In vitro mutagenesis helps to unravel the biological consequences of aspartylglucosaminuria mutation

doi:10.1016/0888-7543(91)90120-4

Genomics

Volume 11, Issue 1, September 1991, Pages 206-211

https://doi.org/10.1016/0888-7543(91)90120-4 Get rights and content

Abstract

Aspartylglucosaminuria (AGU) is a lysosomal storage disease resulting in severe mental retardation. We have recently reported that mutations in the aspartylglucosaminidase (AGA) locus are responsible for this disease. About 90% of reported AGU cases are found in Finland, and we have shown that the vast majority (98%) of AGU alleles in this isolated population contain two point mutations located 5 bp apart. We expressed these Arg₁₆₁ → Gln and Cys₁₆₃ → Ser mutations separately in vitro and demonstrated that deficient enzyme activity is caused by the Cys₁₆₃ → Ser mutation, whereas the Arg₁₆₁ → Gln substitution represents a rare polymorphism. Further analyses of in vitro expressed AGA proteins and the enzyme purified from an AGU patient revealed that Cys₁₆₃ participates in an S-S bridge. The absence of this covalent cross-link in the mutated protein most probably results in disturbed folding of the polypeptide chain and a consequent decrease in its intracellular stability.

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A deep redox proteome profiling workflow and its application to skeletal muscle of a Duchenne Muscular Dystrophy model
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Citation Excerpt :
Also related to phagosome-lysosome function is N(4)-(beta-N-acetylglucosaminyl)-l-asparaginase (ASPG), also known as lysosomal aspartylglucosaminidase (AGA), which is a hydrolase that breaks down glycoproteins in lysosomes. Deficient activity of ASPG results in the lysosomal storage disease aspartylglycosaminuria [71], which is caused by a Cys163Ser mutation that prevents formation of a disulfide bridge, leading to its inability to be properly folded and activated [72,73]. Equally, a Cys179Ser mutation has a similar effect on ASPG folding, leading to its rapid degradation [73].
Perturbation to the redox state accompanies many diseases and its effects are viewed through oxidation of biomolecules, including proteins, lipids, and nucleic acids. The thiol groups of protein cysteine residues undergo an array of redox post-translational modifications (PTMs) that are important for regulation of protein and pathway function. To better understand what proteins are redox regulated following a perturbation, it is important to be able to comprehensively profile protein thiol oxidation at the proteome level. Herein, we report a deep redox proteome profiling workflow and demonstrate its application in measuring the changes in thiol oxidation along with global protein expression in skeletal muscle from mdx mice, a model of Duchenne Muscular Dystrophy (DMD). In-depth coverage of the thiol proteome was achieved with >18,000 Cys sites from 5,608 proteins in muscle being quantified. Compared to the control group, mdx mice exhibit markedly increased thiol oxidation, where a ∼2% shift in the median oxidation occupancy was observed. Pathway analysis for the redox data revealed that coagulation system and immune-related pathways were among the most susceptible to increased thiol oxidation in mdx mice, whereas protein abundance changes were more enriched in pathways associated with bioenergetics. This study illustrates the importance of deep redox profiling in gaining greater insight into oxidative stress regulation and pathways/processes that are perturbed in an oxidizing environment.
Amlexanox provides a potential therapy for nonsense mutations in the lysosomal storage disorder Aspartylglucosaminuria
2018, Biochimica et Biophysica Acta - Molecular Basis of Disease
Citation Excerpt :
Due to a founder effect, the predominant AGU allele in Finland carries a mutation producing an exchange of Cys163 to Ser (C163S), which is the disease-causing substitution. Cys163Ser is always associated with Arg161Gln exchange, and this combination allele is designated as AGUFin-major as it represents about 98% of Finnish AGU alleles [2–4]. However, AGU patients outside Finland usually have their own unique mutations [5–7].
Aspartylglucosaminuria (AGU) is a lysosomal storage disorder caused by mutations in the gene for aspartylglucosaminidase (AGA). This enzyme participates in glycoprotein degradation in lysosomes. AGU results in progressive mental retardation, and no curative therapy is currently available. We have here characterized the consequences of AGA gene mutations in a compound heterozygous patient who exhibits a missense mutation producing a Ser72Pro substitution in one allele, and a nonsense mutation Trp168X in the other. Ser72 is not a catalytic residue, but is required for the stabilization of the active site conformation. Thus, Ser72Pro exchange impairs the autocatalytic activation of the AGA precursor, and results in a considerable reduction of the enzyme activity and in altered AGA precursor processing. Betaine, which can partially rescue the AGA activity in AGU patients carrying certain missense mutations, turned out to be ineffective in the case of Ser72Pro substitution. The Trp168X nonsense allele results in complete lack of AGA polypeptide due to nonsense-mediated decay (NMD) of the mRNA. Amlexanox, which inhibits NMD and causes a translational read-through, facilitated the synthesis of a full-length, functional AGA protein from the nonsense allele. This could be demonstrated as presence of the AGA polypeptide and increased enzyme activity upon Amlexanox treatment. Furthermore, in the Ser72Pro/Trp168X expressing cells, Amlexanox induced a synergistic increase in AGA activity and polypeptide processing due to enhanced processing of the Ser72Pro polypeptide. Our data show for the first time that Amlexanox might provide a valid therapy for AGU.
