Hydrophobic Interactions Contribute to Conformational Stabilization of Endoglycoceramidase II by Mechanism-Based Probes

Article abstract of DOI:10.1021/acs.biochem.6b00363

Small compound active site interactors receive considerable attention for their ability to positively influence the fold of glycosidases. Endoglycoceramidase II (EGCII) from Rhodococcus sp. is an endo-β-glucosidase releasing the complete glycan from ceramide in glycosphingolipids. Cleavage of the β-glycosidic linkage between glucose and ceramide is also catalyzed by glucocerebrosidase (GBA), the exo-β-glucosidase deficient in Gaucher disease. We demonstrate that established β-glucoside-configured cyclophellitol-type activity-based probes (ABPs) for GBA also are effective, mechanism-based, and irreversible inhibitors of EGCII. The stability of EGCII is markedly enhanced by formation of covalent complexes with cyclophellitol ABPs substituted with hydrophobic moieties, as evidenced by an increased melting temperature, resistance against tryptic digestion, changes in ¹⁵N-¹H transverse relaxation optimized spectroscopy spectra of the [¹⁵N]Leu-labeled enzyme, and relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic acid fluorescence. The stabilization of EGCII conformation correlates with the shape and hydrophobicity of the substituents of the ABPs. We conclude that the amphipathic active site binders with aliphatic moieties act as a hydrophobic zipper on the flexible EGCII protein structure.

Full text of DOI:10.1021/acs.biochem.6b00363

Article
pubs.acs.org/biochemistry
Hydrophobic Interactions Contribute to Conformational Stabilization
of Endoglycoceramidase II by Mechanism-Based Probes
Fredj Ben Bdira,† Jianbing Jiang,‡ Wouter Kallemeijn,§ Annett de Haan,∥ Bogdan I. Florea,‡
Boris Bleijlevens,∥ Rolf Boot,§ Herman S. Overkleeft,‡ Johannes M. Aerts,§ and Marcellus Ubbink*,†
†Department of Macromolecular Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands
‡Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands
§Department of Medical Biochemistry, Leiden Institute of Chemistry, Einsteinweg 55, 2333 CC Leiden, The Netherlands
∥Department of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
S
* Supporting Information
ABSTRACT: Small compound active site interactors receive
considerable attention for their ability to positively influence
the fold of glycosidases. Endoglycoceramidase II (EGCII)
from Rhodococcus sp. is an endo-β-glucosidase releasing the
complete glycan from ceramide in glycosphingolipids.
Cleavage of the β-glycosidic linkage between glucose and
ceramide is also catalyzed by glucocerebrosidase (GBA), the
exo-β-glucosidase deficient in Gaucher disease. We demon-
strate that established β-glucoside-configured cyclophellitol-
type activity-based probes (ABPs) for GBA also are effective,
mechanism-based, and irreversible inhibitors of EGCII. The stability of EGCII is markedly enhanced by formation of covalent
complexes with cyclophellitol ABPs substituted with hydrophobic moieties, as evidenced by an increased melting temperature,
resistance against tryptic digestion, changes in 15N−1H transverse relaxation optimized spectroscopy spectra of the [15N]Leu-
labeled enzyme, and relative hydrophobicity as determined by 8-anilino-1-naphthalenesulfonic acid fluorescence. The
stabilization of EGCII conformation correlates with the shape and hydrophobicity of the substituents of the ABPs. We conclude
that the amphipathic active site binders with aliphatic moieties act as a “hydrophobic zipper” on the flexible EGCII protein
structure.
