Biol. Chem., Vol. 389, pp. 1361–1369, November 2008 • Copyright by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.163 2008/171 Article in press - uncorrected proof Review Acid b-glucosidase: insights from structural analysis and relevance to Gaucher disease therapy Yaacov Kacher 1,a , Boris Brumshtein 2,3,a , Swetlana Boldin-Ada msky 1,b , Lilly Toker 2 , Alla Shainskaya 4 , Israel Silman 2 , Joel L. Sussman 3 and Anthony H. Futerman 1, * 1 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel 2 Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel 3 Department of Structural Biology, Weizmann Institute of Scien ce, Rehovot 76100, Israel 4 Biological Mass Spectrometry Unit, Biological Services, Weizmann Institute of Science, Rehovot 76100, Israel * Corresponding author e-mail: tony.fute [email protected]. il Abstract In mammalian cells, glucosylceramide (GlcCer), the sim- plest glycosphingolipid, is hydrolyzed by the lysosomal enzyme acid b-gluco sidase (GlcCerase). In the human metabolic disorder Gaucher disease, GlcCerase activity is significantl y decr eased owing to one of appro ximate ly 200 mutations in the GlcCerase gene. The most common therapy for Gaucher disease is enzyme r eplacement ther- apy (ERT), in which patients are given intravenous injec- tions of reco mbina nt human GlcCerase; the Genz yme product Cerezyme has been used clinicall y for more than 15 years and is administered to approximately 4000 patients worldwi de. Here we revi ew the crystal structur e of Cer ezy me and other recombinant for ms of Glc - Cerase, as well as of their complexes with covalent and non-covalent inhibitors. We also discuss the stability of Cerezyme , which can be altered by modification of its N-glycan chains with possible implications for improved ERT in Gaucher disease. Keywords: Gaucher disease; glucosylceramide; lysosome; X-ray structure. Introduction Gaucher disease, the most prevalent lysosomal storage disea se (Beutler and Grabo wski, 2001; Jmoudia k and Futerman, 2005; Futerman and Zimra n, 2006), occurs These authors contributed equally to this work. a Prese nt address: QBI Enter prises Ltd., Weizmann Scien ce b Park, P.O. Box 4071, Nes Ziona 70400, Israel with a frequency of 1 in 40 000–60 000 in the general population, and 1 in 500–1000 among Ashkenazi Jews (Charr ow et al., 2000; Beu tler and Gra bowski, 200 1). Gau che r dise ase is a genetic disorder of sphingolipid metabolism characterized by markedly decreased cata- lytic activity and/or stability of the enzyme glucocerebro- sidase (GlcCerase, acid b-gluco sidas e, EC 3.2.1.45), which results in intra cellular accumulation of gluco syl- ceramide (GlcCer). The most common and well-charac- teri zed treatment for Gaucher disease is enzyme replacement ther apy (ERT ), in which the defective GlcCerase is supplemented with active enzyme, given to patients by intravenous infusions usually every 2 weeks. ERT using the Genzyme product Cerezy me alleviates many disease symptoms and has proven to be safe and effective over a period of approximately 15 years. Cerezyme is a recombinant human Gl cCerase expr essed in Chinese hamster ovar y cell s. Af ter its expression and purification, Cere zyme is modified by treatment with three glycosidases, a-neuraminidase, b- galactosidase and b-N-acetylglucosaminidase (Friedman and Hayes, 1993), to expose mannose residues that can be recognized by macrophages, a procedure that dra- matically impr oves tar geting to and internalization by mac rop hages, the main cell type aff ect ed in Gau che r disease. Rece ntly , an alternative means of producing GlcCerase has been e stablished by Protalix Biotherapeu- tics, in which the recombinant enzyme is expressed in transgenic carrot cells (prGlcCerase, recombinant plant- derived GlcCerase) grown in suspension culture (Shaal- tiel et al., 2007). The enzyme produced by this method generates a pro tein with exposed ter minal mannose structures (Lerouge et al., 1998; Friedman et al., 1999; Gomord and Faye, 2004), alleviating the need for post- production enzymatic modification. Until recently, the thr ee-dimensional str ucture of GlcCerase was not known. This lack of structural data hampered attempts to establish its catalytic mechanism, to analyze the relationship between mutations, levels of residu al enz yme act ivi ty and dis ease sev eri ty , and to generate more active and/or stable forms for use in ERT. In 2003, the X-ray structure of Cerezyme was solved at a resolution of 2.0 A ˚ (Dvir et al., 2003). To obtain crystals with satisfactory diffracting power, Cerezyme was treat- ed wi th N-gly cos idase F pri or to cry stalliz ati on. Sub- se qu en t to t hi s, and to a ll e vi ate c on ce rn s th at N-glycosidase F-treatment might advers ely af fec t its structure, the structure of intact Cerezyme was solved without N-glyco sidase F-treatment (Brumshtei n et al., 2006). The str ucture of complexes of Glc Cer ase with cov ale nt (Pr emk umar et al., 200 5) and non-covalent (Brumshtein et al., 2007) inhibitors has also been solved,
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Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)
was performed on a Bruker ReflexIII mass spectrometer (Bruker, Bremen, Germany) equipped
with a delayed extraction ion source, a reflectron and a 337-nm nitrogen laser. Nano-LC-ESI-
MS/MS was carried out on a nano-liquid chromatography system incorporating an UltiMateCapillary/Nano LC system consisting of a Famos micro autosampler, a Switchos micro col-
umn switching module (LC Packings, Dionex, Amsterdam, The Netherlands) on line with an API
Q-STAR Pulsar i electrospray-quadrupole TOF tandem mass spectrometer containing a quadru-
pole collision cell (MDS-Sciex, ABI, Toronto, Canada) and equipped with a nanoelectrospray
source (MDS Proteomics, Odense, Denmark). Peptides encompassing N59 and N146 were rou-
tinely detected in the samples after N-glycosidase F treatment, and N270 was detected in two
experiments by MALDI-TOF MS. N19 was never detected after deglycosylation. *Indicates deam-
idation of asparagine (N) to aspartic acid (D) and glutamine (Q) to glutamic acid (E) as a result
of deglycosylation and tryptic digestion. Mox, oxidized methionine.aCalculated for average molecular mass.bDetected by MALDI-TOF MS only.
