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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 Kacher1,a, Boris Brumshtein2,3,a,
Swetlana Boldin-Adamsky1,b, Lilly Toker2,
Alla Shainskaya4, Israel Silman2, Joel L.
Sussman3 and Anthony H. Futerman1,*
1Department of Biological Chemistry, Weizmann
Institute of Science, Rehovot 76100, Israel2Department of Neurobiology, Weizmann Institute of
Science, Rehovot 76100, Israel3Department of Structural Biology, Weizmann Institute
of Science, Rehovot 76100, Israel4Biological Mass Spectrometry Unit, Biological
Services, Weizmann Institute of Science, Rehovot
76100, Israel
* Corresponding author
e-mail: [email protected]
Abstract
In mammalian cells, glucosylceramide (GlcCer), the sim-
plest glycosphingolipid, is hydrolyzed by the lysosomal
enzyme acid b-glucosidase (GlcCerase). In the humanmetabolic disorder Gaucher disease, GlcCerase activity
is significantly decreased owing to one of approximately
200 mutations in the GlcCerase gene. The most common
therapy for Gaucher disease is enzyme replacement ther-
apy (ERT), in which patients are given intravenous injec-
tions of recombinant human GlcCerase; the Genzyme
product Cerezyme has been used clinically for more
than 15 years and is administered to approximately 4000
patients worldwide. Here we review the crystal structure
of Cerezyme and other recombinant forms 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 itsN-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
disease (Beutler and Grabowski, 2001; Jmoudiak and
Futerman, 2005; Futerman and Zimran, 2006), occurs
These authors contributed equally to this work.a
Present address: QBI Enterprises Ltd., Weizmann Scienceb
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
(Charrow et al., 2000; Beutler and Grabowski, 2001).
Gaucher disease 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-glucosidase, EC 3.2.1.45),
which results in intracellular accumulation of glucosyl-
ceramide (GlcCer). The most common and well-charac-
terized treatment for Gaucher disease is enzyme
replacement therapy (ERT), in which the defectiveGlcCerase is supplemented with active enzyme, given to
patients by intravenous infusions usually every 2 weeks.
ERT using the Genzyme product Cerezyme alleviates
many disease symptoms and has proven to be safe and
effective over a period of approximately 15 years.
Cerezyme is a recombinant human GlcCerase
expressed in Chinese hamster ovary cells. After its
expression and purification, Cerezyme 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 improves targeting to and internalization by
macrophages, the main cell type affected in Gaucher
disease. Recently, an alternative means of producing
GlcCerase has been established 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 protein with exposed terminal 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 three-dimensional structure ofGlcCerase was not known. This lack of structural data
hampered attempts to establish its catalytic mechanism,
to analyze the relationship between mutations, levels of
residual enzyme activity and disease severity, 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 with N-glycosidase F prior to crystallization. Sub-
sequent to this, and to alleviate concerns that
N-glycosidase F-treatment might adversely affect its
structure, the structure of intact Cerezyme was solved
without N-glycosidase F-treatment (Brumshtein et al.,2006). The structure of complexes of GlcCerase with
covalent (Premkumar et al., 2005) and non-covalent
(Brumshtein et al., 2007) inhibitors has also been solved,
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Table 1 GlcCerase crystal structures solved to date.
Enzyme source Treatment prior to crystallization Bound molecule Reference
Cerezyme Partial deglycosylationa None Dvir et al., 2003
Cerezyme Partial deglycosylation CBE Premkumar et al., 2005
Cerezyme Partial deglycosylation None Liou et al., 2006
Cerezyme None None Brumshtein et al., 2006
Cerezyme Partial deglycosylation Isofagamine Lieberman et al., 2007Cerezyme Partial deglycosylation Glycerol Lieberman et al., 2007
Cerezyme Partial deglycosylation None Lieberman et al., 2007
prGlcCerase None None Shaaltiel et al., 2007
prGlcCerase None NB-DNJ and NN-DNJ Brumshtein et al., 2007
prGlcCerase None CBE This study
aUsing N-glycosidase F.
as well as that of prGlcCerase; studies from other labo-
ratories have reported structures of various forms of
Cerezyme (Liou et al., 2006; Lieberman et al., 2007)
(Table 1). We now review these structures and discuss
their implications for improved therapeutic options for
Gaucher disease.
