Treatment of Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encoding the lysosomal enzyme, acid-β-glucosidase (acid-beta-glucosidase, glucocerebrosidase, GlcCerase, E.C. 3.2.1.45). The most common treatment for Gaucher disease is enzyme replacement therapy (ERT), in which defective GlcCerase is supplemented with an active enzyme.

The correlation between the ~ 200 mutations in GlcCerase and disease severity is not completely understood, although homozygosity for the common is associated with non-neuronopathic and neuronopathic disease, respectively.

The X-ray structure of GlcCerase (Cerezyme®) at was resolved at 2.0 A resolution (1ogs). The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A family. The distance between the is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the and a separate on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase ^[1].

The crystal structure of the human (colored yellow) with covalently bound irreversible inhibitor (conduritol-B-epoxide; CBE; shown in cyan with its hydroxyl groups are in red) was solved (1y7v). This structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the active-site catalytic nucleophile, because the between the cyclohexitol C1 atom and Glu340 Oε2 is 1.43 Å. The comparison between the active sites of and another representative of the glycohydrolase family - plant (1iev), reveals that CBE bound with this plant enzyme adopted the "chair" conformation, while with human , it is observed in a "boat" conformation, with hydrogen bonds to Asn234 Oδ1 and Nδ2, Glu340 Oε1, Trp179 Nε1, and Asp127 Oδ1 and Oδ2 ^[2]. Only one of two of a pair of flexible loops (L1: Ser345–Glu349, and L2: Val394–Asp399) located at the entrance to the active site in native GlcCerase (1ogs) is observed in the GlcCerase-CBE structure (1y7v), a conformation in which the active site is accessible to CBE (colored blue), while these loops in the second (closed) conformation are colored magenta. In , a major structural change is observed in the positions of , and in a more limited difference is observed in the conformations of . Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. The movies 1 and 2 illustrate the dynamics of the movement of these two loops ^[2][3].

Three-dimensional structure of recombinant plant-derived glucocerebrosidase (prGCD, 2v3f) consists of . Domain I (residues 1–27 and 384–414, colored pink) comprises a 3-stranded anti-parallel β-sheet flanked by a perpendicular amino-terminal strand. Domain II (residues 30–75 and 431–497, colored lime) consists of two β-sheets. Domain III (residues 76–381 and 416–430, colored red) is a (β/α) 8 TIM barrel. with molecule BTB is shown ^[4].

of prGCD (2v3f) with both Cerezyme® (1ogs) and Cerezyme® covalently modified by an irreversible inhibitor, conduritol-B-epoxide (1y7v, colored yellow), revealed highly significant structural identity. The RMSD values for Cα atoms of these structures were of 0.64 and 0.60 Å, respectively. Moreover, there was strict conservation of the ^[4].

of the structure of native human acid β-glucosidase, expressed in cultured plant cells (pGlcCerase, 2v3f) on those of N-butyl-deoxynojirimycin/pGlcCerase (2v3d), N-nonyl-deoxynojirimycin/pGlcCerase (2v3e), and isofagomine/deglycosylated Cerezyme (IFG/DG-Cerezyme, 2nsx) reveals significant structural identity, neither of these ligands causes structural changes upon binding to the enzyme. The imino sugar of N-butyl-deoxynojirimycin forms 7 hydrogen bonds and also makes several hydrophobic interactions with side chains of active site residues (2v3d). The crystal structure of pGlcCerase in complex with N-nonyl-deoxynojirimycin (2v3e) is very similar to that of NB-DNJ/pGlcCerase. The exception is that longer chain of NN-DNJ interacts with 2 additional residues Leu241 (labeled lime) and Leu314 of symmetrically related monomer (not shown). Comparison of the structures of NB-DNJ/pGlcCerase (2v3d) and NN-DNJ/pGlcCerase (2v3e) with that of (2nsx) shows that the pyranose-like ring forms a same number of hydrogen bonds with the enzyme in all three cases (2v3d, 2v3e, and 2nsx) ^[4][5][6].

The of the crystal structure of velaglucerase alfa (colored red) (2wkl) reveals that it is very similar to those of the recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®, colored blueviolet, 2j25) and in transgenic carrot cells (prGCD, 2v3f). of the two individual molecules in the asymmetric unit of velaglucerase alfa and imiglucerase demonstrates striking similarity between positions of catalytic residues E235 and E340 (colored orange) in all 4 molecules. The position of H311 is also very similar in all 4 molecules, whereas the conformations of 3 other active site residues W312, Y313, and, especially N396 are somewhat different. The active site residues (except E235 and E340) of the two individual molecules in the asymmetric unit of velaglucerase alfa are colored: subunit A (red), subunit B (lime) and of imiglucerase: subunit A (blueviolet), subunit B (magenta). Imiglucerase and pr-GlcCerase contain a at residue 495 (blueviolet), whereas velaglucerase alfa contains (red). Mutations which cause Gaucher disease, are close to R495 near the N-terminus of GlcCerase. The (its glycans are colored blue) and (its glycans are colored magenta) have different carbohydrate composition. This difference in glycosylation causes the increased cellular uptake of velaglucerase alfa over imiglucerase and could lead to improvement of treatment of Gaucher disease ^[4][7][8].