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Journal of Biochemistry Oct 1975Two major glycoasparagines (2-acetamido-N-(4'-L-aspartyl)-2-deoxy-beta-D-glycosylamines) were isolated from the urine of patients with aspartylglycosylaminuria (AGU)....
Two major glycoasparagines (2-acetamido-N-(4'-L-aspartyl)-2-deoxy-beta-D-glycosylamines) were isolated from the urine of patients with aspartylglycosylaminuria (AGU). They were composed of equimolar amounts of sialic acid, galactose, glucosamine, and aspartic acid. They were isomeric with respect to the position of sialic acid attachment, since they produced the same glycoasparagine on incubation with the neuraminidase from Clostridium perfringens. The structure of the resulting sialic acid-free glycoasparagine was determined as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based on the following findings. It produced galactose on incubation with beta-galactosidase, and N-acetyllactosamine and aspartic acid on incubation with 4-L-aspartylglycosylamine amindo hydrolase.
Topics: Amidohydrolases; Asparagine; Aspartylglucosaminuria; Galactose; Galactosidases; Glucosamine; Metabolism, Inborn Errors; Neuraminidase; Sialic Acids
PubMed: 1213985
DOI: 10.1093/oxfordjournals.jbchem.a130954 -
The Biochemical Journal Feb 1994The substrate specificity of the core-specific rat liver lysosomal alpha(1-6)-mannosidase was investigated using mannosylated oligosaccharides and glycoasparagines....
The substrate specificity of the core-specific rat liver lysosomal alpha(1-6)-mannosidase was investigated using mannosylated oligosaccharides and glycoasparagines. Hydrolysis of Man(alpha 1-6) linkage hydrolysis was demonstrated to follow the action of endoglycosidases, namely aspartyl-N-acetyl-beta-D-glucosaminidase and endo-N-acetyl-beta-D-glucosaminidase. The results are discussed with respect to the nature of the carbohydrate materials stored in the tissues and excreted in the urine from patients suffering from aspartylglucosaminuria and fucosidosis.
Topics: Acetylation; Animals; Aspartylglucosylaminase; Carbohydrate Sequence; Glycopeptides; Lysosomes; Mannosidases; Mass Spectrometry; Molecular Sequence Data; Oligosaccharides; Rats; Substrate Specificity
PubMed: 8110182
DOI: 10.1042/bj2970463 -
The EMBO Journal Jan 1993Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations...
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.
Topics: Amino Acid Sequence; Animals; Aspartylglucosylaminase; Blotting, Western; Cell Line; DNA; Diethyl Pyrocarbonate; Endoplasmic Reticulum; Enzyme Activation; Humans; Kinetics; Lysosomes; Macromolecular Substances; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Processing, Post-Translational; Transfection
PubMed: 8428587
DOI: 10.1002/j.1460-2075.1993.tb05656.x -
Annals of Medicine Jun 1992
Topics: Amyloidosis; Aspartylglucosaminuria; Aspartylglucosylaminase; Genetic Carrier Screening; Genetic Diseases, Inborn; Genetic Markers; Humans; Lysosomal Storage Diseases; Mutation; Nervous System Diseases; Polymerase Chain Reaction
PubMed: 1627315
DOI: 10.3109/07853899209147820 -
PCR Methods and Applications May 1992We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA...
We present a new method for quantification of mRNA, in which the limitations of the current quantitative PCR methods can be overcome. A known amount of a synthetic RNA standard differing from the mRNA to be quantified by a single nucleotide is reverse-transcribed and amplified together with the mRNA template using a biotinylated primer. The biotinylated PCR product is immobilized on a streptavidin-coated solid support and denatured. The ratio between the two amplified sequences is determined by separate "mini-sequencing" reactions, in which a detection step primer annealing immediately adjacent to the site of the variable nucleotide is elongated by a single labeled dNTP complementary to the nucleotide at the variable site. The ratio between the incorporated labels accurately determines the ratio between the two sequences in the original RNA sample. We applied this method to quantify the mRNA of human aspartylglucosaminidase (AGA) in tissues and cultured cells. AGA is a lysosomal enzyme participating in the degradation of glycoproteins. A mutation in the AGA gene abolishes the enzyme activity and leads to aspartylglucosaminuria (AGU), a recessively inherited metabolic disorder. The mRNA quantification revealed that the normal and mutant genes are expressed at similar levels in kidney, liver, and cultured fibroblast, whereas the amount of AGA mRNA in normal placenta and brain is significantly higher than that found in the corresponding samples from AGU patients. The method presented here is generally applicable for PCR-based quantification of rare mRNAs and DNA as well.
