Characterization of gene-activated human acid-beta-glucosidase: crystal structure, glycan composition, and internalization into macrophages

Abstract

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-beta-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-Activated human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid-base hydrolysis. Kinetic parameters (K(m) and V(max)) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

Fig. 1

Comparison of the crystal structures of velaglucerase alfa and imiglucerase. The three domains of the enzymes are colored pink (domain I, residues 1–29 and 383–414), blue (domain II, residues 30–75 and 431–497), and gray (domain III, residues 76–382 and 415–430).

Fig. 2

Active site of velaglucerase alfa. Stereo representation of an overlay of the active sites of imiglucerase (blue and magenta) and velaglucerase alfa (yellow and green). Catalytic residues are shown as red sticks. Loops near the entrance to the active site are indicated (L1, loop 1; L2, loop 2; L3, loop 3).

Fig. 3

Electron density around the catalytic center. Catalytic residues E235 and E340 are shown as red balls and sticks and surrounding residues are in dark gray. Contours of the 2F_o–F_c map are shown as a blue mesh (at 1.2σ); contours of the F_o–F_c map are shown in green mesh (at 3σ) and in magenta (at −3σ). Several F_o–F_c peaks are visible in the active site, but they did not overlap with the 2F_o–F_c map, nor are they continuous; hence, at this resolution they appear to be noise. A and B show the catalytic centers of molecules A and B, respectively, in the asymmetric unit.

Fig. 4

Mutations at the C-terminus of GlcCerase. Imiglucerase and pr-GlcCerase contain a His at residue 495 (yellow), whereas velaglucerase alfa contains Arg (green). Mutations R496 and D474, which cause Gaucher disease, are shown in magenta. Residues within 4 Å distance of R495 and R496 are shown in cyan.

Fig. 5

Kinetic analysis of velaglucerase alfa and imiglucerase. V_max and K_m values were determined using a natural GlcCer substrate (n = 2).

Fig. 6

Glycosylation sites seen in the crystal structure of velaglucerase alfa. 2F_o–F_c electron density maps are shown, which are contoured at 1.2σ in the vicinity of two of the putative glycosylation sites, N19 and N146 for molecule A, and N19 for molecule B. (A) Glycosylation sites detected in molecule A are shown in green. (B) Glycosylation site detected in molecule B is shown in yellow. (C) Superposition of the two individual molecules in the asymmetric unit reveals their similarity. In all three representations, catalytic residues E235 and E340 are shown as red sticks.

Fig. 7

Glycan structures of velaglucerase alfa and imiglucerase. Predominant N-linked carbohydrate structures on velaglucerase alfa (top) and imiglucerase (bottom) are shown graphically at their relative positions along the protein backbone.

Fig. 8

Glycan map analysis of velaglucerase alfa. Glycans released by N-glycosidase F were analyzed by anion-exchange chromatography with amperometric detection. The method resolves glycans based on negative charge where peak group 1 corresponds to high-mannose type neutral glycans that are resolved into multiple peaks according to the number of mannose units, peak group 2 corresponds to high-mannose type glycans with one M6P that retained its GlcNAc cap (one negative charge), and peak group 3 corresponds to high-mannose type glycans containing one fully processed M6P (two negative charges). In peak group 1, smaller peaks are resolved that correspond to positional isomers of the various oligomannose types observed.

Fig. 9

Velaglucerase alfa and imiglucerase internalization into differentiated macrophages. The ordinate of the graph represents the fluorescence data normalized for the cellular protein concentration and incubation time (RFU/μg/h). The GlcCerase dose is shown on the abscissa.