RESUMEN
Mucopolysaccharidosis IIID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the caprine N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence.
Asunto(s)
ADN Complementario/química , Cabras/genética , Sulfatasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Mucopolisacaridosis III/enzimología , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Sulfatasas/deficienciaRESUMEN
Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5'-region was obtained using the technique of 5'-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.
Asunto(s)
Manosidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Southern Blotting , Bovinos , Clonación Molecular , ADN Complementario , Lisosomas/enzimología , Manosidasas/metabolismo , Datos de Secuencia Molecular , beta-ManosidasaRESUMEN
Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity.
Asunto(s)
Riñón/enzimología , Manosidasas/aislamiento & purificación , Animales , Carbohidratos/análisis , Bovinos , Enfermedades de los Bovinos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Cinética , Lisosomas/enzimología , Manosidasas/genética , Manosidasas/metabolismo , Peso Molecular , Valores de Referencia , alfa-Manosidosis/enzimología , alfa-Manosidosis/genética , alfa-Manosidosis/veterinaria , beta-ManosidasaRESUMEN
Goat beta-mannosidase was purified 120,000-fold in 26% yield from kidney using concanavalin A-Sepharose chromatography followed by immunoaffinity and cation-exchange chromatography. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie Blue staining, the purified enzyme preparation consists of 90- and 100-kDa peptides. Both these peptides react with anti-beta-mannosidase monoclonal antibodies and produce similar electrophoretic peptide patterns when subjected to limited proteolysis. Deglycosylation reduces the size of the 90- and 100-kDa peptides to 86 and 91 kDa, respectively. Goat kidney tissues lacking beta-mannosidase activity, acquired from animals affected with beta-mannosidosis, do not contain detectable quantities of the 90- and 100-kDa peptides as judged by monoclonal antibody reactivity. We postulate that the 90- and 100-kDa peptides represent two related forms of beta-mannosidase.