Topics: Aspartylglucosaminuria; Aspartylglucosylaminase; Base Sequence; Cells, Cultured; Gene Expression Regulation, Enzymologic; Genotype; Humans; Metabolism, Inborn Errors; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; RNA, Messenger; Sequence Analysis, RNA
PubMed: 1477657
DOI: 10.1101/gr.1.4.234 -
Molecular Genetics and Metabolism Jun 2017Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in...
Glycosylasparaginase (GA) is an amidase that cleaves Asn-linked glycoproteins in lysosomes. Deficiency of this enzyme causes accumulation of glycoasparagines in lysosomes of cells, resulting in a genetic condition called aspartylglycosaminuria (AGU). To better understand the mechanism of a disease-causing mutation with a single residue change from a glycine to an aspartic acid, we generated a model mutant enzyme at the corresponding position (named G172D mutant). Here we report a 1.8Å resolution crystal structure of mature G172D mutant and analyzed the reason behind its low hydrolase activity. Comparison of mature G172D and wildtype GA models reveals that the presence of Asp 172 near the catalytic site affects substrate catabolism in mature G172D, making it less efficient in substrate processing. Also recent studies suggest that GA is capable of processing substrates that lack a chitobiose (Glycan, N-acetylchiobios, NAcGlc) moiety, by its exo-hydrolase activity. The mechanism for this type of catalysis is not yet clear. l-Aspartic acid β-hydroxamate (β-AHA) is a non-chitobiose substrate that is known to interact with GA. To study the underlying mechanism of non-chitobiose substrate processing, we built a GA-β-AHA complex structure by comparing to a previously published G172D mutant precursor in complex with a β-AHA molecule. A hydrolysis mechanism of β-AHA by GA is proposed based on this complex model.
Topics: Asparagine; Aspartylglucosaminuria; Aspartylglucosylaminase; Biocatalysis; Crystallization; Crystallography, X-Ray; Disaccharides; Glycopeptides; Humans; Hydrolysis; Lysosomes; Models, Molecular; Mutant Proteins; Mutation; Substrate Specificity
PubMed: 28457719
DOI: 10.1016/j.ymgme.2017.04.008 -
FEBS Letters Aug 1991We have indentified a GT-to-TT transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU)...
We have indentified a GT-to-TT transversion at the splice donor site of intron 8 in the glycosylasparaginase gene from an African American aspartylglucosaminuria (AGU) patient. This mutation causes abnormal splicing of glycosylasparaginase pre-mRNA by joining exon 7 to 9 and excluding 134 bp exon 8. The effect of the mutation is compounded by a frame shift that occurs after the deletion site resulting in premature translational termination. The truncated AGU protein was neither catalytically active nor processed into mature alpha and beta subunits. Both this and a previously characterized Finnish AGU mutation appear to affect folding of the single-chain precursor of glycosylasparaginase and thereby prevent transport of the enzyme to lysosomes.
Topics: Acetylglucosamine; Amino Acid Sequence; Aspartylglucosaminuria; Aspartylglucosylaminase; Base Sequence; Black People; Child; Chromosome Deletion; Exons; Fluorescent Antibody Technique; Glycosuria; Humans; Male; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Protein Conformation; RNA Splicing
PubMed: 1879549
DOI: 10.1016/0014-5793(91)81028-7 -
American Journal of Human Genetics Jun 1995In the present study we explore the attitudes of the Finnish population toward genetic testing by conducting a questionnaire study of a stratified sample of the...
In the present study we explore the attitudes of the Finnish population toward genetic testing by conducting a questionnaire study of a stratified sample of the population as well as of family members of patients with a severe hereditary disease, aspartylglucosaminuria (AGU). The questionnaire evaluated attitudes toward gene tests in general and also respondents' preparedness to undergo gene tests for predictive testing, carrier detection, prenatal diagnosis, and selective abortion, in theoretical situations. The results of the study indicate that both the Finnish population in general and family members of AGU patients have a favorable attitude toward genetic testing. However, a commonly expressed reason against testing was that test results might lead to discrimination in employment or insurance policies. Based on the responses, we predict that future genetic testing programs will most probably be met with a high acceptance rate by the Finnish population.