humans, collectively named glycosphingolipidoses. Most prev-
lycosphingolipids, an important class of cellular membrane
Glipids, contain a ceramide, and its primary hydroxyl is alent among these disorders is Gaucher disease, in which
deficiency of the lysosomal β-glucosidase, named glucocere-
brosidase (EC 3.2.1.45, GBA), a member of glycosidase family 30
(www.cazy.org), causes accumulation of the substrate glucosyl-
ceramide. A number of GBA active site binders, called
pharmacological chaperones, are being investigated as enzyme
stabilizers with envisioned therapeutic application in Gaucher
disease patients. One of the best studied compounds in this
respect is the iminosugar isofagomine (IFG), a competitive GBA
inhibitor.6,7 The retaining β-glucosidase GBA employs a
glycosylated with either a glucose or galactose residue to which
further sugars may be attached. Two pathways for degradation of
glycosphingolipids are known to exist in nature. Nonmammalian
endoglycoceramidases are able to remove in one step the entire
oligosaccharide chain from the ceramide moiety. Endoglycocer-
amidase II (EGCII) from Rhodococcus sp.1 was the first
endoglycoceramidase identified (grouped in glucosyl-hydrolase
family 5). Later, other nonhomologous endoglycosidases
degrading glycosphingolipids from leeches2 and earthworms3
were discovered. No mammalian endoglycoceramidase has been
identified so far, although a similar enzymatic activity has been
reported for rat and rabbit mammary glands4 and human cancer
cells.5 In mammalian cells, the degradation of glycosphingolipids
takes place via a very different pathway. Inside lysosomes, the
subcellular compartments that specialize in the breakdown of
macromolecules, terminal sugars of glycosphingolipids are
removed stepwise by specific exoglycosidases, ultimately
generating the free lipid, ceramide. The physiological relevance
of lysosomal degradation of glycosphingolipids is illustrated by
associated pathologies. Inherited defects in glycosphingolipid-
degrading exoglycosidases cause a number of storage diseases in
1
Koshland double-displacement mechanism following a S3 4H3
4C1 reaction itinerary.8 In the first displacement event, a covalent
α-enzyme glucosyl intermediate is formed, with the glucopyr-
anose moiety adopting a 4C1 conformation. After binding of the
substrate β-glucoside, a transition state emerges with high
oxocarbenium ion character with a concomitant conformational
change of the substrate in the Michaelis complex (1S3) to a 4H3
half-chair.9 It is the enzyme glucosyl intermediate state of the
Received: April 18, 2016
Revised: July 21, 2016
© XXXX American Chemical Society
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Figure 1. (a) Structures of β-glucoside-configured cyclophellitols used and (b) mechanism-based inactivation of β-glucosidases by covalent binding to a
catalytic nucleophile.
hydrolysis reaction that is emulated by cyclophellitol 1 (see
Figure 1a for the compounds used in this study). Thus,
cyclophellitol binds with high specificity to the β-glucosidase
active site. Cyclophellitol 1, first isolated from a Phellinus sp.
culture by Atsumi and co-workers,10 irreversibly inhibits GBA by
forming a stable, covalent adduct to residue Glu340.11 The
cyclophellitol epoxide is ideally positioned for acid-catalyzed
nucleophilic opening involving the active side residues. As a
result, a covalent enzyme−inhibitor complex is formed (with the
inhibitor now also adopting a 4C1 conformation), with the ester
bond much more stable and thereby irreversible compared to the
acylal linkage that characterizes the enzyme−substrate covalent
intermediate (Figure 1b).9 Substitution of the epoxide oxygen
with nitrogen yields cyclophellitol aziridine 2, an equally strong
inhibitor.12 Cyclophellitol 1 and cyclophellitol aziridine 2
functionalized to contain a fluorophore have been applied as
activity-based glycosidase probes for sensitive in vitro and in situ
detection of GBA molecules.11,13 Conduritol B epoxide (CBE)
10, an analogue of cyclophellitol lacking the C8 methylene,
likewise irreversibly inhibits GBA but with lower affinity and
specificity.14 Freezing of the enzyme midway through its catalytic
reaction through covalent inhibition offers an important tool for
investigating the characteristics of the formed complex and
elucidating which parts of the inhibitors are important for
efficient stabilization activity.