Figure 4 Overview of the GlcCerase structure and possible modes of binding to the lipid bilayer.
(A) 3D structure of GlcCerase. The ( b / a )8 (TIM) barrel (domain III) is shown in green. Oligosaccharide chains are shown as sticks, and
sulfate ions as sticks and spheres. Domain I is shown in olive and domain II in blue. (B) Possible mode of binding of GlcCerase to
the membrane lipid bilayer. GlcCerase is oriented such that the entrance to the active site is adjacent to the membrane bilayer. Loop
1 is rich in aromatic residues and might therefore partially penetrate into the lipid layer. The orientation of the oligosaccharide chainsis such that they do not interfere with catalytic activity and/or with binding to the bilayer.
Catalytic residues
The retaining mechanism in glycosyl hydrolases involves
two catalytic residues, one functioning as the acid/base
catalyst and the other as the nucleophile. Site-directed
mutagenesis and homology modeling of GlcCerase
(Fabrega et al., 2000, 2002) had suggested that E235
was the acid/base catalyst, and tandem mass spectrom-
etry had identified E340 as the nucleophile (Miao et al.,
1994). These two residues are located near the C-termini
of strands 4 and 7 in domain III, with a distance betweentheir carboxyl oxygen atoms of approximately 5 A ˚ , con-
sistent with retention of the anomeric carbon configura-
tion upon cleavage rather than with an inversion
mechanism (Davies and Henrissat, 1995). Moreover, in
crystals of Cerezyme soaked with an irreversible inhib-
itor, conduritol B epoxide (1,2-anhydro- myo-inositol;
CBE), E340 was confirmed as the catalytic nucleophile
(Premkumar et al., 2005). The epoxide oxygen of CBE,
oriented similarly to the cyclohexitol ring, is within hydro-
gen-bonding distance of E235O´, consistent with the
role of E235 as the acid/base catalyst (Henrissat et al.,
1995).
Catalytic mechanism
Although there is significant evidence to support a
nucleophilic role for Glu340 in the GlCerase catalytic
cycle, several studies (e.g., Davies and Henrissat, 1995)
have implied direct attack of the carboxylate oxygen of
Glu340 on the anomeric carbon of GlcCer. Upon reso-
lution of the structure of prGlcCerase complexed with
either of two non-covalent inhibitors, N-butyl-deoxynoji-
rimycin (NB-DNJ) or N-nonyl-deoxynojirimycin (NN-DNJ)
(Brumshtein et al., 2007), we obtained evidence that
does not support such a straightforward mode of attack.
Modeling of the binding of the natural substrate on thebasis of the structures of the complexes with DNJ-based
inhibitors demonstrates that the apical hydrogen on the
anomeric carbon of the glucose moiety is positioned
between the carbon atom and the attacking oxygen atom
of Glu340 in such a way that it would block direct nucleo-
philic attack by steric hindrance. If, however, there is an
intermediate involving a planar anomeric carbon, which
would result from distortion of the sugar moiety, then
nucleophilic attack by Glu340 should be possible (Legler,
1990; Sinnott, 1990). In the case of CBE, direct nucleo-
philic attack on the epoxide carbon by Glu340 is possi-
ble, since the hydrogen atom is not apical, rendering the
carbon susceptible to such attack.
Resolution of the structures of the complexes of pr-
GlcCerase with NB-DNJ or NN-DNJ raised another issue,
namely the protonation state of Glu235, the acid/base
catalyst. This residue corresponds to Glu35 in lysozyme,
which supplies a proton to the leaving group in the initial
stage of the reaction, and thus must be protonated in the
resting state of the enzyme (Legler, 1990; Sinnott, 1990).
The basic limb of the pH/activity profile of lysozyme, with
a pK a of approximately 6.5, is attributed to deprotonation
of Glu35. The high pK a of this residue in lysozyme has
been explained by it being partially buried. GlcCerase
has a similar pH/activity profile, with pK a values of 4.5
and 6.5 (Erickson and Radin, 1973; Osiecki-Newman etal., 1988). However, in GlcCerase (Brumshtein et al.,
2006), Glu235 is near His311 Nd1 (3.2 A ˚ ), Asn234 (3.3 A ˚ )
and Gln284 (3.6 A ˚ ). His311 is part of a hydrogen bond
network involving Asp282, Arg120, and the catalytic res-
idue Glu340. Given its proximity to Asp282, it is presum-
ably protonated, despite being buried in the active site.
The close proximity of polar residues, particularly if
His311 is charged, should only serve to lower the pK a of
Glu235 rather than increasing it to pH 6.5. One possible
explanation is pK a cycling (McIntosh et al., 1996), where-
by a charge on the side chain of one of the glutamic
acids in the active site would effect the pK a of the other
proximal glutamic acid through electrostatic forces. Thus,a negative charge on the nucleophile would make it ener-
getically unfavorable for the proximal glutamic acid to
carry a negative charge. Upon binding of the covalent
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