Effect of N-glycosidase F-treatment onCerezyme structure and stability
As mentioned above, the initial structure of Cerezyme
(Dvir et al., 2003; Premkumar et al., 2005) was obtained
after treatment with N-glycosidase F, which removes car-
bohydrate chains from proteins and peptides by cleaving
the amide bonds between Asn residues and N-acetylglu-
cosamine (GlcNAc) (Han and Martinage, 1992), but does
not necessarily remove all carbohydrate chains from
native proteins. The structure revealed that GlcCerasecomprises three non-contiguous domains that could not
be predicted from the primary amino acid sequence, as
discussed in detail in the next section.
After publication of the structure, some concern was
raised that N-glycosidase F treatment might modify the
Cerezyme structure in such a way that it might not
reflect that of the native untreated Cerezyme. This con-
cern was based on earlier studies in which removal of
sugar residues from GlcCerase irreversibly affected its
catalytic activity and hence, presumably, its structure
(Berg-Fussman et al., 1993). GlcCerase has five potential
glycosylation sites, four of which are occupied. Site-
directed mutagenesis demonstrated that only N19 isrequired for catalytic activity, although the role of N19
glycosylation is somewhat complicated by the differing
activities observed depending on the mutation and sub-
strate used (Grace and Grabowski, 1990; Grace et al.,
1990; Berg-Fussman et al., 1993). Interestingly, glyco-
sylation at N19 was detected in the original crystal struc-
ture (Dvir et al., 2003). However, to alleviate the concern
that N-glycosidase F treatment might nevertheless affect
the Cerezyme structure by removing essential oligo-
saccharide chains, we performed mass spectrometry
analysis of Cerezyme prior and subsequent to N-gly-
cosidase F treatment. According to matrix-assisted laser
desorption time-of-flight mass spectrometry (MALDI-TOFMS), the molecular mass of Cerezyme decreased by an
average of 1347.9"367.7 Da after N-glycosidase F treat-
ment (Figure 1), implying loss of approximately 6–7 sugar
residues. No information is available concerning the pre-
cise number of sugar residues remaining on Cerezyme
after a-neuraminidase, b-galactosidase and b-N-acetyl-
glucosaminidase treatment. However, assuming a mini-
mum of 4–5 sugars per chain, this implied loss of no
more than 1–2 sugar chains after N-glycosidase F treat-ment. Moreover, there may be considerable heteroge-
neity in the oligosaccharide composition of Cerezyme,
since MALDI-TOF MS analyses revealed significant dif-
ferences in the molecular mass of Cerezyme from dif-
ferent batches, ranging from 60 106.4 Da to 62 109.6 Da
(ns12), with a mean of 60 888.9"677.6 (the molecular
mass of the Cerezyme polypeptide chain is 55 576 Da).
Subsequent analyses by nano-liquid chromatography
electrospray ionization tandem mass spectrometry
(nano-LC-ESI-MS/MS) (Table 2) revealed that N59 and
N146 were deglycosylated by N-glycosidase F treatment,
but that the essential glycosylation site, N19, was never
deglycosylated, confirming results obtained by X-raycrystallography (Dvir et al., 2003). In some cases, N270
was also deglycosylated, although this did not occur
consistently (Table 2). Thus, we conclude that treatment
with N-glycosidase F only partially deglycosylates Cere-
zyme, removing 2 and occasionally 3 sugar chains, but
leaving N19 glycosylated.