hydrolases (Figure S1), GBA and EGCII share a similar folding
topology of their polypeptide chains. Both of them display a TIM
barrel (triose-phosphate isomerase barrel) catalytic domain
formed by (α/β)8, which makes them both members of GH-A
clan (www.cazy.org). The superposition of the three-dimen-
sional (3D) structure of the TIM barrels has a root-mean-square
deviation (rmsd) of 3.1 Å for the Cα atoms [Protein Data Bank
(PDB) entry 2v3e for GBA and PDB entry 2osx for EGCII] yet
shows a remarkable superposition of the acid/base and the
nucleophile catalytic residues, which are separated by an average
distance of ∼5.2 Å, consistent with their role in a double-
displacement mechanism with net retention of stereochemistry
(see Figure 2a,c). The two proteins also have the aglycan sites in
the same position relative to the active site. The analysis of the
GBA crystal structure in the presence of nonyl-deoxynojirimycin
(NN-DNJ), a potent inhibitor of GBA, shows that the aliphatic
tail of the substrate is accommodated within a narrow
hydrophobic channel where it interacts with residues Leu241,
Phe246, Tyr313, Leu314, and Tyr244. Similarly, the ceramide
moiety of GM3 ganglioside, the natural substrate for EGCII,
makes several hydrophobic interactions with residues Leu180,
Tyr182, Ile183, Phe235, Ala275, Ile276, Tyr306, and Leu308
(Figure 2b,c). In view of the similarities between the two protein
binding sites, it is not surprising that NN-DNJ can also inhibit
EGCII and fits well in the active site of the EGCII crystal
structure (PDB entry 2OSX)15 (Figure 2c). The GBA glycan
binding site is much narrower than that of EGCII, which allows
Despite the significant differences in their amino acid
sequences, as they belong to different families of glycoside
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Figure 2. Comparison of the folding topology and aglycan binding sites of GBA and EGCII. (a) Structural alignment of the TIM barrel catalytic domain
of EGCII (orange cartoon) and GBA (cyan cartoon) using the jFATCAT algorithm59 (RCSB comparison tool). The acid/base and nucleophile catalytic
residues are shown as sticks and colored red for EGCII and blue for GBA. For the sake of clarity, nonoverlapping loops were hidden. (b) Aglycan binding
site cavities of EGCII (orange) and GBA (cyan) as observed in the superposition shown in panel a. The surface of the residues interacting with the
aliphatic tail of the ligands is shown; β-D-glucose ceramide (in EGCII) is colored red and NN-DNJ (in GBA) blue. (c) Primary sequences of EGCII and
GBA with the structurally homologous parts highlighted in orange and cyan, respectively. Acid/base catalytic residues are marked with stars and a black
box. Hydrophobic residues interacting with the NN-DNJ alkyl chain and the ceramide moiety of GM3 are marked with cyan and orange circles,
respectively. Nonhydrophobic interacting residues within the aglycan binding sites are marked by black triangles.
GBA to accommodate only a single glucose ring, precluding the
binding of longer saccharide moieties and suggesting an
explanation for the different exo and endo hydrolysis specificities
for GBA and EGCII, respectively. The location of the reactive
residues and primary sugar is, however, essentially the same in
both active sites, suggesting that the mechanism of cleavage of
the β-glycosidic linkage connecting glucose and ceramide is the
same.
The analogies between the two retaining β-glucosidases
stimulated us to test the impact of a series of mechanism-based
irreversible inhibitors known to bind covalently in the active site
of GBA on EGCII conformational stability. Here, we
demonstrate that in its free state in solution EGCII is
thermolabile and highly flexible. An equilibrium between
monomeric and dimeric states is observed for the resting state
enzyme, because of the large solvent-exposed hydrophobic
surface area of EGCII. Upon formation of the complex with the
amphiphilic cyclophellitol analogues (4−8), marked stabilization
and rigidification are observed, correlating with the lipophilic
potential of the compounds. No changes are observed in the
protein stability or rigidity after incubation with nonhydrophobic
compounds (1 and 2). These findings demonstrate the
importance of the length and shape of the non-glycan moiety
of substituted cyclophellitols for the enzyme stabilization.
EXPERIMENTAL PROCEDURES
■
General Synthesis. All reagents and solvents were of
commercial grade and used as received unless stated otherwise.
Tetrahydrofuran and dichloromethane were stored over flame-
dried 3 Å molecular sieves. All reactions were performed under
an inert atmosphere unless stated otherwise. Solvents used for
flash chromatography were of pro analysis quality. Reactions
were monitored by TLC analysis using aluminum sheets
precoated with silica gel 60 with detection by UV absorption
(254 nm) and by spraying with a solution of (NH4)6Mo7O24·
H2O (25 g/L) and (NH4)4Ce(SO4)4·H2O (10 g/L) in 10%
sulfuric acid, followed by charring at ∼150 °C or by spraying with
20% sulfuric acid in ethanol followed by charring at ∼150 °C.
Column chromatography was performed using Screening Device
silica gel in the indicated solvents. 1H nuclear magnetic resonance
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(NMR), 13C NMR, COSY, and HSQC spectra were recorded on
Bruker DMX-400 (400/100 MHz), Bruker AV-400 (400/100
MHz), and Bruker AVIII-600 (600/150 MHz) spectrometers in
the given solvent. Chemical shifts are reported as δ values in parts
per million relative to the chloroform residual solvent peak,
tetramethylsilane (TMS) as an internal standard, or the
deuterated solvent signal for CD3OD. All given 13C spectra
were proton-decoupled. High-resolution mass spectra were
recorded with an LTQ Orbitrap instrument (ThermoFinnigan).
