RESUMEN
Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.
Asunto(s)
Errores Innatos del Metabolismo de los Carbohidratos/genética , Dolicoles/metabolismo , Oligosacáridos/biosíntesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , Secuencia de Carbohidratos , Línea Celular , Cartilla de ADN , Fosfatos de Dolicol/metabolismo , Femenino , Fibroblastos , Prueba de Complementación Genética , Glicosilación , Humanos , Masculino , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Linaje , Monosacáridos de Poliisoprenil Fosfato/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Deficiency of the endoplasmic reticulum enzyme dolichyl-phosphate mannose (Dol-P-Man):Man(7)GlcNAc(2)-PP-dolichyl mannosyltransferase leads to a new type of congenital disorder of glycosylation, designated type Ig. The patient 1 presented with a multisystemic disorder with microcephaly, developmental retardation, convulsions and dysmorphic signs. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains. In skin fibroblasts from the patient, the activity of Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase was severely reduced leading to the accumulation of Man(7)GlcNAc(2)-PP-Dol, which was transferred to newly synthesized glycoproteins. Sequencing of the Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA revealed a compound heterozygosity for two point mutations, leading to the exchange of leucine(158) for a proline residue and a premature translation stop with loss of the C-terminal 74 amino acids. The parents were heterozygous for one of the two mutations. Retroviral expression of the wild-type Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA in patient's fibroblasts normalized the mannosyltransferase activity.
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Glicosilación , Manosiltransferasas/deficiencia , Manosiltransferasas/fisiología , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Femenino , Fibroblastos/enzimología , Prueba de Complementación Genética , Humanos , Focalización Isoeléctrica , Mutagénesis Sitio-Dirigida , Oligosacáridos/metabolismo , Fenotipo , Mutación Puntual , Retroviridae/metabolismo , Transferrina/metabolismoRESUMEN
Deficiency of the Golgi enzyme UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase I (beta4GalT I) (E.C.2.4.1.38) causes a new congenital disorder of glycosylation (CDG), designated type IId (CDG-IId), a severe neurologic disease characterized by a hydrocephalus, myopathy, and blood-clotting defects. Analysis of oligosaccharides from serum transferrin by HPLC, mass spectrometry, and lectin binding revealed the loss of sialic acid and galactose residues. In skin fibroblasts and leukocytes, galactosyltransferase activity was reduced to 5% that of controls. In fibroblasts, a truncated polypeptide was detected that was about 12 kDa smaller in size than wild-type beta4GalT I and that failed to localize to the Golgi apparatus. Sequencing of the beta4GalT I cDNA and gene revealed an insertion of a single nucleotide (1031-1032insC) leading to premature translation stop and loss of the C-terminal 50 amino acids of the enzyme. The patient was homozygous and his parents heterozygous for this mutation. Expression of a corresponding mutant cDNA in COS-7 cells led to the synthesis of a truncated, inactive polypeptide, which localized to the endoplasmic reticulum.