Partial deglycosylation of Cerezyme by N-glycosidase
F did not change Cerezyme activity, as determined by
analysis of K m and V max values using a synthetic fluores-
cent GlcCer analog (Figure 2). In contrast, when Cere-
zyme (passed through a Centricon YM-30 filter to
remove formulation additives) was incubated for the
same length of time and in the same incubation buffer(20 mM sodium phosphate, pH 7.4) used for partial degly-
cosylation, but without N-glycosidase F, a significant loss
of activity was obtained (Figure 2), implying that removal
of N59 and N146 might enhance Cerezyme stability, at
least in vitro in aqueous solution. Similarly, treatment of
Cerezyme with endoglycosidase F1 (Endo F1), which
cleaves between the two GlcNAc residues in the diace-
tylchitobiose core of the oligosaccharide to generate a
truncated glycan with one GlcNAc residue, did not result
in any loss of activity (Figure 3). In contrast, treatment
with endoglycosidase H (Endo H) resulted in rapid and
complete loss of activity. The latter result is surprising,
since both Endo F1 and Endo H cleave the oligosaccha-ride chain between the two GlcNAc residues, and have
nearly identical capacities to hydrolyze high-mannose oli-
gosaccharides (Trimble and Tarentino, 1991). The most
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Acid b-glucosidase activity and structure 1363
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Figure 1 Decrease in Cerezyme Mr subsequent to N-glycosidase F treatment.
(A) SDS-PAGE analysis of Cerezyme with and without N-glycosidase F treatment. (B) MALDI-TOF MS spectra of Cerezyme before
and after N-glycosidase F treatment.
likely explanation for these differences is that Endo H,
but not Endo F1, cleaves the oligosaccharide chain on
N19, but this has not been examined experimentally.
In summary, modifying the oligosaccharide chains on
Cerezyme may have substantial effects on enzyme sta-
bility, at least in aqueous solution. Unfortunately, no infor-
mation is available from Genzyme on the stability of
Cerezyme compared to unmodified GlcCerase (prior to
mannose exposure by enzymatic modification; see
above). Moreover, it is possible that enzymatic treatment
of the expressed GlcCerase to expose the terminal man-
nose residues present in Cerezyme may result in a loss
of enzyme stability. Our data have shown that further
modification of the oligosaccharide chains on Cerezyme
can have substantial effects on its activity and stability,
which might be of therapeutic significance. For instance,
if a more stable form of Cerezyme could be produced,
this might be of benefit in ERT by extending the half-life
of the enzyme in the circulation or in macrophages, and
might even allow the enzyme to penetrate the
blood–brain barrier, as has been shown, albeit at small
levels, for administration of a high dose of recombinant
human b-glucuronidase in mouse models of mucopoly-
saccharidosis over a relatively long period (Vogler et al.,
2005).
Key structural features of GlcCerase
Upon resolution of the Cerezyme structure, we dem-
onstrated that it consisted of a characteristic ( b / a )8 (TIM)
barrel containing the catalytic residues, designated as
domain III (residues 76–381 and 416–430), and two
closely-associated b-sheets designated as domain II
(residues 30–75 and 431–497) (Figure 4). Structures sim-
ilar to domain II have been described in several other
glycosidases, such as xylanase (Larson et al., 2003) and
endo-glycoceramidase II (Caines et al., 2007), although
their function is unknown. In addition, the enzyme has an
unusual small domain (domain I) containing one 3-
stranded anti-parallel b-sheet that is flanked by a per-
pendicular N-terminal b-strand and loop (residues 1–27
and 383–414). This domain is formed by the two b-
strands from the N-terminus and the two anti-parallel b-
strands from an insertion between b-strand 8 and a-helix
8 of the TIM barrel. Inter-domain interactions between
domains I and II, which are connected by a long loop,
and between domains II and III, which are connected bya hinge, do not seem to be significant in the crystal struc-
ture, whereas domain I interacts tightly with catalytic
domain III and comprises one of the major loops shaping
the entrance to the active site.