LC/MS analysis was performed on an LCQ Adventage Max
(ThermoFinnigan) ion trap spectrometer (ESI+) coupled to a
Surveyor high-performance liquid chromatography (HPLC)
system (ThermoFinnigan) equipped with a C18 column
(Gemini, 4.6 mm × 50 mm, 3 μm particle size, Phenomenex)
using buffers A (H2O), B (acetonitrile in MeCN), and C (50 mM
NH4OAc). For reverse phase HPLC purifications, an Agilent
Technologies 1200 series instrument equipped with a semi-
preparative Gemini C18 column (10 mm × 250 mm) was used.
The applied buffers were 50 mM NH4HCO3 in H2O (buffer A)
and MeCN (buffer B). Compound 6 was synthesized according
to a published report.16 Compounds 1−5 and 7 and cyclo-
phellitol-aziridine ABP 9 were prepared as described previ-
ously.17
Production and Purification of Recombinant EGCII. The
gene encoding EGCII from Rhodococcus sp. strain M777 lacking
the N-terminal signal peptide sequence was subcloned into
pET21a using NdeI/NotI restriction sites, introducing a C-
terminal His tag. Escherichia coli BL21(DE3) pLysS cells were
transformed with the obtained vector and cultured at 37 °C in
lysogency broth containing 50 μg/mL ampicillin and 30 μg/mL
chloramphenicol. Gene expression was induced with 0.5 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) when the culture
reached an OD600 of ≈0.6, and the incubation was continued
overnight at 20 °C. The cells were harvested by centrifugation,
resuspended in extraction buffer [20 mM Tris-HCl, 150 mM
NaCl, and 20 mM imidazole (pH 7.6)], and sonicated. The
resulting EGC six-His supernatant was further purified by Ni(II)
affinity chromatography to >95%, as determined by sodium
dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−
PAGE). All the purification steps were conducted at 4 °C.
[15N]Leucine isotope selective labeling of EGCII was started by
culturing the transformed E. coli BL21(DE3) in a 50 mL culture
in M9 medium overnight at 37 °C, supplemented with each
amino acid (at 0.1 g/L) except leucine, as well as 125 mg/L
adenosine, 125 mg/L guanosine, 125 mg/L cytosine, 50 mg/L
thymine, 50 mg/L uracil, 50 mg/L nicotinic acid, and 2 g/L
succinic acid. The next day, 0.5 L of this medium was inoculated
with the overnight preculture and incubated at 37 °C until the
OD600 reached ≈0.6. Fifteen minutes before induction, a mixture
of 0.5 g of each unlabeled amino acid except leucine and 50 mg of
[15N]leucine were added to the culture.18 Protein production
was started by inducing gene expression with 0.5 mM IPTG, and
the culture was continued overnight at 20 °C. The purification
procedure was as described above.
Synthesis of Compound 8. N-Ethoxycarbonyl-2-ethoxy-
1,2-dihydroquinoline (EEDQ) (79 mg, 0.32 mmol) and 6-O-
adamantanemethyl-6-hydroxyhexanoic acid X (85 mg, 0.32
mmol) were dissolved in anhydrous DMF (0.32 mL) and stirred
at room temperature for 2 h (Scheme 1). The resulting dissolved
Scheme 1. Synthesis of Compound 8
NMR Spectroscopy. Two-dimensional 15N−1H TROSY19
spectra were recorded at 298 K on a Bruker Avance III HD 850
MHz spectrometer equipped with a TCI-Z-GRAD cryoprobe on
a sample of 150−200 μM [15N]Leu EGCII in McIlvaine’s buffer
[110 mM Na2HPO4 and 40 mM citric acid (pH 5.5)] and 6%
D2O. Data were processed with Topspin (Bruker Biospin) and
analyzed using the Sparky software package.20
EGCII Activity Assay and Cyclophellitol Complex
Formation. The enzymatic activity of the purified enzyme was
measured in the presence of the substrate 4-methylumbelliferyl
β-D-lactopyranoside (4MU-Lac) (Marker Gene Technologies,
Inc.). The enzyme (153 nM) was incubated with different
concentrations of 4MU-Lac for 1 h at 37 °C in McIlvaine’s
(citrate/phosphate) buffer and 0.1% BSA. The reaction was
quenched by adding 2.5 mL of a 0.3 M glycine solution (pH
10.6). The reaction rates were monitored by measuring the
fluorescence of the released 4MU at its emission wavelength of
445 nm on an LS55 fluorimeter. For each enzymatic reaction, a
blank was used, in the absence of EGCII, to measure the
spontaneous hydrolysis of 4MU-Lac, and this rate was subtracted
from the enzymatically catalyzed reaction rate. For covalent
complex formation, EGCII and the irreversible inhibitors were
incubated at a ratio of 1:30 in McIlvaine’s buffer for 3 h at 25 °C.