Activation and oligomerization of aspartylglucosaminidase
1998, Journal of Biological Chemistry
Secretory, membrane, and lysosomal proteins undergo covalent modifications and acquire their secondary and tertiary structure in the lumen of the endoplasmic reticulum (ER). In order to pass the ER quality control system and become transported to their final destinations, many of them are also assembled into oligomers. We have recently determined the three-dimensional structure of lysosomal aspartylglucosaminidase (AGA), which belongs to a newly discovered family of homologous amidohydrolases, the N-terminal nucleophile hydrolases. Members of this protein family are activated from an inactive precursor molecule by an autocatalytic proteolytic processing event whose exact mechanism has not been thoroughly determined. Here we have characterized in more detail the initial events in the ER required for the formation of active AGA enzyme using transient expression of polypeptides carrying targeted amino acid substitutions. We show that His¹²⁴ at an interface between two heterodimers of AGA is crucial for the thermodynamically stable oligomeric structure of AGA. Furthermore, the side chain of Thr²⁰⁶ is essential both for the proteolytic activation and enzymatic activity of AGA. Finally, the proper geometry of the residues His²⁰⁴–Asp²⁰⁵ seems to be crucial for the activation of AGA precursor polypeptides. We propose here a reaction mechanism for the activation of AGA which could be valid for homologous enzymes as well.
Primary folding of aspartylglucosaminidase. Significance of disulfide bridges and evidence of early multimerization
1996, Journal of Biological Chemistry
Aspartylglucosaminidase (AGA) is a lysosomal enzyme involved in the degradation of N-linked glycoproteins in lysosomes. AGA is synthesized as an inactive precursor molecule, which is rapidly activated in the endoplasmic reticulum by a proteolytic cleavage into α- and β-subunits. We have recently determined the three-dimensional structure of AGA and shown that it is a globular molecule with a heterotetrameric (αβ)₂ structure. On the basis of structural and functional analyses, AGA seems to be the first mammalian protein belonging to a newly described protein family, the N-terminal nucleophile hydrolases. Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA. To gain further information on the structural determinants influencing the early folding of AGA, we used site-specific mutagenesis of cysteine residues to define the role of intrachain disulfide bridges in the folding and activation of the enzyme. The N-terminal disulfide bridges in both the α- and β-subunits seem to have only a stabilizing role, whereas the C-terminal disulfide bridge in both subunits evidently plays an important role in the early folding and activation of AGA.
Molecular cloning, chromosomal assignment, and expression of the mouse aspartylglucosaminidase gene
1995, Genomics
Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to human lysosomal storage disease aspartylglucosaminuria. Here, we describe isolation, chromosomal location, genomic structure, and tissue-specific expression of the mouseAgagene as well as the intracellular processing of the mouse Aga polypeptide and compare these characteristics to human AGA. The mouseAgagene was localized to the central area of the B region of chromosome 8, which represents the synteny group in the human chromosome 4q telomeric region where the humanAGAgene is located. The mouse gene spans an 11-kb genomic region and contains nine exons and eight introns, which is analogous to the human gene. Furthermore, the exon–intron boundaries of the mouse and human genes are identically positioned. The nucleotide sequence identity of the cDNA and deduced amino acid sequence identity of the protein are 84.4 and 82.4%, respectively. However, the mouse Aga cDNA contains untranslated regions that are shorter than those in the human cDNA, and only one 1.2-kb mRNA transcript is produced in mouse versus two transcripts in human. Expression of the mouse Aga cDNA in COS-1 cells showed that the mouse Aga polypeptide was processed similarly to the human counterpart.
Lysosomal storage diseases
1995, BBA - Molecular Basis of Disease

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In vitro mutagenesis helps to unravel the biological consequences of aspartylglucosaminuria mutation

Abstract

Lancet

Gene

Adv. Protein Chem

Biochem. Biophys. Res. Commun

Lancet

J. Biol. Chem

J. Biol. Chem

Anal. Biochem

Aspartylglucosaminuria

Aspartylglucosaminuria: Deficiency of aspartylglucosaminidase in cultured fibroblasts of patients and their heterozygous parents

Clin. Genet