2008 kacher biol chem

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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,



1362 Y. Kacher et al.


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



Acid b-glucosidase activity and structure 1363


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





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.





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





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





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 conjugate 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





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

2008 kacher biol chem

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