Thereafter, the samples were incubated for 1 h at 37 °C as a final
step to promote complex formation and to discard aggregated
protein. The samples were centrifuged for 30 min at 16000g, and
the excess of inhibitor was removed by buffer exchange. The
samples were kept at 4 °C for further experiments.
mixed anhydride (160 μL) was added to cyclophellitol-aziridine
217 (35 mg, 0.2 mmol) in dry DMF (1.2 mL) at 0 °C and stirred
for 30 min. At this time, an additional batch of mixed anhydride
(160 μL) was added. The resulting mixture was stirred at 0 °C for
2 h. The reaction was quenched with MeOH (1 mL), and the
mixture was concentrated in vacuo. Then the crude product was
purified by semipreparative reverse HPLC [linear gradient from
38 to 47% B in A, 12 min; solutions used, H2O (A) and MeCN
(B)], and the fractions were lyophilized yielding 8 as white
1
powder (21 mg, 0.05 mmol, 25% yield): H NMR (400 MHz,
MeOD) δ 4.09 (d, J = 10.4, 4.4 Hz, 2H), 3.72−3.66 (m, 2H),
3.414 (t, J = 6.4 Hz, 2H), 3.23 (dd, J = 10.0, 8.0 Hz, 1H), 3.10−
3.02 (m, 2H), 2.74 (d, J = 5.6 Hz, 1H), 2.55−2.51 (m, 2H),
2.01−1.95 (m, 4H), 1.77−1.66 (m, 8H); 1.64−1.56 (m, 8H);
13C NMR (100 MHz, MeOD) δ 188.5, 83.0, 79.1, 73.4, 72.2,
69.4, 63.6, 45.3, 42.4, 41.1, 40.8, 38.3, 36.6, 35.2, 30.1, 29.9, 29.8,
22.9; LC/MS tR 7.87 min (linear gradient from 10 to 90% B in 15
min); ESI-MS m/z 424.4 (M + H)+; HRMS calcd for
C14H17NO4 (M + H+) 424.26936, found 424.26921 (see the
Supporting Information).
Circular Dichroism (CD) Spectroscopy. The far-UV
region (200−280 nm) CD spectra were recorded in a 0.1 cm
path length quartz cuvette in a Jasco 810 CD spectrometer using
a 5 μM protein solution. The spectra were recorded using four
D
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Figure 3. Thermodynamic stability of EGCII. (a) Thermal denaturation curves of EGCII at varying scanning rates (1−5 °C/min), at a protein
concentration of 5 μM. (b) Temperature dependence of EGCII activity toward 4MU-Lac in 150 mM McIlvaine’s buffer (pH 5.5), with an incubation
time of 1 h. The activity at 25 °C is highlighted by the green rectangle; the error bars represent the standard deviations of a duplicate experiment. (c)
Decays over time of the ellipticity at 222 nm (red empty circle and curve), the specific activity (blue filled circle and curve), and SDS−PAGE of ABP
labeling of EGCII samples at 37 °C in 150 mM McIlvaine’s buffer (pH 5.5) (bottom panel). The red and blue curves are exponential decay fits of the
ellipticity and enzymatic activity (EA), respectively. The half-life decays are 12 min (ellipticity) and 7 min (enzymatic activity). The error bars represent
standard deviations of a duplicate experiment. (d) EGCII kinetic stability at 25 °C monitored by circular dichroism spectra (blue lines), recorded every
10 min for 90 min, and SDS−PAGE of ABP selective labeling of EGCII (bottom panel).
scans with a bandwidth and a wavelength step of 1 nm. The
obtained spectra were background-corrected and smoothed
using Jasco Spectra Manager. The unfolding transition point
(Tm) was measured by following the dichroic signal decay at 222
nm, by applying heating rates from 1 to 5 °C/min, over a
temperature gradient from 20 to 80 °C in McIlvaine’s buffer. The
obtained CD ellipticity at each point (θt) was assumed to be the
linear combination of the folded (θF) and unfolded (θU)
ellipticity. At each point of the melting curve, the unfolded
fraction was calculated using the formula f U = (θt − θF)/(θU −
θF). The unfolding transition curves were plotted and fitted using
GraphPad Prism, and the Tm was extracted from the obtained fits
as the temperature for which fU = 0.5.
