mice expressing the human mutant FGFR-3 under the transcriptional control of the mouse gene. Mice that are hemizygous for the mutant human gene display disproportionate dwarfism with skeletal phenotypes remarkably similar to those of human achondroplasia. Mice that are homozygous for the transgene suffer from a profound delay in skeletal development and die at birth, similar in that respect to humans homozygous for the achondroplasia mutant gene. Microscopic analysis of long bones demonstrates growth plate morphology compatible with that of human achondroplasia cases, sharing endochondral growth inhibition with restrained chondrocyte proliferation and maturation, penetration of ossification tufts and aberrant vascularization.
Asunto(s)
Huesos/anomalías , Condrocitos/patología , Placa de Crecimiento/anomalías , Placa de Crecimiento/irrigación sanguínea , Ratones Transgénicos/anomalías , Ratones Transgénicos/genética , Proteínas Tirosina Quinasas , Receptores de Factores de Crecimiento de Fibroblastos/genética , Animales , Diferenciación Celular/genética , División Celular/genética , Desarrollo Embrionario y Fetal/genética , Factores de Crecimiento de Fibroblastos/genética , Placa de Crecimiento/química , Humanos , Ratones , Osteogénesis/genética , Receptor Tipo 3 de Factor de Crecimiento de FibroblastosRESUMEN
Fluorescent glycolipids were utilized for detection of the intracellular, activator-dependent, activities of beta-glucocerebrosidase and arylsulphatase A. Activities were measured in primary skin fibroblasts from normal individuals, from patients with Gaucher disease who had mutations within the beta-glucocerebrosidase gene, and from a prosaposin-deficient patient. Fluorescent microscopy demonstrated that glucosylceramide or sulphatide labelled with a fluorescent probe (lissamine-rhodamine) were endocytosed and reached the lysosomes. There, in the presence of active enzyme and the corresponding saposin, they were hydrolysed to fluorescent ceramide, which changed its intracellular localization. When these substrates were labelled with pH-sensitive lissamine-rhodamine, which loses its fluorescence at neutral or alkaline pH, the transport of the product, i.e. fluorescent ceramide, from the lysosomes resulted in disappearance of the cellular fluorescence. In cells of patients having mutations within the genes encoding the glucocerebrosidase or the prosaposin, there was a considerable reduction in the intracellular rate of substrate hydrolysis that could be followed by fluorescence microscopy or measured quantitatively in cell extracts.
Asunto(s)
Cerebrósido Sulfatasa/metabolismo , Colorantes Fluorescentes/metabolismo , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/metabolismo , Glucosilceramidas/metabolismo , Rodaminas/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Activación Enzimática , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/genética , Glicoproteínas/deficiencia , Glicoproteínas/metabolismo , Humanos , Líquido Intracelular/metabolismo , Lisosomas/enzimología , Lisosomas/metabolismo , Mutación , Saposinas , Proteínas Activadoras de EsfingolípidosRESUMEN
The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.
Asunto(s)
Empalme Alternativo , Glicoproteínas/genética , Precursores de Proteínas/genética , Northern Blotting , Línea Celular , Cerebrósido Sulfatasa/metabolismo , Clonación Molecular , ADN Complementario/genética , Activación Enzimática , Vectores Genéticos , Glucosilceramidasa/metabolismo , Glicoproteínas/biosíntesis , Humanos , Lisosomas/metabolismo , Microscopía Fluorescente , Mutación Puntual , Pruebas de Precipitina , Precursores de Proteínas/biosíntesis , ARN/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Saposinas , Proteínas Activadoras de Esfingolípidos , Vaccinia/genéticaRESUMEN
Gaucher disease is a heterogeneous disease characterized by impaired activity of the lysosomal enzyme glucocerebrosidase. This heterogeneity is attributed to a large number of mutations in the corresponding gene. In order to test the biochemical properties of some mutations prevalent among Israeli populations, the normal human glucocerebrosidase cDNA and cDNAs carrying mutations N370S, L444P, D409H, recTL, recNcil, P415R and 84GG were coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). Recombinant viruses were produced and used to infect human tissue culture cells. RNA and protein stability, recognition by anti-glucocerebrosidase monoclonal antibodies and intracellular enzymatic activity were measured. The results demonstrated that the D409H allele directed synthesis of cytoplasmic RNA with decreased stability compared with its normal counterpart or other mutated forms. The D409H and L444P mutated proteins had lower stability than that of their normal counterpart, while the recNcil-mutated protein was more stable. Only glucocerebrosidase forms harboring leucine at position 444 were recognized by the anti-glucocerebrosidase monoclonal antibodies used (8E4 and 2C7). Measurements of enzymatic activity of the recombinant proteins in cells loaded with a fluorescent glucosylceramide demonstrated that the N370S mutated enzyme had activity similar to that of the normal enzyme. The other mutated enzymes exhibited varying degrees of activities, generally corresponding to the phenotypes with which they are associated. The results presented demonstrate the use of the vaccinia virus-derived expression system and of loading living cells with fluorescent substrate as efficient tools for studying mutants in Gaucher disease and in other lysosomal diseases.