Aspartoacylase (EC 3.5.1.15; UniProt ID: P45381) (ASPA) – also known as aminoacylase II (ACY2) [1] or N-acetyl-L-aspartate amidohydrolase [2] in older literature, is a 35.7 kDa, 313 residue enzyme that catalyzes the hydrolysis of N-acetyl-L-aspartic acid (NAA) into acetate and aspartate [3,4,5]. In humans, the ASPA gene is located on the short arm of chromosome 17, spans 29 kb and contains 6 exons [3, 6]. The ASPA mRNA is widely distributed, but particular expressed in kidney and in oligodendrocytes of the brain [7,8,9]. The encoded enzyme is a single domain homo-dimeric protein complex, with a highly specific active site buried within a channel in the native protein [10]. Insufficient ASPA activity caused by germline ASPA variants is linked to Canavan disease (CD) (OMIM: 271900), a recessive, neurodegenerative leukodystrophy, where oligodendrocytes fail to correctly myelinate neuroaxons [11]. While the precise pathogenic mechanism remains elusive, several non-mutually exclusive hypotheses have been proposed [12,13,14,15].

Clinically, CD patients display poor muscle control, decreased cognitive capabilities and other severe conditions. Typically, these symptoms appear early, already within the first 3–6 months of life, and gradually progress over time eventually leading to an early death [16]. Various attempts at ameliorating the symptoms [17,18,19] and curing the disease [20,21,22] have been reported, including more recently promising attempts and ongoing trials with gene therapy [21, 23, 24]. Accordingly, clinical classification and a detailed understanding of how pathogenic ASPA gene variants operate are highly warranted. Using deep mutational scanning technologies, it was recently shown that most loss-of-function missense variants cause a structural destabilization of the ASPA protein structure, leading to the formation of non-native ASPA proteins that negatively affect cell fitness and are subject to rapid degradation by the cellular protein quality control (PQC) system [25]. Accordingly, studies on ASPA, therefore also provide a model system for understanding intracellular protein folding, misfolding and the PQC system, which ultimately may further our understanding of CD and other protein misfolding diseases.

Here we comprehensively summarize the physiological, cellular, and molecular details of the ASPA enzyme, its substrate NAA and Canavan disease. We focus particularly on the pathogenic ASPA gene variants, the importance of variant classification and gaining a mechanistic understanding of how loss of function gene variants operate for future implementation of gene therapy or other forms of precision medicine.

While ASPA gene expression is elevated in the brain and even higher in the kidneys [1, 2, 7, 26,27,28,29], the protein also seems to be present to a lesser extent in several other tissues, including liver, intestine and lung [1, 7, 30,31,32]. Indeed, skin fibroblasts have also been used to obtain ASPA for activity assays [32,33,34].

However, the most important site of ASPA expression is the brain white matter (WM), where the enzyme plays an essential role in NAA catabolism, as evident by the following investigations with rats. Some studies, using antibodies, detected ASPA protein clearly in oligodendrocytes, and faintly in neurons and microglial-like cells, but not in astrocytes [35, 36]. These observations were corroborated by two other studies of both ASPA mRNA [8] and protein [37], which found it to be restricted [37] or primarily restricted [8] to oligodendrocytes. Similarly, one study detected ASPA enzymatic activity in oligodendrocytes, but not astrocytes [38]. However, yet another study found ASPA activity in O2A progenitor cells and both their differentiated cell types (astrocytes and oligodendrocytes), but not in neurons [39]. It has been reported that rat Schwann cells (the peripheral nervous system equivalent of oligodendrocytes) do not express ASPA mRNA [8], while a later study in mice found Schwann cells to express ASPA. However, the authors noted that the peripheral nerves looked grossly normal in CD mice, thus emphasizing the role of ASPA primarily within the central nervous system (CNS) [30]. This was corroborated by a study of the auditory processing in the same mouse strain, showing functional and morphological deterioration was limited to the CNS, with the cochlear nerve fibers being unaffected [40].

Regardless of these discrepancies, there seems to be consensus that ASPA is mainly associated with oligodendrocytes and their myelin sheaths. A notion supported by the fact that oligodendrocyte-specific ASPA knock-out in mice causes the same – albeit milder – phenotype in the CNS as whole body ASPA knock-out [41]. Further supporting this idea, is the low ASPA activity observed in grey matter (GM) [39], and the fact that the brain periphery remains unaffected in CD patients [29]. Even within the brain WM, ASPA expression exhibits spatiotemporal regulation [35, 39]. Temporally, little to no ASPA seems to be present in neonatal rats, with levels starting to rise postnatally, coinciding with the myelination of the brain for it to then decrease somewhat while maintaining a detectable level in adult rats [8, 37, 39, 42].