Limited Proteolysis. Proteolysis of free EGCII and EGCII
complexes by trypsin (Boehringer, trypsin sequencing grade)
was conducted at 25 or 37 °C using a trypsin:EGCII ratio of
1:100 (by weight) in the absence and the presence of 1% Triton
X-100 and lauryl glucoside. A time course analysis of the
proteolytic events was conducted using SDS−PAGE, and the
peptic fragments were further analyzed by in-gel digestion LC−
MS on a Thermo LTQ-orbitrap mass spectrometer following the
described protocol.21 To analyze the mass of the produced
EGCII tryptic fragments, the proteolytic reaction was quenched
by adding phenylmethanesulfonyl fluoride (PMSF) after
incubation for 10 min and analyzed on a SYNAPT G2-Si mass
spectrometer (Waters).
A stock solution of 10 mM ANSA was prepared in McIlvaine’s
buffer. A variable amount of EGCII or EGCII−inactivator
complex was mixed with a 2 mM ANSA solution. The mixture
was incubated for 30 min in the dark at room temperature. The
fluorescence was measured using an LS55 fluorimeter. The
excitation and emission slits were both 5 nm, and the excitation
and emission wavelengths were set at and 366 and 490 nm,
respectively. The fluorescence of a blank solution was subtracted
from each protein sample. The slope of the net fluorescence
intensity (percent) plotted versus the protein concentration was
calculated by linear regression analysis with GraphPad Prism and
used as an index of the protein surface hydrophobicity.
Size Exclusion Chromatography and Multiangle Laser
Light Scattering. Size exclusion chromatography in con-
junction with multiangle laser light scattering (SEC−MALLS)
was used to determine the absolute molecular mass of EGCII
samples. EGCII (1 mg/mL) in 20 mM Tris-HCl (pH 7.6) or
McIlvaine’s buffer was injected into the column. The refractive
index and multiangle light scattering were measured simulta-
neously as the solute eluted from the size exclusion column. The
concentration of the solute was determined from the refractive
index, the excess Rayleigh scattering from the light scattering.
These two parameters were entered into the Rayleigh−Gans−
Debye approximation to determine the molar mass of the solute
using SEC−MALLS Astra 473 software manager.23
RESULTS
EGCII Solvent-Accessible Hydrophobic Surface Deter-
mination by 8-Anilino-1-naphthalenesulfonic Acid
(ANSA). Protein surface hydrophobicity was determined using
ANSA according to the method described by Kato and Nakai.22
■
Readouts for Active EGCII Molecules. We first identified
convenient readouts for active EGCII molecules. We determined
whether 4-methylumbelliferyl lactoside (4MU-Lac) is a suitable
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Figure 4. Time course of tryptic proteolysis of EGCII analyzed by SDS−PAGE. Tryptic proteolysis was performed at an E:S ratio of 1:100 (by weight) in
150 mM McIlvaine’s buffer (pH 5.5) at 25 °C (a), at 25 °C in the presence of 1% lauryl glucoside (b), at 37 °C (c), and at 25 °C in the presence of 1%
Triton X-100 (d). Samples (5 μg) were taken from the reaction mixture every 10 min and analyzed via 10% SDS−PAGE followed by protein staining.
(e) Major trypsin cleavage sites (Arg321 and Arg332 in the red helix) identified by mass spectrometry and depicted in EGCII 3D structure (PDB entry
2osw).27 N-Terminal and C-terminal protein fragments are colored blue and green, respectively.
fluorogenic substrate for recombinant EGCII. This proves to be
the case. The enzyme exhibits an optimal activity at pH 5.5
(Figure S2a), with hydrolysis occurring in a time-dependent
manner (Figure S2b).
Next, we demonstrated that EGCII can be labeled in a
mechanism-based manner by a β-glucoside-configured cyclo-
phellitol 10- and cyclophellitol-aziridine substituted with a
BODIPY fluorophore 9 (Figure S2c).
Thermal Stability of EGCII. Thermal unfolding of EGCII
under optimal conditions for enzymatic activity [150 mM
McIlvaine’s buffer (pH 5.5)]24 was monitored by recording the
temperature dependence of the dichroic signal at 222 nm and
applying a range of heating rates from 1 to 5 °C/min (Figure 3a).
