A stable GH31 α-glucosidase as a model system for the study of mutations leading to human glycogen storage disease type II

Abstract

GH31 glycosidases are widespread across organisms, but remarkably, less than 1% of them have been biochemically characterised to date. Among them, human lysosomal acid α-glucosidase (GAA) stands out due to its link to Pompe disease, a rare lysosomal storage disorder caused by its deficiency. This disease results in glycogen accumulation, severe cellular damage, motor impairment, and premature death. Structural and functional studies of GAA mutants are challenging due to their instability and lack of activity, hindering their expression and purification. The GH31 enzyme MalA from a hyperthermophilic archaeon is explored here as a stable homolog of GAA. MalA is highly expressible, easy to purify, and structurally characterised. The R400H mutant in MalA, corresponding to the pathogenic GAA R600H mutation, revealed here a 1200-fold drop in specificity constant and >8 °C reduction in thermal stability. We propose MalA's as a robust model for studying GAA mutations and developing therapeutic chaperones.

Keywords: Pompe disease; enzymatic model; extremozymes; pharmacological chaperon therapy.

Figure 1.

Structural comparison between the human acidic α-glucosidase (GAA) and the α-glucosidase from Saccharolobus solfataricus (MalA). (A) Pathogenic and likely pathogenic residues mapped on the MalA monomer. The figure shows 39 residues linked to Pompe disease mapped on the MalA monomer. Colours indicate their classification: pathogenic (red), pathogenic/likely pathogenic (orange), and likely pathogenic (yellow). (B) Close-up view (8 Å) of the active sites of MalA (PDB ID 2G3M, blue backbone, left panel) and GAA (PDB ID 5KZW grey backbone, right panel). Both the catalytic nucleophile and acid/base residues are highlighted in blue (D320/D518 as the nucleophile and in red D416/D616 as the acid/base in MalA and GAA, respectively). Conserved residues between MalA and GAA’s active sites, associated with Pompe disease mutations, are highlighted in magenta; (C) structural prediction of the MalA active site mutations.

Figure 2.

Analysis of the stability of wild-type and mutants by DSF. (A) Thermal scan profile and (B) melting temperature of MalA wild-type and their mutants.

Figure 3.

Comparison of the N-acetyl cysteine binding sites of human GAA (light blue) and MalA WT (grey). The functional and surface representation is shown in (A) and (B), respectively. Detail of the full affinity site (NAC1) and low-affinity site (NAC2) for NAC.