RESUMEN
A seven-month-old, female domestic shorthair cat was presented to the Veterinary Teaching Hospital, University of Zurich, with abnormal facial features, retarded growth and progressive hindlimb paresis. On physical examination the cat had a flat, broad face with hypertelorism, frontal bossing, small ears and thickened upper and lower eyelids. The corneas of both eyes were clear and the pupils were dilated. The skin was generally thickened, most prominently on the dorsal aspect of the neck. Radiography of the entire skeleton revealed a severely deformed spinal column, bilateral hip luxation with hip dysplasia, an abnormally shaped skull and generalised decreased bone opacity. The clinical features and radiographic changes were suggestive of mucopolysaccharidosis. The toluidine blue spot test on a urine sample, however, was negative for glycosaminoglycans. Further biochemical investigations revealed a deficiency of the enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) in peripheral leukocytes and an elevation of many lysosomal enzymes in the serum of the cat which is diagnostic for mucolipidosis type II. Histology and electron microscopy of different tissues are briefly summarised. The findings of this cat, the first reported case of mucolipidosis type II are compared with other similar storage diseases described in the cat.
Asunto(s)
Enfermedades de los Gatos/diagnóstico , Mucolipidosis/veterinaria , Animales , Axones/ultraestructura , Huesos/anomalías , Huesos/diagnóstico por imagen , Huesos/ultraestructura , Cartílago/ultraestructura , Enfermedades de los Gatos/metabolismo , Enfermedades de los Gatos/patología , Gatos , Tejido Conectivo/ultraestructura , Femenino , Leucocitos/enzimología , Leucocitos/ultraestructura , Microscopía Electrónica/veterinaria , Mucolipidosis/diagnóstico , Mucolipidosis/patología , Mucopolisacaridosis I/diagnóstico , Mucopolisacaridosis I/metabolismo , Mucopolisacaridosis I/veterinaria , Mucopolisacaridosis VI/diagnóstico , Mucopolisacaridosis VI/metabolismo , Mucopolisacaridosis VI/veterinaria , Mucopolisacaridosis VII/diagnóstico , Mucopolisacaridosis VII/metabolismo , Mucopolisacaridosis VII/veterinaria , Radiografía , Piel/ultraestructuraRESUMEN
A 7-month-old female cat was seen for abnormal facial features and abnormality of gait. Facial dysmorphism, large paws in relation to body size, dysostosis multiplex, and poor growth were noted, and mucopolysaccharidosis was suspected. A negative urine test for sulfated glycosaminoglycans and extreme stiffness of skin indicated a mucolipidosis hitherto unknown in animals. Deficiency of UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase, EC 2.7.8.17) activity was demonstrated in leukocytes and cultured fibroblasts, which had the appearance of inclusion cells (I-cells). Activities of a set of lysosomal hydrolases were abnormally low in fibroblasts and excessive in blood plasma. Postmortem morphology revealed lysosomal inclusions predominantly in fibroblasts but also in endothelial cells and chondrocytes, i.e., in cells of mesenchymal origin. Storage lysosomes contained oligosaccharides, mucopolysaccharides, and lipids. Tissues most affected were bones, cartilage, skin, and other connective tissues such as those in heart valves, aortic wall, and vocal cords. Parenchymal cells of liver and kidney were unaffected, as was skeletal muscle. Only a few of the cerebral cortical neurons had lipid inclusions; in sciatic nerve some axons were affected, but other peripheral nerves were normal. There were striking clinical, biochemical, and morphologic similarities between the disorder in this cat and the human I-cell disease.
Asunto(s)
Enfermedades de los Gatos/diagnóstico , Enfermedades de los Gatos/metabolismo , Gatos/metabolismo , Mucolipidosis/veterinaria , Animales , Aorta/patología , Constitución Corporal/fisiología , Huesos/diagnóstico por imagen , Huesos/patología , Enfermedades de los Gatos/patología , Gatos/crecimiento & desarrollo , Gatos/fisiología , Modelos Animales de Enfermedad , Femenino , Marcha/fisiología , Glicosaminoglicanos/orina , Riñón/patología , Riñón/ultraestructura , Leucocitos/química , Leucocitos/patología , Hígado/patología , Hígado/ultraestructura , Mucolipidosis/diagnóstico , Mucolipidosis/metabolismo , Radiografía , Degeneración Retiniana/patología , Degeneración Retiniana/fisiopatología , Nervio Ciático/patología , Nervio Ciático/ultraestructura , Piel/patología , Piel/ultraestructura , Transferasas (Grupos de Otros Fosfatos Sustitutos)/análisis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/fisiología , Uridina Difosfato N-Acetilglucosamina/análisis , Uridina Difosfato N-Acetilglucosamina/deficiencia , Uridina Difosfato N-Acetilglucosamina/fisiologíaRESUMEN
The glycosylation and phosphorylation of the lysosomal enzyme arylsulfatase A was analyzed by a combination of metabolic labeling, tryptic fragmentation, mass spectrometry, and radiosequencing. The results demonstrate that all three potential N-glycosylation sites at Asn residues 158, 184, and 350 are utilized in arylsufatase A and carry high mannose or hybride type oligosaccharides. Phosphorylation of mannose residues is restricted to oligosaccharides at the first and third N-glycosylation site (Asn-158 and Asn-350). Both are phosphorylated with comparable efficiency. An earlier study had shown that a mutant arylsulfatase A containing only the second N-glycosylation site at Asn-184 folds correctly and is phosphorylated (Gieselmann, V., Schmidt, B., and von Figura, K. (1992) J. Biol. Chem. 267, 13262-13266). The lack of phosphorylation at Asn-184 in wild type arylsulfatase A therefore indicates that in vivo the presence of oligosaccharides can interfere with phosphorylation of other sites or that phosphorylation occurs in an ordered manner whereby phosphorylation of one site can affect the phosphorylation of another site.