The obtained unfolding curve at a heating rate of 1 °C/min
shows an apparent Tm of 42 °C, close to the enzyme optimal
catalytic temperature (37 °C) (Figure 3b).1 The apparent Tm
value depends on the scanning rate, suggesting an irreversible
denaturation process toward aggregation, which is visible by eye
due to the turbidity of the protein solution. The unfolding
kinetics of EGCII at 37 °C (pH 5.5) were determined by
recording a CD spectrum every 10 min for 50 min. EGCII shows
an exponential decay of its ellipticity signal at 222 nm with a half-
life of ∼10 min and forms a visible aggregate. The enzymatic
activity was also tested with 4MU-Lac as a substrate. A parallel
decay was observed (Figure 3c), correlating unfolding kinetics
with loss of activity. During the kinetic unfolding, the amount of
residual active protein was also followed by labeling with the
activity-based probe compound 9 (Figure 3c, bottom panel). A
similar decay of the fluorescently labeled EGCII over the
incubation time was observed. EGCII denaturation was also
followed at 25 °C by CD (Figure 3d). At this temperature, the
enzyme’s activity is close to optimal (Figure 3b). At 25 °C [150
mM McIlvaine’s buffer (pH 5.5)], EGCII is stable with no
changes in its secondary structure and its active state as
monitored by ABP (Figure 3d, bottom panel). These results
show that EGCII is a thermolabile protein with an apparent Tm of
42 °C at a heating rate of 1 °C/min and tends to form an
aggregate as a final step in the irreversible unfolding process.
Therefore, for experiments requiring prolonged incubations, it is
practical to work at 25 °C.
EGCII Flexibility and Conformations in Solution.
Limited proteolysis with trypsin at a trypsin:EGCII ratio of
1:100 was employed to investigate the flexibility of EGCII in
solution. Proteolysis under different conditions and as a function
of incubation time was analyzed via SDS−PAGE. At 25 °C,
directly after trypsin addition, an additional band rapidly appears
on the gel close to the intact protein. This could result from
cleavage of one of the flexible ends of the protein. After
incubation for 10 min, approximately 50% of the protein has
been digested with the appearance of two major protein
fragments with apparent molecular weight of 34 and 25 kDa
(Figure 4a). These fragment species remain relatively resistant to
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Figure 5. EGCII solution state analysis by size exclusion chromatography and native PAGE. (a) EGCII size exclusion chromatogram in 20 mM Tris-HCl
and 150 mM NaCl (pH 7.6). The elution volumes of the peaks are indicated. (b) Enzymatic activity of eluted fractions P1 and P2 using 4MU-Lac as a
substrate. The error bars represent the standard deviations of two experiments. (c) Size exclusion chromatograms of eluted fractions P1 (blue) and P2
(red). (d) Native gel analysis of the EGCII sample in Tris buffer, stained with Coomassie Brilliant Blue (CBB) and the activity-based probe (ABP) 9.
further proteolysis and accumulate in the reaction mixture. At 37
°C, the protein was fully digested into these two bands within 10
min, the digestion already being observed immediately upon
addition of the protease (time zero) (Figure 4c). The peptic
fragment pattern is similar at 37 and 25 °C, suggesting that
digestion occurs at the same position in the polypeptide. To
correct for the difference in trypsin activity at 25 and 37 °C, the
digestion at 25 °C was conducted by adding 3 and 10 times more
trypsin. The tryptic pattern of EGCII was not affected by the
additional trypsin activity (Figure S3). The subsequent rapid
disappearance of the tryptic fragments at 37 °C, as compared to
that at 25 °C, suggests that at the lower temperature the nicked
polypeptide remains folded, whereas at higher temperature it
unfolds, allowing further proteolysis of the two fragments. We
tried to identify the site of digestion by mass spectrometry.
The in-gel digestion and analysis by LC/MS of the produced
tryptic fragments suggested that the major digestion occurs after
Arg332. However, LC/MS of the intact fragments (that is,
without further digestion into smaller peptides) suggested that
the major digestion site is located after the preceding arginine
residue at position 321 (observed mass for C-terminal fragment
18622 Da, expected 18624 Da). We conclude that either or both
of these arginines represent the primary cutting site. Interest-
ingly, in the EGCII crystal structure, both arginines are located in
a long, well-structured α-helix (Figure 4e). In a modeling study,
Hubbard et al. concluded that a local disorder or unfolding of the
protein polypeptide chain of at least 12 residues is required to
allow access into a protease active site.25 Therefore, our results
suggest that this part of the protein is less structured in solution
than in the crystalline state. In the presence of the detergent
lauryl glucoside (Figure 4b) or Triton X-100 (Figure 4d), the
protein is strikingly resistant to tryptic digestion. It has been
reported that the used surfactants have no inhibitory effects on
trypsin.26 This tryptic resistance of the protein in the presence of
detergents could be due to interaction with the micelles,
shielding the protein surface, leading to a more rigid and
compact structure. Moreover, lauryl glucoside has an amphiphilic
structure quite similar to that of EGCII natural substrates 11
(Figure 1a), and thus, it might interact with the binding pocket of
the protein, stabilizing it in a rigid conformation (see below).
