Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Aspartylglucosylaminase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1977

Purification and some properties of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.

Amidohydrolases↗
PubMed1978

Effect of different compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase from human liver.

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.

Acetylcysteine↗
PubMed1977

Measurement of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase activity in human tissues.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.

Acetylglucosamine↗
PubMed1996

Large-scale purification and preliminary x-ray diffraction studies of human aspartylglucosaminidase.

Aspartylglucosaminidase (AGA) is a lysosomal asparaginase that takes part in the ordered degradation of glycoproteins and a deficiency of which results in a lysosomal accumulation disease aspartylglucosaminuria in human. The mature enzyme consists of 24-kDa and 17-kDa subunits, which are both heterogeneously glycosylated. Activation of the enzyme from a single precursor polypeptide into two subunits is accomplished in the endoplasmic reticulum (ER). The relative lack of this proteolytic capacity in several tested high-producing expression systems has complicated the production of active recombinant enzyme in high quantities, which would be an alternative for purification of this molecule for crystallization. Consequently, the AGA enzyme has to be purified directly from cellular or tissue sources for crystallographic analysis. Here we describe a large-scale purification method to produce milligram amounts of homogeneous AGA from human leukocytes. The purified AGA enzyme represents a heterogeneous pool of molecules not only due to glycosylation, but also heterogeneity at the polypeptide level, as demonstrated here. We were able to isolate a homogeneous peptide pool that was successfully crystallized and preliminary X-ray data collected from the crystals. The crystals diffract well to 2.0 angstroms and are thus suitable for determination of the crystal structure of AGA.

Aspartylglucosylaminase↗
PubMed1984

Prenatal diagnosis and fetal pathology of aspartylglucosaminuria.

The prenatal diagnosis of aspartylglucosaminuria (AGU), a lysosomal storage disorder of glycoprotein degradation, was made by demonstrating the deficiency of N-aspartylglucosaminidase on cultured cells from a midterm amniotic fluid sample. Four other amniotic fluid studies from at-risk pregnancies gave a normal or a heterozygote level of enzyme activity. These pregnancies have gone to term and the delivery of healthy babies. The pregnancy with the affected fetus was terminated and the prenatal diagnosis was verified by enzyme assays on cord blood lymphocytes, cultured cells from skin biopsy, and from placental villi. Electron microscopic evidence of lysosomal storage was seen in several organs of the fetus with the notable exception of the central nervous system. The undifferentiated mesenchymal fibroblasts particularly were heavily loaded with cytoplasmic inclusions in skin, liver, kidney, and placenta.

Acetylglucosamine↗
PubMed1988

Aspartylglucosaminuria in a Puerto Rican family: additional features of a panethnic disorder.

We report on 3 Puerto Rican brothers with the clinical and laboratory findings of aspartylglucosaminuria (AGU). Their parents were first cousins. The affected sibs have the "cardinal" manifestations of AGU, including developmental disabilities, progressive "coarsening" of the face, and early onset of hepatosplenomegaly. Biochemical studies showed elevated levels of urinary aspartylglucosamine and very low activity of aspartylglucosaminidase(AGA) in cultured fibroblasts. With long term follow-up, previously undescribed manifestations were noted, including radiographic evidence of spondylolysis and spondylolisthesis in early childhood and development of macro-orchidism during puberty. This family shows that AGU is not limited to individuals of Finnish background, but that the gene is panethnic in distribution and that additional changes, not previously noted, may present with advancing age.

Acetylglucosamine↗
PubMed1991

Interstitial tandem direct duplication of the long arm of chromosome 4 (q23-q27) and possible assignment of the structural gene encoding human aspartylglucosaminidase to this segment.

We report on a girl with a previously undescribed de novo direct tandem duplication 4q involving the segment q23----q27. Clinical manifestations included postnatal growth and psychomotor retardation, microcephaly, hirsute forehead, epicanthic folds, strabismus, depressed nasal bridge, long philtrum, small mouth, tetralogy of Fallot, and sacral dimple. Her phenotype is compared with that of previously reported cases of duplication 4q. An increased activity of the enzyme aspartylglucosaminidase (AGA) in cultured fibroblasts was demonstrated. This suggests possible assignment of the AGA gene to the chromosomal segment 4q23----4q27.

