Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

Aspartylglucosaminuria caused by a novel homozygous mutation in the AGA gene was identified by an exome-first approach in a patient from Japan.

BACKGROUND: Aspartylglucosaminuria (AGU) is an autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme, aspartylglucosaminidase (AGA). This disorder is rare in the general population except in Finland. Since the most characteristic feature of this disorder is a progressive developmental regression, patients often show no specific symptoms in the initial stages, and thus early diagnosis is often challenging. CASE REPORT: We encountered a 16-year-old boy who began to show difficulties in his speech at the age of 6years. Due to a mild regression in his development, he gradually lost common daily abilities. His diagnosis was first obtained through exome sequencing that identified a novel homozygous mutation in the AGA gene. This result was reasonable because of parental consanguinity. Reduced enzymatic activity of AGA was then confirmed. His urine was retrospectively screened by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and a specific pattern of abnormal metabolites was identified. CONCLUSIONS: Because both exome sequencing and MALDI-TOF-MS screening are adaptable and comprehensive, future combinatory use of these methods would be useful for diagnosis of rare inborn errors of metabolism such as AGU.

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.

Activation and oligomerization of aspartylglucosaminidase.

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.

Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase.

Aspartylglucosaminidase (AGA, EC 3.5.1.26) is a dimeric lysosomal hydrolase involved in the degradation of glycoproteins. The synthesized precursor polypeptide of AGA is rapidly activated in the endoplasmic reticulum by proteolysis into two subunits. Expression of the alpha- and beta-subunits of AGA in separate cDNA constructs showed that independently folded subunits totally lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer of the enzyme. Both of the subunits are required for the enzyme activity, and the immediate interaction of the subunits in the endoplasmic reticulum is necessary for the correct folding of the dimeric enzyme molecule. The specific amino acid residues essential for the active site of the AGA enzyme were further analyzed by site-directed mutagenesis and in vitro expression of mutagenized constructs. Replacement of Thr206, the most amino-terminal residue of the beta-subunit, with Ser resulted in a complete loss of enzyme activity without influencing intracellular processing or transport of the mutant polypeptide to the lysosomes. Analogously, replacement of the most amino-terminal tryptophan, Trp34 with Phe or Ser in the alpha-subunit, resulted in a totally inactive enzyme without influencing the intracellular processing or stability of the polypeptide. These results suggest that the catalytic center of this amidase is formed by the interaction of the amino-terminal parts of two subunits and requires both Trp34 in the alpha-subunit and Thr206 in the beta-subunit.

Dissection of the molecular consequences of a double mutation causing a human lysosomal disease.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency in which leads to human storage disease aspartylglucosaminuria (AGU). AGUFin is the most common AGU mutation in the world and is found in 98% of AGU alleles in Finland, where the population displays enrichment of the disease allele. The AGUFin allele actually contains a double mutation, both individual mutations resulting in amino acid substitutions: Arg-161-->Gln and Cys-163-->Ser. The separate consequences of these two amino acid substitutions for the intracellular processing of the AGA polypeptides were analyzed using a stable expression of mutant polypeptides in Chinese hamster ovary (CHO) cells. The synthesized polypeptides were monitored by metabolic labeling, followed by immunoprecipitation, immunofluorescence, and immunoelectron microscopy. The Arg-161-->Gln substitution did not affect the intracellular processing or transport of AGA and the fully active enzyme was correctly targeted to lysosomes. The Cys-163-->Ser substitution prevented the early proteolytic cleavage required for the activation of the precursor AGA polypeptide and the inactive enzyme was accumulated in the endoplasmic reticulum (ER). The precursors of the translation products of the AGUFin double mutant and the Cys-163-->Ser mutant were also observed in the culture medium. When cells expressing the normal AGA or AGUFin double mutation were treated with DTT to prevent the formation of disulfide bonds, both normal and mutated AGA polypeptides remained in the inactive precursor form and were not secreted into the medium. These results indicate that correct initial folding is essential for the proteolytic activation of AGA.(ABSTRACT TRUNCATED AT 250 WORDS)