Glycobiology vol. 20 no. 1 pp. 24–32, 2010 doi:10.1093/glycob/cwp138 Advance Access publication on September 9, 2009 Characterization of gene-activated human acid-β-glucosidase: Crystal structure, glycan composition, and internalization into macrophages Boris Brumshtein2,3, Paul Salinas4, Brian Peterson4,Victor Futerman and Zimran 2006). The most common treatment for Chan4, Israel Silman2, Joel L Sussman3, Philip J Savickas4, Gaucher disease is enzyme replacement therapy (ERT), in which Gregory S Robinson4, and Anthony H Futerman1,5 defective GlcCerase is supplemented with an active enzyme. ERT using imiglucerase, a recombinant analog of human Glc- 2Department of Neurobiology; 3Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel; 4Shire Human Genetic Therapies, Cerase expressed in Chinese hamster ovary (CHO) cells has Inc., Cambridge, MA, USA; and 5Department of Biological Chemistry, been available for ∼15 years. After expression and purifica- Weizmann Institute of Science, Rehovot 76100, Israel tion, imiglucerase is modified by exo-glycosidase treatment Received on June 1, 2009; revised on August 30, 2009; accepted on September (Friedman and Hayes 1996) to expose the core mannose 1, 2009 residues that can be recognized by macrophages. Glycan re- modeling greatly improves targeting to and internalization by Gaucher disease, the most common lysosomal storage dis- macrophages, the main cell type affected in Gaucher disease ease, can be treated with enzyme replacement therapy (Futerman and Zimran 2006). An alternative means of pro- (ERT), in which defective acid-β-glucosidase (GlcCerase) ducing GlcCerase (prGCD) in transgenic carrot root cells has is supplemented by a recombinant, active enzyme. The been developed (Aviezer et al. 2009). The X-ray structures of X-ray structures of recombinant GlcCerase produced in imiglucerase and prGCD have been previously reported (Dvir Chinese hamster ovary cells (imiglucerase, CerezymeR ) and et al. 2003; Shaaltiel et al. 2007). in transgenic carrot cells (prGCD) have been previously In the current study, we have used gene activation in solved. We now describe the structure and characteristics a well-characterized, continuous human cell line to pro- of a novel form of GlcCerase under investigation for the duce gene-activated human acid-β-glucocerebrosidase (ve- treatment of Gaucher disease, Gene-ActivatedTM human laglucerase alfa). Gene activation refers to targeted recombi- GlcCerase (velaglucerase alfa). In contrast to imiglucerase nation with a promoter that activates the endogenous GlcCerase and prGCD, velaglucerase alfa contains the native human gene in the selected human cell line. Velaglucerase alfa is se- enzyme sequence. All three GlcCerases consist of three creted as a monomeric glycoprotein of approximately 63 kDa domains, with the active site located in domain III. The and is composed of 497 amino acids with a sequence identical distances between the carboxylic oxygens of the catalytic to that of the natural human protein (Zimran et al. 2007). Glyco- residues, E340 and E235, are consistent with distances pro- sylation of velaglucerase alfa is altered by using kifunensine, a posed for acid–base hydrolysis. Kinetic parameters (Km and mannosidase I inhibitor, during cell culture, which results in the Vmax) of velaglucerase alfa and imiglucerase, as well as their secretion of a protein containing predominantly high-mannose specific activities, are similar. However, analysis of glycosy- type glycans (Elbein et al. 1990). lation patterns shows that velaglucerase alfa displays dis- Herein we describe the crystal structure of velaglucerase alfa, tinctly different structures from imiglucerase and prGCD. using a preparation that had been partially deglycosylated, and The predominant glycan on velaglucerase alfa is a high- show that it is similar to that of imiglucerase (Dvir et al. 2003) mannose type, with nine mannose units, while imiglucerase and prGCD (Shaaltiel et al. 2007). Velaglucerase alfa differs contains a chitobiose tri-mannosyl core glycan with fuco- from imiglucerase and prGCD as the latter two enzymes contain sylation. These differences in glycosylation affect cellular a mutation at residue 495 (an Arg to His substitution: R495H), internalization; the rate of velaglucerase alfa internaliza- and prGCD contains seven additional residues at the C terminus tion into human macrophages is at least 2-fold greater than (DLLVDTM) and two additional residues at the N terminus that of imiglucerase. (EF). Moreover, the kinetic parameters and specific activity of velaglucerase alfa are very similar to those of imiglucerase. We Keywords: Gaucher disease/gene activation/ also compare the