Aspartylglucosylaminase↗
PubMed1992

Mutations causing aspartylglucosaminuria (AGU): a lysosomal accumulation disease.

This article provides a review of the mutations reported so far in the lysosomal storage disease aspartylglucosaminuria (AGU). The clinical symptoms, biochemical findings, and diagnostic possibilities of the disease are introduced. The prevalence and biological consequences of the found mutations are then described, as well as the availability of a new rapid DNA test suitable for carrier screening. This test will be especially applicable in the genetically isolated Finnish population, where the carrier frequency of AGU was found to be as high as 1:36. Finally, future prospects dealing with the foreseeable therapeutic interventions of the disease are discussed.

Acetylglucosamine↗
PubMed1995

Identification of a novel mutation causing aspartylglucosaminuria reveals a mutation hotspot region in the aspartylglucosaminidase gene.

Aspartylglucosaminuria (AGU) is a recessively inherited metabolic disorder caused by the deficiency of a lysosomal enzyme, aspartylglucosaminidase. The worldwide most common mutation causing the disease is the AGUFin, enriched in Finland; all the other known AGU mutations are family-specific. We developed exon-specific primers to facilitate mutation search directly from the genomic DNA and identified a novel mutation, designated AGUFin minor, in seven Finnish AGUFin compound heterozygote patients. This deletion/frameshift mutation creates a premature translational termination codon and was shown to result in severely reduced transcript levels as quantified by the solid-phase minisequenching method. Genealogical data on this novel mutation suggest its relatively recent introduction into the population. The AGU mutations identified so far have been reported to be evenly distributed throughout the 1 kb coding region of the AGA cDNA. We identified a mutation hotspot region of 40 bp within the 12.5 kb AGA gene containing two previously identified mutations and the novel AGUFin minor mutation characterized in this study.

Acetylglucosamine↗
PubMed2004

A novel aspartylglucosaminuria mutation affects translocation of aspartylglucosaminidase.

The AGA gene is mutated in patients with aspartylglucosaminuria (AGU), a lysosomal storage disease enriched in the Finnish population. The disease mechanism of AGU and the biochemistry and cell biology of the lysosomal aspartylglucosaminidase (AGA) enzyme are well characterized. Here, we have investigated a novel AGU mutation found in a Finnish patient. The mutation was detected as a compound heterozygote with the Finnish major mutation in the other allele. The novel point mutation, c.44T>G, causes the L15R amino acid substitution in the signal sequence of the AGA enzyme. The mutated AGA enzyme was here analyzed by over expression in BHK and COS-1 cells. The L15R AGA protein was only faintly detectable by immunofluorescence analysis and observed in the endoplasmic reticulum. Metabolic labeling and immunoprecipitation revealed only a small amount of AGA polypeptides but the specific activity of the mutant enzyme was surprisingly high, 37% of the wild type. The amino acid substitution probably affects translocation of AGA polypeptides by altering a critical hydrophobic core structure of the signal sequence. It appears that the small amounts of active enzyme are not able to reach the lysosomes thus explaining the development of AGU disease in the patient.

Amino Acid Sequence↗
PubMed1991

Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease.

We have isolated a 2.1 kb cDNA which encodes human aspartylglucosaminidase (AGA, E.C. 3.5.1.26). The activity of this lysosomal enzyme is deficient in aspartylglucosaminuria (AGU), a recessively inherited lysosomal accumulation disease resulting in severe mental retardation. The polypeptide chain deduced from the AGA cDNA consists of 346 amino acids, has two potential N-glycosylation sites and 11 cysteine residues. Transient expression of this cDNA in COS-1 cells resulted in increased expression of immunoprecipitable AGA protein. Direct sequencing of amplified AGA cDNA from an AGU patient revealed a G----C transition resulting in the substitution of cysteine 163 with serine. This mutation was subsequently found in all the 20 analyzed Finnish AGU patients, in the heterozygous form in all 53 carriers and in none of 67 control individuals, suggesting that it represents the major AGU causing mutation enriched in this isolated population. Since the mutation produces a change in the predicted flexibility of the AGA polypeptide chain and removes an intramolecular S-S bridge, it most probably explains the deficient enzyme activity found in cells and tissues of AGU patients.