Most mutations in GlcCerase appear to either partially
or entirely decrease catalytic activity and/or GlcCerase
stability (reviewed by Futerman et al., 2004). Prior to elu-
cidation of the 3D structure of GlcCerase, no clear cor-
relation was apparent between the location of particular
mutants within the sequence and the severity of clinical
symptoms. Even now that the 3D structure is available,
no clear relationship is immediately discernible between
the spatial location of most of the approximately 200known GlcCerase mutations and disease severity, with
one or two exceptions. A number of mutations (e.g.,
H311R, A341T and C342G) located near the active site
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Table 2 Determination of the degree of deglycosylation of Cerezyme by N-glycosidase
F treatment using mass spectrometry after in-gel tryptic digestion.
Peptide Sequence Molecular mass (Da)
Calculated Detected
48–74 R-RMELSMGPIQ*AN*59HTGTGLLLTLQPEQK)F 2964.45 2964.38
48–74 R-RMELSMGPIQAN*59
HTGTGLLLTLQ*PEQ*K)F 2965.45 2965.4948–74 R-RMoxELSMGPIQAN*59HTGTGLLLTLQPEQK)F 2979.46 2979.52
48–74 R-RMELSMoxGPIQAN*59HTGTGLLLTLQ*PEQ*K)F 2981.46 2981.51
48–74 R-RMoxELSMoxGPIQAN*59HTGTGLLLTLQPEQK)F 2995.49 2995.51
49–74 R-MELSMGPIQAN*59HTGTGLLLTLQPEQK)K 2807.44 2807.49
49–74 R-MELSMoxGPIQAN*59HTGTGLLLTLQPEQK)K 2823.41 2823.25
49–74 R-MELSMoxGPIQ*AN*59HTGTGLLLTLQPEQK)K 2824.41 2824.38
49–74 R-MoxELSMoxGPIQ*AN*59HTGTGLLLTLQ*PEQK)K 2841.48 2841.62
132–155 R-TYTYADTPDDFQLHN*146FSLPEEDTK)L 2847.26 2847.24
132–155 R-TYTYADTPDDFQ*LHN*146FSLPEEDTK)L 2848.26 2848.20
132–157a,b R-TYTYADTPDDFQ*LHN*146FSLPEEDTKLK)I 3091.33 3091.24
263–277b R-DLGPTLAN*270STHHNVR)L 1632.77 1632.80
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 2 Partial deglycosylation enhances Cerezyme stability
in vitro.
Cerezyme was passed through a Centricon YM-30 centrifugal
filter device with a relative molecular mass cut-off of ca. 30 kDa
to remove formulation additives, and then incubated with or
without N-glycosidase F for 88 h at 258C. Samples were then
passed through a Centricon YM-30 filter device again to remove
glycosidase. The activity was compared to that of freshly dis-
solved Cerezyme (i.e., Cerezyme taken from new bottles and
not passed through the Centricon YM-30 filter) using
N-w6-w(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)aminoxhexanoylx-glu-
cosylsphingosine (C6-NBD-GlcCer) as substrate (Meivar-Levy et
al., 1994; Shaaltiel et al., 2007) at pH 5.5 in 50 mM MES. TheV max of Cerezyme prior to treatment was 0.58 mmol C6-NBD-
ceramide formed per min per mg of Cerezyme, compared to
0.59 after treatment, with K m values of 10.8 and 10.5 mM,
respectively.
Figure 3 Effect of glycosidases on Cerezyme stability in vitro.
Cerezyme (1–1.5 mg/ml) was treated with 45 U/ml glycosidase
at 258C. After the incubation times indicated, samples were
passed through a Centricon YM-30 centrifugal filter device with
a relative molecular mass cut-off of approx. 30 kDa to remove
glycosidase and other formulation additives. The in vitro activity
of Cerezyme (1 mg/ml) was measured using N-w6-w(7-nitroben-
zo-2-oxa-1,3-diazol-4-yl)aminox hexanoylx-glucosylsphingosine(C6-NBD-GlcCer) (7.5 mM, 5 min, 378C) as substrate (Meivar-
Levy et al., 1994; Shaaltiel et al., 2007) at pH 5.5 in 50 mM MES.