Topics: Adolescent; Adult; Attitude to Health; Employment; Female; Finland; Genetic Diseases, Inborn; Genetic Testing; Humans; Insurance; Intellectual Disability; Interpersonal Relations; Lysosomal Storage Diseases; Male; Middle Aged; Patient Acceptance of Health Care; Population; Pregnant Women; Prejudice; Sampling Studies; Sex Determination Analysis; Surveys and Questionnaires
PubMed: 7762573
DOI: No ID Found -
The Journal of Biological Chemistry May 1992Aspartylglucosaminuria (AGU) is a lysosomal storage disease due to mutations in the aspartylglucosaminidase (AGA) gene. The deficient enzyme activity in patients' cells...
Aspartylglucosaminuria (AGU) is a lysosomal storage disease due to mutations in the aspartylglucosaminidase (AGA) gene. The deficient enzyme activity in patients' cells blocks one of the final steps in the degradation of N-linked glycoproteins. All the AGU mutations identified so far affect the coding region of the AGA gene. Here we report a homozygous 876-base pair deletion, which removes the 3'-noncoding area but leaves the coding region of the AGA mRNA intact. This deletion does not prevent transcription termination or polyadenylation of the patient's truncated mRNA, and the steady state level of the mRNA is comparable with the control. However, the quantity of AGA polypeptide chains in the patient's fibroblasts is negligible. This suggests that the deletion interferes with the translational efficiency in vivo and provides a unique model to pursue the biological significance of untranslated regions of human mRNAs.
Topics: Aspartylglucosylaminase; Base Sequence; Blotting, Southern; Blotting, Western; Cells, Cultured; Chromosome Deletion; DNA; Homozygote; Humans; Introns; Lysosomal Storage Diseases; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Protein Biosynthesis; RNA, Messenger; Transcription, Genetic
PubMed: 1577713
DOI: No ID Found -
The Biochemical Journal Mar 1993Aspartylglycosaminuria (AGU) is a lysosomal storage disease principally occurring in Finland that results from mutations in the structural gene for glycosylasparaginase...
Aspartylglycosaminuria (AGU) is a lysosomal storage disease principally occurring in Finland that results from mutations in the structural gene for glycosylasparaginase (AGU). This work characterizes the inheritance of two previously reported AGU mutations in a British patient [Ikonen, Aula, Grön, Tollersrud, Halila, Manninen, Syvänen and Peltonen (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11222-11226]. Use of the PCR determined the glycosylasparaginase cDNA sequence from both parents of the British patient and his AGU-affected brother. The father of the British AGU-affected siblings was found to be a heterozygote carrier for a C-->T point mutation which causes an Ala-->Val amino-acid substitution, while the mother was heterozygous for a 7 bp deletion that results in premature translational termination. The brother of the previously studied patient was similarly shown to be a compound heterozygote. Expression in COS-1 cells revealed the paternal Ala-->Val amino-acid substitution destroyed glycosylasparaginase catalytic activity, prevented transport of the mutant protein to the lysosome, and prevented maturation of the enzyme precursor to its native subunit structure. The Ala-->Val mutation therefore affects glycosylasparaginase in a manner similar to the Finnish AGU Cys-->Ser substitution, further supporting a linkage of glycosylasparaginase catalytic activity to its lysosomal transport and subunit processing [Fisher and Aronson (1991) J. Biol. Chem. 266, 12105-12113]. In addition, a 5 bp deletion mutation from an American patient with AGU has been characterized. The deleted sequence occurs at the beginning of the glycosylasparaginase coding sequence, resulting in an extremely truncated polypeptide. The American 5 bp deletion and the British maternal 7 bp deletion possibly decrease mRNA stability.
Topics: Acetylglucosamine; Alleles; Amino Acid Sequence; Aspartylglucosaminuria; Aspartylglucosylaminase; Base Sequence; Cell Line; DNA; Female; Fluorescent Antibody Technique; Gene Deletion; Heterozygote; Humans; Male; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Transfection; United Kingdom; United States
PubMed: 8457202
DOI: 10.1042/bj2900735