Ritva Tikkanen, PhD, Professor of Biochemistry & Antje Banning, PhD
Institute of Biochemistry, Medical Faculty, University of Giessen, Giessen, Germany

Aspartylglucosaminuria (AGU)
Aspartylglucosaminuria (AGU) is a lysosomal storage disorder that is characterized mainly byprogressive mental retardation. AGU patients are born seemingly normal, but within the first years oflife, the symptoms of the disease manifest with progressive loss of mental capabilities such asspeech. In their early adulthood, most AGU patients are severely mentally retarded, but upon goodmedical care, the life expectancy is relatively long. AGU is a rare disease, with an unknown
prevalence in most countries. However, due to the peculiar population history of Finland, the diseaseis enriched in the Finnish population.
Aspartylglucosaminidase (AGA)
AGU is caused by recessive mutations in the gene coding for a lysosomal enzyme,
aspartylglucosaminidase (AGA). This enzyme participates in one of the final steps of lysosomal
degradation of proteins that are N-glycosylated, that is: decorated with specific sugar chains. In theabsence of AGA activity, the substrates of AGA, the so-called glycoasparagines, accumulate in thelysosomes and are even excreted in the urine, which can be used as a diagnostic measure for AGU.
The AGA enzyme is synthesized as a single polypeptide chain, but enzyme activity is only observed
after two precursors soon after their synthesis join together and become cleaved into two subunits
called α and β. Thus, the active enzyme is a heterotetramer consisting of two α and two β subunits.
Effect of AGU mutations on AGA
The most common AGU mutation worldwide is the AGUFin-major mutation found in the vast majority ofFinnish AGU patients. This mutation is actually a combination of two amino acid substitutions, anexchange of cysteine 163 to serine and arginine 161 to glutamine. However, only the Cys163Ser
exchange is pathologically relevant, as it results in destabilization of the AGA enzyme structure andthus prevents the processing of the precursor into the active form. A second typical AGU mutation, adeletion called AGUFin-minor, is present in some Finnish patients. Patients from elsewhere in the world
primarily have their individual gene defects that range from point mutations to deletions andinsertions.
The three-dimensional structure of the human AGA enzyme at atomic resolution has been solved.
This has facilitated a detailed analysis of the consequences of AGU mutations on the AGA enzyme
structure. Therefore, we are now able to understand in which way the AGU mutations impair thefunction of the enzyme. Some of the mutations result in a profound lack of any AGA protein at all,since a part of the gene is missing (deletion), or the protein synthesis is prematurely interrupted (socalled nonsense mutations). However, some mutations, including AGUFin-major, alter the enzymestructure, but the amount of the AGA protein is not severely reduced. In most of these cases, theactivation of the AGA precursor is impaired and only a very low degree of residual AGA activity isdetected in the cells of the affected patients. Depending on where in the enzyme structure thesemutations reside, the degree of functional impairment may be relatively moderate, and it may evenbe possible to correct or reactivate these mutated enzymes.
1Animal models and therapy for AGUTwo different AGU mouse models were created in the 1990s, both of which lack AGA expression due
to disruption of the AGA gene. Adult mice show massive accumulation of glycoasparagines and very
similar pathological changes as found in human AGU patients. No mouse strain carrying a specificdisease-causing mutation, such as AGUFin-major, has been created yet. The existing AGU mice havebeen used to test different treatment options, e.g. gene therapy and enzyme replacement therapy(ERT). The gene therapy trials in which a virus carrying the AGA gene injected into the brain of adult
AGU mice gave promising results. The amount of storage material in the lysosomes was clearly
reduced and the AGA enzyme was even found in cells that had not been infected with the virus,
indicating that the AGA protein can be transported from one cell to another. However, for the genetherapy to become available as a treatment option for AGU, more time is needed to test the safety
and to establish this method for use in patients.
In further studies, AGU mice were subjected to ERT. For this, AGA protein purified from AGA
overexpressing cells was injected into AGU mice of different age. In all studies, the level of enzyme
uptake was tissue-dependent. In general, non-neuronal tissues, especially liver and spleen, gained
high AGA activity and showed a profound (> 60%) reduction in intracellularly stored
glycoasparagines. However, in the brain, ERT was less successful in adult mice, but promising in
neonatal mice, stressing that early start of ERT is important to prevent the progression of AGU.
Despite the beneficial effects of ERT in mice, no human trials have been performed yet. This is due tothe fact that it has been difficult to produce sufficient amounts of AGA protein for ERT in patients. Sofar, no pharmaceutical company has launched this therapy option for clinical use.
In the 1990s, seven AGUFin-major patients (1-10 years of age) were subjected to bone marrowtransplantation (BMT). Only in 5 patients, the procedure was successful and initially led to promisingresults. Transplanted patients had increased AGA activity in blood cells and improved brain scandata. However, the patients suffered from different post-transplant complications and had to endure
long hospital stays. Unfortunately, after 4-7 years of follow-up, no mental improvement was noted,
and the patients still experienced AGU-specific health problems. However, further progression of thedisease appeared to be slowed down. Altogether, BMT was not recommended as a treatment option
due to the complications associated with the therapy and the relatively benign therapeutic effect.
Recently, our group has developed a pharmacological chaperone therapy (PCT) as a putative
treatment option for AGU. Pharmacological chaperones in general are small molecules that directlybind and stabilize their target protein. By doing so, they may help to stabilize the mutated enzymes
and thus increase their activity. Our group could show that the small molecule betaine (a componentof beet root) and the amino acid glycine were able to reactivate AGUFin-major and another mutatedform of AGA with a single amino acid exchange. The PCT can only be effective in patients who exhibit
mutations that do not affect the active site of the enzyme, but rather produce milder defects in thestructure of the AGA enzyme and thus affect its processing into subunits. However, this treatment
option will not work for nonsense mutations, splicing mutations or deletions.
Currently, no curative therapy is available for AGU patients. However, we have decided to convert
our promising findings on PCT for AGU into clinics. A clinical trial using Cystadane (trade name ofbetaine) with 21 AGU patients who are homozygous for the AGUFin-major mutation has started inJanuary 2018 in Finland (EU Clinical Trial number 2017-000645-48).
2