Results are the mean"SD of three representative experiments,
each of which gave similar results.
result in severe disease, as might be predicted, but the
majority of the mutations are spread throughout all threedomains. No 3D structures are yet available for any
mutant GlcCerase. Conjecture as to the mechanism by
which catalytic activity might be compromised by a givenmutation is thus currently limited to structural predictions
and in silico mutational analysis.
Additional structural features are described below.
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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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intermediate, i.e., glucose, the effect of the negative
charge would be obliterated, and the pK a of glutamic acid
would be restored to its normal value, so that it would
be readily able to donate its proton. Further investigation
is required to determine if this or other explanations can
account for the anomalous protonation states of these
residues.
Another issue concerns the role of water in the cata-
lytic mechanism; a water molecule is required for hydrol-
ysis of the covalent intermediate. In all GlcCerase
structures of high enough resolution, a cavity near
Glu235 has been shown to contain water molecules. One
particular water molecule at an equivalent location is vis-
ible in other glycosidase structures, such as endo-GCase
II (Caines et al., 2007) and xylanase (Larson et al., 2003).
These conserved water molecules may be involved in the
catalytic cycle, since the solvent cavity is located next to
the catalytic residues and is accessible from the active
site.
Loops in GlcCerase structures
A number of loops that display alternative conformations
have been detected in GlcCerase. Prominent among
these are residues 345–349 (loop 1), 394–399 (loop 2)
and 312–319 (loop 3), whose conformations control
access to the active site (Premkumar et al., 2005;
Brumshtein et al., 2006, 2007; Shaaltiel et al., 2007). Of
these loops, loop 1, which is rich in hydrophobic resi-
dues, shows the most limited range of structural move-
ments. In contrast, loops 2 and 3 occur in two different
conformations with respect to each other, and a number
of different combinatorial conformations of these loops
are observed. Loop 3 shows considerable flexibility,adopting a helical structure in some cases and a coil in
others. One residue on this loop that appears relevant to
catalysis is Tyr313. In the three structures resolved to
date in which a complex is formed with a reversible inhib-
itor (prGlcCerase-NB-DNJ, prGlcCerase-NN-DNJ, and
Cerezyme-IFG) (Table 1), the hydroxyl group of Tyr313
forms a hydrogen bond with Glu340. This requires loop
3 to be in a helical conformation. In the conjugate formed
with CBE (Cerezyme-CBE), in which CBE is covalently
attached to Glu340, Tyr313Oz forms a hydrogen bond
with Glu235 and loop 3 assumes a coil conformation. If
the two interactions observed for Tyr313 are adopted by
the enzyme during catalysis, a conformational change inloop 3 may be involved in the reaction mechanism of
GlcCerase.
Anion-binding sites
Analysis of a surface plot of GlcCerase reveals some
interesting features that may help to explain how the
enzyme interacts with the membrane lipid bilayer to
hydrolyze GlcCer (Figure 4). First, there is an annulus of
hydrophobic residues surrounding the entrance to the
active site (Dvir et al., 2003). Second, substrate modeling
reveals that the glucose moiety and the adjacent glyco-
side bond of GlcCer fit into a cavity at the entrance tothe active site (Brumshtein et al., 2007) that does not
appear deep enough to encompass all of the hydropho-
bic chains of GlcCer, implying that at least the distal parts
of the hydrophobic chains remain embedded in the
membrane lipid bilayer during catalysis. Third, a number
of anion-binding sites exist at the interface of the
domains of GlcCerase (Brumshtein et al., 2006, 2007).
Interestingly, crystallization requires sulfate or phosphate
ions in the crystallization media, which may bind to the
same anion-binding sites involved in binding to phos-
pholipid membranes. One of these sites, comprising res-
idues 12, 44, 45, 353, and 356–358, is located near the
active site, and the second one is near residues 79, 228,
277 and 306.