ASPA consists of an N-terminal (residues 1–212) and a C-terminal domain (resides 213–313) (Fig. 1). The N-terminal region consists of a central six-stranded mixed β-sheet surrounded by eight helices of variable size and multiple connecting loops. The C-terminal domain consists of two antiparallel β-sheets that wrap around the substrate-binding side of the N-terminal domain with a globular portion between the two β-sheets [10]. The two domains connect though various interactions, including a β-sheet anchor formed between β1 and β13 at the very N- and C-terminal ends of the domain (based on the solved structure of rat ASPA and the AlphaFold predictions for human ASPA). Additionally, the C-terminal region of ASPA wraps around the N-terminal region via the antiparallel β-strands β8 and β12 [10]. Consequently, the N-terminal domain is not stable when expressed on its own [25, 43], and the two domains should thus be considered as one joint unit. Together, the N- and C-terminal regions form a channel leading to the active site.

The ASPA protein structure. The structure of the human ASPA homodimer (PDB: 2O53) [44] is shown with the two subunits in blue and yellow. The N-terminal region covering residues 1–212 (upper panel) and C-terminal region spanning residues 212–313 (lower panel) are highlighted

Sequence alignments [45] and structural analyses [10, 46] have demonstrated similarities between the N-terminal part of ASPA and a range of Zn2+-dependent carboxypeptidase A-related hydrolases [10, 45]. However, carboxypeptidase A has a ~ 60 residue N-terminal extension of the central β-sheet by two strands and carboxypeptidases completely lack the C-terminal extension [10]. More specifically, ASPA belongs to the succinyl glutamate desuccinylase/aspartoacylase family (AstE/AspA, PFAM04952) [10]. The C-terminal region in ASPA likely reflects a requirement for high substrate specificity for ASPA, which is localized in the cytosol, compared to carboxypeptidase A, which cleaves a range of peptides in the small intestine.

Unlike aminoacylase I, which hydrolyses N-acetyl groups from all amino acids, ASPA (also known as aminoacylase II) exhibits high substrate specificity towards N-acetyl-L-aspartic acid [16, 47].

The C-terminal extension of ASPA shields the active site, restricting access to it. In the entry channel R71, K228, K291, K292 and E293 provide a positive electrostatic potential, which may guide NAA to the active site while repelling positively charged metabolites. Although some peptides may enter the channel, the C-terminal region would orientate them in a position that does not enable hydrolysis to occur [10, 48]. A tight pocket consisting of residues T118, Q184, F282, E285, A287, and Y288 accommodates the acetyl group of NAA, while restricting compounds with acyl groups longer than acetate. ASPA also shows high selectivity towards the aspartate-side of NAA [49], possible due to a hydrogen bond between R168 and the β-carbonyl group of NAA [10]. The key catalytic core residues include: R63, N70, R71, R168, E178 and Y288 as well as H21, E24 and H116, which coordinate the catalytic Zn2+ ion (Fig. 2).

The active site. A zoom in on the active site within one subunit of the human ASPA structure (PDB: 2O53) [44] and residues (H21, E24, H116) coordinating the Zn2+ ion (red). Residues Arg63, Asn70, Arg71, Arg168, and Tyr288 interact with the substrate

A “promoted-water pathway” mechanism similar to that of carboxypeptidase A, has been proposed for the hydrolysis catalyzed by ASPA (Fig. 3). In this model, E178 deprotonates a water molecule, which is stabilized by Zn2+. The resulting hydroxide then attacks the β-carbonyl group of NAA, which is stabilized by R63 and possibly also the Zn2+ ion (Fig. 3A), to allow the formation of a tetrahedral intermediate (Fig. 3B). Lastly, the intermediate collapses with aspartate being eliminated as the leaving group [10] (Fig. 3C). The model is supported by computational analyses, which also indicated that substrate release, rather than bond cleavage, is the rate limiting step of the reaction [50].

Overview of the proposed catalytic mechanism of aspartoacylase. A First, water initiates a nucleophile attack on the NAA carbonyl group leading formation of B a tetrahedral intermediate and finally C the products. Green indicates residues coordinating Zn2+, purple indicates residues interacting with the substrate, and yellow indicates the catalytic active E178 residue

Solved crystal structures of human and rat ASPA shows that they form similar homodimers. In humans, the dimer interface covers ~ 1200 Å2 of surface accessible solvent area and involves 12 hydrogen bonds and two salt bridges [10, 44]. This structural evidence of dimerization has been supported by various biochemical assays performed on human and rat ASPA [10, 37, 46, 49]. However, some of these observations could be explained by aggregation [49] or antibody cross-reactivity [26,