Asunto(s)
Cerebrósido Sulfatasa/metabolismo , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cricetinae , Glicosilación , Humanos , Hidrólisis , Espectrometría de Masas , Datos de Secuencia Molecular , Fosforilación , Espectrofotometría UltravioletaRESUMEN
Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of a patient affected with late infantile metachromatic leukodystrophy revealed that the patient is a compound heterozygote of two alleles carrying two deleterious mutation each. One allele bears a splice donor site mutation together with two polymorphisms and an additional missense mutation (Gly122 > Ser). The splice donor site mutation and the Gly122 > Ser substitution have been described recently but on different alleles. The other allele carries two missense mutations causing a Gly154 > Asp and a Pro167 > Arg substitution. When arylsulfatase A cDNAs carrying these mutations separately or in combination were transfected into baby hamster kidney cells expression of arylsulfatase A activity could not be detected. Linkage of mutations was verified by sequencing of the parental DNAs. Biosynthesis studies performed with the patients' fibroblasts show that the enzyme carrying both mutations is synthesized in almost normal amounts but is rapidly degraded in an early biosynthetic compartment. The occurence of two disease causing mutations on the same allele is a novel phenomenon in metachromatic leukodystrophy and as far as lysosomal storage diseases are concerned have so far only been described in Fabry disease and in the complex glucocerebrosidase alleles associated with Gaucher disease.
Asunto(s)
Cerebrósido Sulfatasa/genética , Leucodistrofia Metacromática/genética , Mutación Puntual , Alelos , Secuencia de Bases , Preescolar , ADN Complementario , Frecuencia de los Genes , Humanos , Leucodistrofia Metacromática/enzimología , Datos de Secuencia Molecular , Sondas de OligonucleótidosRESUMEN
The critical step in the sorting of lysosomal enzymes is their recognition by a phosphotransferase in the Golgi apparatus. The topogenic sequences responsible for the recognition by this enzyme have so far only been defined for the lysosomal protease cathepsin D. We have generated four monoclonal antibodies directed against lysosomal arylsulphatase A (ASA). These antibodies inhibit the recognition of ASA by the phosphotransferase in vitro and thus define a region of topogenic sequences in the ASA polypeptide. The antibodies do not interfere with the enzymic activity nor with pH-dependent dimerization of ASA. The epitopes recognized by the antibodies have been located in the second quarter of the ASA polypeptide using chimeric mouse-human ASA molecules. Three of the monoclonal antibodies bind to identical or closely adjacent epitopes, which are formed by the interaction of amino acid residues 165-184 and 202-240. The fourth antibody recognizes a different epitope within amino acids 256-265.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Cerebrósido Sulfatasa/metabolismo , Lisosomas/enzimología , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Cerebrósido Sulfatasa/química , Cerebrósido Sulfatasa/inmunología , Cricetinae , Epítopos/inmunología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Concentración de Iones de Hidrógeno , Técnicas de Inmunoadsorción , Hígado/enzimología , Sustancias Macromoleculares , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , FosforilaciónRESUMEN
We have studied the convergence of the biosynthetic lysosomal route marked by the newly synthesized lysosomal enzyme arylsulfatase A (ASA) with the endosomal/prelysosomal compartment in ASA overexpressing baby hamster kidney (BHK) cells. A monoclonal antibody against ASA conjugated to transferrin (Tf-alpha ASA) was used to load the endocytic pathway via the transferrin receptor. Subsequent internalization of [125I]labeled ASA and Tf-alpha ASA conjugates at 18 degrees C followed by rewarming to 37 degrees C showed that immunocomplexes were formed within the recycling pathway and released into the medium. Furthermore, in cells labeled with [35S]methionine for 10 min about 54% of newly synthesized ASA passed into Tf-alpha ASA accessible compartments during a 4 hour chase period and accumulated in the medium. These data indicate that in overexpressing BHK cells the majority of newly synthesized ASA is transported to the lysosome via transferrin receptor-containing early endosomes.