Interaction with saposin C
GlcCerase requires the coordinate action of saposin C
and negatively charged lipids for maximum activity (Gra-
bowski et al., 1990; Vaccaro et al., 1997). Saposin C is
an 80-aa, heat-stable glycoprotein. Its overall sequence
and structure resemble those of other saposins (Bruhn,
2005; John et al., 2006), which are all cleavage products
of a single prosaposin precursor (Leonova et al., 1996).They are all believed to interact with lipids, leading to
enhanced accessibility of the lipid headgroups to their
cognate hydrolases (Leonova et al., 1996; Ahn et al.,
2003; Ciaffoni et al., 2003).
Recent structural studies on saposin C (de Alba et al.,
2003; Hawkins et al., 2005; Abu-Baker et al., 2007) have
shown that it can undergo conformational changes in the
presence of detergents or phospholipids and that chang-
es in pH can modulate its interactions with phospholip-
ids. Saposins A and C are both monomeric in solution at
pH 7.0. At pH 4.8 in the presence of detergent, saposin
A assembles into dimers, whereas saposin C forms tri-
mers (Ahn et al., 2006). However, saposin B was foundto be a dimer under all conditions tested, so that it forms
an enclosed shell for lipid binding (Ahn et al., 2003). The
crystal structure of saposin D (Rossmann et al., 2008)
reveals that it too is a dimer that interacts with lipids in
a pH-dependent manner (Ciaffoni et al., 2001, 2003).
The precise mode of activation of GlcCerase by sapo-
sin C is still unclear, although evidence has been pre-
sented for direct interaction between the two proteins
(Alattia et al., 2007). A computational study predicted
mutations in GlcCerase that might disrupt this interaction
(Alattia et al., 2007).
Sugar residues
Subsequent to our initial crystallization success using N-
glycosidase F-treated Cerezyme, we were able to
obtain Cerezyme crystals without the need for N-gly-
cosidase F-treatment. The crystals had the same space
group (Brumshtein et al., 2006) as the partially deglyco-
sylated Cerezyme and there were no fundamental dif-
ferences between the two structures, although some
novel conformations were observed in loop 3. The asym-
metric unit of Cerezyme was shown to contain two cop-
ies of the GlcCerase molecule, molecules A and B,
which, although very similar, are not completely identical
to each other in conformation. They also differed in thenumber of sugar molecules for which electron density
was visible (Brumshtein et al., 2006). Thus, in molecule
B, a core glycan chain containing five sugar residues
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Acid b-glucosidase activity and structure 1367
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Figure 5 Refined structure of the prGlcCerase-CBE conjugate.
(A) The two enantiomers of CBE. The 2R,3S,4S,5R enantiomer
is that which interacts with GlcCerase. (B) CBE bound at the
active site of GlcCerase. The previous structure of the CBE con-
jugate (PDB code 1Y7V; Premkumar et al., 2005) is shown in
blue, and the new structure (PDB code 1VT0) in green. Catalytic
residues are indicated as sticks. Loops 1–3 are indicated by
L1–L3, respectively.
Table 3 Data collection and refinement statistics for the
prGlcCerase-CBE conjugate.
Data collection
Space group P21
Cell dimensions
a, b, c (A ˚ ) 68.18, 96.88, 83.17
a, b, g ( 8 ) 90.00, 103.73, 90.00
Resolution (A ˚ ) 20.00–2.15 (2.20–2.15)
Rsym (%) 9.2 (41)
s 10.4 (1.7)
Completeness (%) 97.1 (82.7)
Redundancy 3.6 (1.9)
Refinement
Resolution (A ˚ ) 20.00–2.15
Number of reflections 53238
Rwork / Rfree 15.4/20.0
R.m.s deviations
Bond length (A ˚ ) 0.025
Bond angle ( 8 ) 1.991
Number of refined atoms
Protein 7723
Carbohydrate 87Solvent 464
Ligand 22
Ramachandran outliers (%) 0.2
prGlcCerase was crystallized in microbatch using 0.2 M
ammonium sulfate, 0.1 M Tris, pH 6.5, 25% (w/v) PEG
3350 under Al’s oil (1:1 silicon/paraffin oils) (D’Arcy et al.,
1996) using an IMPAX I-5 crystallization robot. CBE was
purchased from Sigma-Aldrich (St. Louis, USA) and dis-
solved in water to a final concentration of 10 mM; 0.5 ml
was added to crystallization drops after crystals had been
detected. X-Ray diffraction data were collected on the
ID29 beamline at the ESRF (Grenoble, France). Diffraction
data were processed using the HKL2000 software pack-
age, and the structure was solved by molecular replace-ment using the PDB 2V3F structure as the starting point
and refined with REFMAC5 (Murshudov et al., 1997). The
structure was deposited in PDB as code 1VT0. The high-
est resolution shell is shown in parentheses.
