Structure of human lysosomal acid α-glucosidase-a guide for the treatment of Pompe disease

Abstract

Pompe disease, a rare lysosomal storage disease caused by deficiency of the lysosomal acid α-glucosidase (GAA), is characterized by glycogen accumulation, triggering severe secondary cellular damage and resulting in progressive motor handicap and premature death. Numerous disease-causing mutations in the gaa gene have been reported, but the structural effects of the pathological variants were unknown. Here we present the high-resolution crystal structures of recombinant human GAA (rhGAA), the standard care of Pompe disease. These structures portray the unbound form of rhGAA and complexes thereof with active site-directed inhibitors, providing insight into substrate recognition and the molecular framework for the rationalization of the deleterious effects of disease-causing mutations. Furthermore, we report the structure of rhGAA in complex with the allosteric pharmacological chaperone N-acetylcysteine, which reveals the stabilizing function of this chaperone at the structural level.

Fig. 1

The effect of pharmacological chaperones on misfolded lysosomal enzymes and on recombinant enzymes used in ERT. Lysosomal enzymes are assisted by molecular chaperones during synthesis. Mutated enzymes fail to fold correctly and are intercepted by the quality control (QC) system of the endoplasmic reticulum (ER). a Pharmacological chaperones favor proper folding of mutated enzymes, prevent their recognition by the quality control system and stabilize the enzyme during transport to their destination. b Pharmacological chaperones can enhance the effect of recombinant enzymes administered in ERT by favoring trafficking to lysosomes and increasing enzyme stability

Fig. 2

Structure of mature rhGAA. a Proteolytic treatment of rhGAA. b Schematic representation of the sequence of GAA. Myozyme^® rhGAA used in ERT starts at residue Q57. Domains corresponding to the rhGAA structure are colored as in c, with the regions removed by treatment with α-chymotrypsin colored in white. c Cartoon representation of the structure of rhGAA consisting of the trefoil type-P domain (salmon), the N-terminal β-sheet domain (slate), the catalytic GH31 (β/α)₈ barrel domain (green) with insert I (gold) and insert II (pink), and the proximal (orange) and distal (teal) β-sheet domains. Catalytic residues (magenta) and glycan chains (grey) are depicted as sticks. Dashed lined circles highlight peptide portions removed by proteolysis with α-chymotrypsin before crystallization. (SP, signal peptide; PP, propeptide)

Fig. 3

Ligand binding to rhGAA. DNJ (a), NHE-DNJ (c) and acarbose (e) colored in orange bound to rhGAA, color-coded as in Fig. 2c, overlapped onto unbound rhGAA (grey) in (a) and (c). Hydrogen bonding interactions are represented as dashed lines. In e, substrate-binding subsites are numbered and residues of a symmetry-related molecule are depicted in white sticks. Unbiased F _o –F _c difference electron density maps, calculated before incorporation of the ligands into the models and contored at 3.0 σ, are shown in b, d and f

Fig. 4

rhGAA substrate recognition and specificity and allosteric chaperone binding sites. a Model of isomaltose (steelblue), derived from an overlap with B. obeum α-glucosidase in complex with isomaltose (PDB ID 3MKK), superposed onto the rhGAA-acarbose complex (rmsd of 1.50 Å for 318 aligned Cα positions). b Model of isomaltose bound to rhGAA in surface representation. c The secondary substrate-binding site of rhGAA with unbiased F _o –F _c difference electron density map calculated before incorporation of acarvosine (orange) into the model and contoured at 2.0 (lightblue) and 3.0 (blue) σ. The surface loop removed by proteolytic treatment is represented by dashed lines. d NAC1 (orange) in the fully occupied binding site. e NAC2 (orange) in the partially occupied binding site. In d and e, unbiased F _o –F _c difference electron density maps, calculated before incorporation of NAC into the model, are shown in lightblue (2.0 σ) and blue (3.0 σ)