Amino Acid Sequence↗
PubMed1993

Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations have been identified in AGU patients, elucidation of the molecular pathogenesis of the disease has been hampered by the missing information on the cellular events resulting in the maturation and activation of the enzyme. Here we used the expression of in vitro mutagenized constructs of the AGA cDNA to define three specific proteolytic trimming steps resulting in mature AGA. Removal of the signal peptide is immediately followed by proteolytic cleavage of the precursor into two subunits and results in biologically active enzyme already in the endoplasmic reticulum. This early activation has not previously been described for lysosomal enzymes. The subsequent lysosomal trimming does not influence the enzymatic activity of AGA. It consists only of a single proteolytic cleavage which removes 10 amino acids from the C-terminal end of the larger subunit, in contrast to the multistep lysosomal processing observed in several other hydrolases.

Amino Acid Sequence↗
PubMed2003

Aspartylglucosaminidase (AGA) is efficiently produced and endocytosed by glial cells: implication for the therapy of a lysosomal storage disorder.

BACKGROUND: Aspartylglucosaminuria (AGU) represents diseases affecting the central nervous system and is caused by a deficiency of a lysosomal enzyme, aspartylglucosaminidase (AGA). AGA, like lysosomal enzymes in general, are good targets for gene therapy since they move from cell to cell using the mannose-6-phosphate receptor. Consequently, only a minority of target cells need to be corrected. Here, we wanted to determine which cell type, neurons or glia would better produce AGA to be transported to adjacent cells for use in possible treatment strategies. METHODS: Adenoviruses containing tissue-specific glial fibrillary acidic protein (GFAP) promoter and neuron-specific enolase (NSE) promoter were generated to target expression of AGA in Aga-deficient mouse primary glial and neuronal cell cultures. In addition an endogenous AGA promoter was used. The experimental design was planned to measure the enzymatic activities in the cells and media of neurons and glia infected with each specific virus. The endocytosis of AGA was analyzed by incubating neuronal and glial cells with media produced by each virus-cell combination. RESULTS: AGA promoter was shown to be a very powerful glia promoter producing 32 times higher specific AGA activity in glia than in neurons. GFAP and NSE promoters also produced a clear overexpression of AGA in glia and neurons, respectively. Interestingly, both the NSE and GFAP promoters were not cell-specific in our system. The amount of exocytosed AGA was significantly higher in glial cells than neurons and glial cells were also found to have a greater capacity to endocytose AGA. CONCLUSIONS: These data indicate the importance of glial cells in the expression and transport of AGA. Subsequently, new approaches can be developed for therapeutic intervention.

Adenoviridae↗
PubMed2006

Use of nonviral promoters in adenovirus-mediated gene therapy: reduction of lysosomal storage in the aspartylglucosaminuria mouse.

BACKGROUND: Aspartylglucosaminuria (AGU) is a lysosomal storage disease with severe neurodegenerative clinical features resulting from the deficiency of lysosomal aspartylglucosaminidase (AGA). The AGU knockout mouse is a good model to test different therapy strategies, as it mimics well the human pathogenesis of the disease exhibiting storage vacuoles in all tissues. In this study we investigated the efficiency of nonviral promoters in adenovirus-mediated gene therapy. METHODS: The deficient corrective enzyme, AGA, was expressed using two tissue-specific promoters, neuron-specific enolase (NSE), astrocyte-specific (GFAP) and the endogenous AGA promoter. An intrastriatal injection site was chosen due to its wide connections in the central nervous system (CNS). The expression of AGA was analyzed 1 week, 2 weeks, 4 weeks, 2 months and 4 months after the virus injection by lysosomal AGA-specific immunostaining. A correction of the lysosomal storage in the brain of treated mice was also studied using toluidine blue stained thin sections. RESULTS: The overexpressed AGA enzyme was detected in addition to the injection site, also in the ipsilateral parietal cortex indicating migration of AGA in the brain tissue. Duration of AGA expression was markedly longer with all the viruses used compared to the green fluorescent protein (GFP) expression driven by the viral cytomegalovirus (CMV) promoter. In most animals the storage was decreased by at least 50% as compared to untreated AGU mouse brains. Remarkably, >90% correction of storage at the ipsilateral cortex was found with the NSE promoter at 4 weeks and 2 months after injection. Additionally, partial clearance of storage was demonstrated also in the contralateral side of the brain. CONCLUSIONS: These data implicate that tissue-specific promoters are especially useful in virus-mediated gene therapy aiming at long-term gene expression.