attached to N19 was detected, as well as three sugars
attached to N59, two to N146, and none to N270. In mol-
ecule A only two sugars were detected on N19, one on
N146 and none on N59 and N270. The glycans attached
to residue N19 of molecule B and to N59 of molecule B
are in contact with each other, causing the glycan chains
to become ordered and thus visible in the electron den-
sity map. The two sugar moieties on the glycan of N146
of molecule B do not make crystal contacts, consistent
with the low number of sugar residues visible on N146.
Binding of a covalent inhibitor to the active site of
GlcCerase
Crystals were recently obtained in which prGlcCerase
formed a covalent conjugate with CBE (Table 3), the
structure of which could be compared with that of the
conjugate previously obtained using Cerezyme (Prem-
kumar et al., 2005). As reported for Cerezyme, CBE was
covalently bound to Glu340, the catalytic nucleophile.
However, the higher resolution of the prGlcCerase con- jugate clearly revealed that CBE is in a chair conforma-
tion (Figure 5) rather than in a boat conformation, as had
been initially assigned for the Cerezyme conjugate (Pre-
mkumar et al., 2005). We have now modified the struc-
ture of the Cerezyme conjugate in the PDB databasewith the updated chair conformation of CBE. In addition,
the higher resolution of the current structure (Table 3)
allowed us to determine that it is the (2R,3S,4S,5R )-7-
oxabicycloheptane-2,3,4,5-tetrol enantiomer of CBE
(Figure 5) that binds to GlcCerase, whereas the
(2S,3R,4R,5S ) enantiomer does not. This is consistent
with early studies that showed that the (2R,3S,4S,5R )
enantiomer inactivates glucosidases, whereas the
2S,3R,4R,5S enantiomer is inactive (Braun et al., 1977).
Concluding comments
In this brief review we have discussed the X-ray struc-
tures of GlcCerase expressed in either Chinese hamster
ovary cells or transgenic carrot cells and the stability of
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1368 Y. Kacher et al.
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Cerezyme in vitro. Unfortunately, very little information
is available in the literature on the stability of Cerezyme
or on the effect of modifying its glycan chains on its sta-
bility. Moreover, few attempts have been made to
enhance GlcCerase stability by either modifying its poly-
peptide backbone or altering its 3D structure or its mode
of binding to the membrane bilayer; the availability of the
3D structure now makes such approaches feasible.
Whether a more stable GlcCerase would be of benefit to
Gaucher disease patients is a matter of discussion. How-
ever, since the half-life of Cerezyme in the circulation is
relatively short (of the order of 10–15 min; reviewed by
Futerman et al., 2004) and only very small amounts of
injected Cerezyme are actually internalized by macro-
phages, it seems plausible that engineering either a more
stable enzyme or an enzyme with higher catalytic activity
could reduce the number of infusions and potentially also
reduce the cost of ERT in Gaucher disease.
Acknowledgments
Work in the authors’ laboratories was supported by the Mag-
neton Program, Office of the Chief Scientist, Ministry of Industry
and Commerce, Israel, the National Gaucher Foundation, the
Gaucher Disease Divot Classic of Chicago, and the Benoziyo
Center for Neuroscience. We thank Tevie Mehlman for help with
MS analysis. J.L. Sussman is the Morton and Gladys Pickman
Professor of Structural Biology, and A.H. Futerman is the Joseph
Meyerhoff Professor of Biochemistry at the Weizmann Institute
of Science.
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Received May 8, 2008; accepted August 4, 2008
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