ABSTRACT
Two sibs are described with an unbalanced 4;6 translocation resulting in partial trisomy 6p and monosomy for distal 4p. Growth retardation, psychomotor retardation, and characteristic facial appearance are present. The facial anomalies include high prominent forehead, blepharoptosis, blepharophimosis, high nasal bridge, bulbous nose, long philtrum, small mouth with thin lips, and low-set ears. Both children have small kidneys and have had proteinuria since early childhood. The older boy developed progressive renal disease including hypertension and renal failure necessitating renal transplantation at age 18 years. Renal biopsy of the younger girl also indicates significant renal involvement. Progressive renal disease is likely an important part of the trisomy 6p phenotype.
Subject(s)
Abnormalities, Multiple/genetics , Chromosomes, Human, Pair 4 , Chromosomes, Human, Pair 6 , Craniofacial Abnormalities/genetics , Kidney Diseases/genetics , Translocation, Genetic , Abnormalities, Multiple/diagnosis , Adolescent , Adult , Chromosome Deletion , Craniofacial Abnormalities/pathology , Cytogenetic Analysis , Disease Progression , Female , Humans , Kidney Transplantation , Male , Nuclear Family , Phenotype , Proteinuria/genetics , Psychomotor Disorders/genetics , Trisomy/pathologyABSTRACT
The alpha- and beta-subunits of the hetero-dimeric glucosidase II complex from human liver were cloned and expressed in COS-1 cells. The 4106 bp full-length cDNA for the alpha-subunit contained a 2835 bp ORF encoding a 107 kDa polypeptide. The 2095 bp cDNA for the beta-subunit encodes a approximately 60 kDa protein in a continuous 1605 bp ORF. The alpha- and beta-subunits each contain two potential Asn-Xaa-Thr/Ser acceptor sites, with only one site in the alpha-subunit (Asn97) being glycosylated. Additional lambda-clones were isolated for each subunit containing in-frame insertions/deletions within the coding region, indicating alternative splicing. Analysis of different human tissues revealed approximately 4.4 kb and approximately 2.4 kb transcripts for alpha- and beta-subunit, respectively, consistent with their full-length cDNA. Coexpression of the alpha- and beta-subunits in COS-1 cells resulted in >4-fold increase of glucosidase II activity. An inactive protein was obtained, however, after transfection with the alpha-subunit alone, showing that both subunits are essential for expression of active glucosidase II. The observation that the enzyme, previously purified from pig liver and lacking the beta-subunit, was catalytically active indicates that the beta-subunit is involved in alpha-subunit maturation rather than being required for enzymatic activity once the alpha-subunit has acquired its mature form. The alpha-subunit is expressed in COS-1 cells as an ER-located protein, whether inactive or part of a catalytically active complex. This suggests that ER-localization of the alpha-subunit, when associated with the dimeric enzyme complex, is mediated by the C-terminal HDEL-signal in the beta-subunit, whereas the apparently incompletely folded form of the inactive alpha-subunit could be retained in the ER by the putative "glycoprotein-specific quality control machinery. "
Subject(s)
Liver/enzymology , alpha-Glucosidases/chemistry , alpha-Glucosidases/metabolism , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , COS Cells , Catalysis , Cloning, Molecular , DNA, Complementary/chemistry , Dimerization , Gene Expression , Humans , Microscopy, Fluorescence , Molecular Sequence Data , Open Reading Frames , RNA Precursors , Transfection , alpha-Glucosidases/geneticsABSTRACT
Glucosidase II has been purified from crude pig liver microsomes by a convenient procedure involving DEAE-Sephacel, Con A-Sepharose and affinity chromatography on N-5-carboxypentyl-1-deoxynojirimycin-AH-Sepharose. Specific binding of glucosidase II to the affinity matrix required its prior separation from glucosidase I, which was accomplished by fractional Con A-Sepharose chromatography. The three-step procedure yielded, with approximately 15% enzyme recovery, a > 190-fold enriched glucosidase II, consisting of two proteins (107 kDa and 112 kDa). Both polypeptides are N-glycosylated with probably one glycan chain, in line with their binding to Con A-Sepharose. Immunological cross-reactivity and other experimental data indicate that the 107 kDa N-glycoprotein is derived from the 112 kDa species by partial proteolysis. The occasional presence of a 60 kDa peptide co-eluting with the catalytic activity suggests that glucosidase II may be associated with other protein subunit(s) in a heteromeric membrane complex. Glucosidase II hydrolyzes the alpha1,3-glucosidic linkages in Glc(2-1)-Man9-GlcNAc2, as well as synthetic alpha-glucosides, efficiently but does not remove the distal alpha1,2-linked glucose in Glc3-Man9-GlcNAc2. The enzyme has a pH optimum close to 6.5 and is not metal ion-dependent. Catalytic activity is strongly inhibited by basic sugar analogues including 1-deoxynojirimycin (dNM; app. Ki approximately 7.0 microM), N-5-carboxypentyl-dNM (app. Ki approximately 32 microM) and castanospermine (app. Ki approximately 40 microM). Substitution of the 3-OH or 6-OH group in dNM by a fluoro group reduces the inhibitory potential drastically. We conclude from these observations that the two hydroxy groups are essential for inhibitor/substrate binding due to their ability to interfere as hydrogen bond donors. A polyclonal antibody raised against the 107 kDa polypeptide reacted specifically with two proteins from different cell types on Western blots. Their molecular masses were identical with those from pig liver microsomes, pointing to a highly conserved amino acid sequence of glucosidase II. This suggests that the variance in molecular mass for glucosidase II reported for the enzyme from other tissues and species may be due to partial proteolysis.