Adenoviridae↗
PubMed2001

Antenatal gene tests in low-risk pregnancies: molecular screening for aspartylglucosaminuria (AGU) and infantile neuronal ceroid lipofuscinosis (INCL) in Finland.

Approximately one in five subjects in Finland carries some gene defect associated with 30 diseases belonging to the Finnish disease heritage, and about one in 500 children born is affected. Almost all carriers, women and men, are unaware of their condition. Recent advances in molecular medicine have offered the possibility of population-based carrier screening for recessive disorders. We studied acceptance and attitudes to antenatal screening for aspartylglucosaminuria (AGU) and infantile neuronal ceroid lipofuscinosis (INCL). From January 1995 until December 1996 carrier tests were offered at Kuopio City Health Center, free of charge to all pregnant women attending maternity care units. Women found to be carriers of AGU (n = 47) or INCL (n = 14) underwent detailed genetic counseling, and their male partners were also offered the test. If both partners appeared to be carriers we offered prenatal testing (n = 1). No affected fetuses were detected. Attitudes towards the gene test were elicited by means of a questionnaire. Altogether 87% of pregnant women elected to undertake the gene tests. Antenatal screening for gene defects was feasible and well accepted, and it provided an effective way to find carriers of genetic diseases and to incorporate prenatal testing into this process.

Adult↗
PubMed1998

Crystal structure of glycosylasparaginase from Flavobacterium meningosepticum.

The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.

Aspartylglucosylaminase↗
PubMed1995

Molecular cloning and sequence analysis of Flavobacterium meningosepticum glycosylasparaginase: a single gene encodes the alpha and beta subunits.

A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.

Amino Acid Sequence↗
PubMed1996

Glycosylation and phosphorylation of lysosomal glycosylasparaginase.

Glycosylasparaginase (EC 3.5.1.26) is a lysosomal amidase which hydrolyzes the bond between asparagine and the sugar moiety in N-linked glycoproteins. Deficiency of the enzyme results in aspartylglycosaminuria (AGU), the most common disorder of glycoprotein degradation. Mature enzyme is formed by two proteolytic cleavage steps subsequent to removal of its signal peptide: (1) an activation cleavage in the ER of the initial single-chain 49-kDa polypeptide into a 27-kDa alpha- and 19-kDa beta-subunit; (2) a cleavage in lysosomes which removes 10 amino acids from the C-terminus of the alpha-subunit without affecting enzyme activity. Each subunit of glycosylasparaginase contains one N-linked oligosaccharide (N38, alpha-subunit; N308, beta-subunit). Both oligosaccharides were phosphorylated and releasable by Endo-H digestion, indicating they were of the high-mannose type. These glycosylation sequenons were mutagenized to determine the role of the oligosaccharide at each site in proper folding and transport of glycosylasparaginase. An N38D mutant underwent the lysosomal processing step, indicating that targeting to lysosomes can be via the phosphorylated beta-subunit oligosaccharide alone. Deletion of the beta-subunit oligosaccharide oat N308 by an aspartic acid substitution resulted in very little protein or enzyme activity in the transfected cells, reemphasizing that glycosylation of the beta-subunit site is important for efficient folding and/or targeting. A different mutation to eliminate the same N-glycosylation sequenon (T310A) yielded more protein and enzyme activity, and a double mutant N38D/T310A yielded the same results as the single beta-subunit substitution. Yield of enzyme for all mutants was increased in cells treated with brefeldin A. The N308 glycosylation site of the beta-subunit appears to be more important in maintaining normal transport and stability of human glycosylasparaginase.

Animals↗