glycosylation patterns of velaglucerase alfa glucocerebrosidase/glycans/mannose-6-phosphate receptor/ and imiglucerase by use of LC-MS and assess the impact of the site-specific glycosylation/X-ray structure different glycosylation patterns by analyzing internalization in human macrophages. Introduction Results and discussion Gaucher disease is caused by mutations in the gene encod- ing the lysosomal enzyme, acid-β-glucosidase (glucocerebrosi- X-ray structure dase, GlcCerase, E.C. 3.2.1.45) (Beutler and Grabowski 2001; Diffraction-quality crystals of velaglucerase alfa were obtained after partial deglycosylation using N-glycosidase F, by a proce- 1To whom correspondence should be addressed: Tel: +972-8-9342704; dure similar to that previously described for imiglucerase (Dvir Fax: +972-8-9344112; e-mail: [email protected] et al. 2003). Velaglucerase alfa crystallized in the same space c 2009 The Author(s). Published by Oxford University Press. All rights reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5),which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 24 Characterization of gene-activated human acid-β-glucosidase Table I. Data collection and refinement statistics B(<0.3 A)˚ shows that they are virtually identical. A compari- Velaglucerase alfa son of the structures of imiglucerase, prGCD, and velaglucerase alfa demonstrates that these structures are very similar, with an Data collection RMSD of 0.35–0.46 A˚ (Table II). Space group C2221 Velaglucerase alfa thus consists of three noncontiguous do- Cell dimensions mains, with the catalytic site located in domain III (residues 76– a, b, c (A)˚ 109.37, 285.55, 91.69 ◦ β α abg ( ) 90.00, 90.00, 90.00 381 and 416–430), which is a ( / )8 (TIM) barrel (Figure 1). Resolution (A)˚ 19.9–2.7 (2.75–2.70)a A more detailed analysis of the active site reveals that it is Rsym(%) 15.7 (51.0) virtually identical to that of imiglucerase (Figure 2), with the dis- I/s<I> 14.4 (4.6) tances between the carboxylic oxygens of the catalytic residues, Completeness (%) 100 (100) ˚ ˚ Redundancy 7.5 (7.6) E340 and E235 (5.2 A in molecule A and 5.1 A in molecule B), Refinement similar to those obtained previously (Brumshtein et al. 2006) Resolution (A)˚ 19.9–2.7 and in agreement with the distances proposed for acid–base hy- Number of reflections 39,776 drolysis (Davies and Henrissat 1995). Moreover, the three loops Rwork/Rfree 17.3/23.4 (loop 1, residues 345–350; loop 2, residues 393–399; and loop Rms deviations 3, residues 312–319) observed in previous structures (reviewed Bond lengths (A)˚ 0.012 Bond angles (◦) 1.486 in Kacher et al. (2008)) are also seen in velaglucerase alfa. Number of refined atoms Similarly to prGCD (Shaaltiel et al. 2007), loops 2 and 3 show Protein 7871 differences in their backbone angles and side chain orientations Carbohydrates 70 in the two molecules of the asymmetric unit, whereas loop 1, Ions 90 Solvent 326 since it makes crystal contacts, exhibits less pronounced con- Ramachandran outliers (%) 0.4 formational changes (Figure 2). In the case of loop 3, a helical conformation is seen in molecule B, whereas a coiled confor- aThe highest resolution shell is shown in parentheses. mation is seen in molecule A (Figure 3), as previously reported for imiglucerase (Brumshtein et al. 2006). Although the crystal was cryo-protected with 25% ethylene glycol, we did not detect group, C2221, as imiglucerase (Table I), and unit cell parameters were similar to the previously published GlcCerase structures any ethylene glycol molecules in the electron density map. (Dvir et al. 2003; Premkumar et al. 2005; Brumshtein et Imiglucerase and prGCD both contain an Arg to His muta- al. 2006). The asymmetric unit contained two copies of ve- tion at residue 495, with H495 making an H-bond (2.6 A)˚ with laglucerase alfa, designated as molecules A and B. The root the peptide carbonyl of F31. In contrast, velaglucerase alfa con- mean square deviation (RMSD) value between molecules A and tains a sequence identical to that of the natural human enzyme, Table II. RMS deviations of velaglucerase alfa compared to imiglucerase and prGCD. RMS deviations (A)˚ are shown for each of the two copies of the molecules in the asymmetric unit and were calculated using PyMol (www.pymol.org). The PDB codes for imiglucerase and pr-GlcCerase are 2J25 and 2V3F, respectively Imiglucerase-A Imiglucerase–B prGCD-A prGCD-B Velaglucerase alfa-A 0.39 0.35 0.36 0.40 Velaglucerase alfa-B 0.38 0.43 0.46 0.46 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). 25 B Brumshtein et al. 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). with an Arg at residue 495, which does not make a similar Kinetic analysis H-bond.