AGU patients with a nonsense mutation in their AGA gene, i.e. a mutation that results in a prematurestop codon and therefore synthesis of either truncated AGA proteins or no protein at all , may
benefit from treatment with Amlexanox. Recently, our group was able to show that this substance isable to stabilize the mutated mRNA, which would normally be degraded by a process called
nonsense-mediated decay (NMD). Furthermore, Amlexanox induces a translational read-through at
the nonsense codon, which allows the synthesis of a full-length, functional AGA enzyme. In somecountries, Amlexanox already is an approved drug for e.g. asthma, and it is currently being tested inclinical trials for Diabetes Type II and obesity.
Recommended reading:
Arvio & Mononen https://www.ncbi.nlm.nih.gov/pubmed/27906067
Banning et al. 2016 https://www.ncbi.nlm.nih.gov/pubmed/27876883
Banning et al. 2017 https://www.ncbi.nlm.nih.gov/pubmed/29247835
Kaartinen et al. 1996 https://www.ncbi.nlm.nih.gov/pubmed/8946839
Jalanko et al. 1998 https://www.ncbi.nlm.nih.gov/pubmed/9425233
Virta et al. 2006 https://www.ncbi.nlm.nih.gov/pubmed/16518877
Peltola et al. 1998 https://www.ncbi.nlm.nih.gov/pubmed/9930336
Kelo et al. 2005 https://www.ncbi.nlm.nih.gov/pubmed/15342551
Dunder et al. 2010 https://www.ncbi.nlm.nih.gov/pubmed/20607610
Laitinen et al. 1997 https://www.ncbi.nlm.nih.gov/pubmed/9137882
Arvio et al. 2001 https://www.ncbi.nlm.nih.gov/pubmed/11174635
Malm et al. 2004 https://www.ncbi.nlm.nih.gov/pubmed/15316370
Autti et al. 1999 https://www.ncbi.nlm.nih.gov/pubmed/10706021

3