X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease

Abstract

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-beta-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the approximately 200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 A resolution. The catalytic domain consists of a (beta/alpha)(8) TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest alpha-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain 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.

Figure 1

Reaction catalysed by acid-β-glucosidase. Acid-β-glucosidase (GlcCerase) hydrolyses the β-glucosyl linkage of glucosylceramide (GlcCer), to yield ceramide and glucose.

Figure 2

The refined X-ray structure of acid-β-glucosidase. (A) Domain I is shown in magenta and contains the two disulphide bridges, the sulphur atoms of which are shown as green balls. The glycosylation site at N19 is shown as a ball-and-stick model. Domain II, which is an immunoglobulin-like domain, is shown in green. The catalytic domain (domain III), which is a TIM barrel, is shown in blue, and the active-site residues E235 and E340 are shown as ball-and-stick models. The six most common acid-β-glucosidase (GlcCerase) mutations are shown as balls, with those that cause predisposition to severe (types 2 and 3) and mild (type 1) disease in red and yellow, respectively. (B) Two-dimensional topology of GlcCerase. The diagram is consistent with a three-dimensional view, looking down the opening of the active-site pocket, as in (A). All connecting loops in the diagram are of an arbitrary length. α-Helices and β-strands of domain III are numbered according to their position in the sequence. For clarity, sequence numbers for certain positions are shown in the connecting loops, and secondary structural elements that consist of four residues or less are not shown. NAG, N-acetylglucosamine.

Figure 3

Active site of acid-β-glucosidase. (A) The catalytic and glucone-binding site of acid-β-glucosidase (GlcCerase). The catalytic glutamates are shown as ball-and-stick models and amino-acid residues nearby are shown as sticks. Hydrogen bonds are shown as dashed lines for those residues close enough to contact the glutamates. These residues may be involved directly in catalysis or may modulate the protonation states of the carboxyl groups. The others residues are near the docked glucosyl moiety (see (B)), and these may thus stabilize its interaction with GlcCerase. (B) Three-dimensional surface diagram of GlcCerase (created using PyMOL (http://www.pymol.org)), with a model of the docked substrate (based on the coordinates of galactosylceramide (Nyholm et al., 1990) and modified for GlcCer). Hydrophobic residues (W, F, Y, L, I, V, M and C; Hopp & Woods, 1981) are shown in blue, and the activesite residues (E235 and E340) in yellow. GlcCer is shown in CPK format (carbon atoms in green, and oxygen atoms in red).

Figure 4

Mutations in acid-β-glucosidase. (A) The sequence of the 497 residues of acid-β-glucosidase (GlcCerase). Mutations reported to cause severe disease (http://www.tau.ac.il/~racheli/genedis/gaucher/gaucher.html) are shown in red, those that cause mild disease in yellow, and those for which clinical data documenting severity of the disease are lacking in blue. Only single amino-acid substitutions are included, with frameshifts and splices excluded as the enzyme is not expressed in most of these cases. Helices are indicated by cylinders, and β-strands are indicated by arrows of colours corresponding to those of the domains shown in Fig. 2. (B) Distribution in the three-dimensional structure of GlcCerase of single amino-acid substitutions that lead to Gaucher disease. Colour coding is the same as in (A). In some cases, assignment of phenotypes as mild (type 1) or severe (types 2 and 3) is based on a few individuals, and sometimes only on one. The phenotypes of several mutations are not known, as the mutations were detected in genomic DNA, and data about disease severity may not have been available. The activesite glutamate residues are shown as black sticks. Cerezyme® differs from GlcCerase by a single amino-acid substitution at residue 495 (His for Arg).

Figure 5

A cluster of mutations in α-helix 7 that cause Gaucher disease. Transparent ribbon diagram showing the three domains of acid-β-glucosidase as in Fig. 1A, but rotated ∼90° around the x axis to look down helix 7, which is shown in red. The amino acids on this helix that are mutated in Gaucher disease (R359, Y363, S366, T369 and N370) are shown as red balls and sticks. E235 and E340 (the activesite residues) are shown with carbon atoms as yellow balls and oxygen atoms as red balls.