Crystals of ECGII were obtained in the presence of 1% (v/v)
Triton X-100, 10% (v/v) glycerol, and 25% (w/v) PEG.27 The
possibility that under these conditions, and because of crystal
packing effects, the protein could adopt a rigid conformation that
is not representative of the lowest-energy state present in
solution cannot be excluded.
Next, EGCII in solution was studied by size exclusion
chromatography (SEC) to monitor changes in the hydro-
dynamic dimensions accompanying unfolding, dimerization, or
conformational changes of globular proteins in solution.28,29 We
observed that EGCII elutes as two fractions from the SEC
column (P1 and P2) with elution volumes of 8.5 and 9.5 mL,
respectively. The integration of the chromatogram peaks area
yields percentages of 30 and 70% for peaks 1 and 2, respectively
(Figure 5a). The re-injection of the extremity of the collected
fractions of the two peaks shows an exchange between the two
states of the protein even at low concentrations (Figure 5c). The
enzymatic activity of both peak fractions was the same within
error (Figure 5b). They also exhibit the same molecular weight
on SDS−PAGE. Therefore, both fractions represent a native and
active state of EGCII. Native PAGE analysis of EGCII shows two
bands, and staining of the gel with ABP 9 confirms that the
enzyme in both of the bands is active (Figure 5d). The analysis of
the molecular weight of the eluted peaks with SEC−MALLS
yields an Mw of 100 kDa for P1 and an Mw of 50 kDa for P2
(Figure S4), providing more evidence of a monomeric state and a
dimeric state of the protein. Thus, we conclude that EGCII is in
equilibrium between monomeric and dimeric states in solution.
EGCII Complexed with N-Alkyl Azide-Substituted
Cyclophellitol Aziridine (compound 6). Because EGCII
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Figure 6. Effects of compound 6 on EGCII structure and thermal stability. (a) Thermal denaturation at pH 5.5 monitored by CD spectroscopy with a
heating rate of 1 °C/min. The fitted apparent Tm values of the EGCII free state (black) and the EGCII−6 complex (red) were 43 and 55 °C, respectively.
(b) 15N−1H TROSY spectra of [15N]Leu-labeled EGCII in the free (black) and bound (red) states. (c) Relative solvent-accessible hydrophobic surface
of EGCII free (black) and bound (red) states, determined by ANSA fluorescence. The lines represent a linear fit through the origin with the indicated
fitted slopes. (d) Size exclusion chromatograms of EGCII free (black) and covalently bound (red) states.
was found to irreversibly react with cyclophellitol aziridines, we
studied the impact of cyclophellitol derivatives on the biophysical
properties of EGCII. For this, we first used compound 6, an N-
alkylated cyclophellitol aziridine functionalized with an azide.
The effects of compound 6 on the thermodynamic properties of
EGCII were investigated by measuring the apparent Tm of the
complex. A pronounced increase in the apparent Tm of 12 °C was
observed (Figure 6a). Such an increase is usually accompanied by
changes in the protein structure and rigidity.30,31 The occurrence
of conformational changes of EGCII upon complex formation
was probed by comparing 15N−1H TROSY spectra of the free
and bound states. EGCII contains 38 leucine residues that were
used as probes to detect conformational changes. In the free
form, only a few resonances are observed (Figure 6b), which are
very intense, suggesting that some leucine residues are in highly
flexible regions whereas many others experience slower mobility,
on the millisecond to microsecond time scale, which results in
broadening of their resonances due to chemical exchange
processes. For the EGCII−6 complex, additional peaks with
more spectral dispersion are observed, suggesting rigidification of
regions of the enzyme. The effect of these conformational
changes on the solvent-accessible hydrophobic surface of EGCII
was monitored by ANSA, as an extrinsic fluorescence probe,
which interacts with the exposed hydrophobic areas of the
protein. The results show an 8-fold decrease in exposed protein
hydrophobicity between the enzyme in the free form and that
bound to compound 6, as determined from the changes in ANSA
fluorescence curve slopes (Figure 6c). The change in the
accessible hydrophobic surface is accompanied by a shift in the
protein equilibrium between the dimeric and monomeric state
toward the monomeric state, as demonstrated by the single
elution peak in SEC (Figure 6d). This suggests that the
dimerization is due to intermolecular interactions between the
exposed hydrophobic surfaces of the protein. Additionally, the
effect of complex formation on EGCII flexibility was tested by
trypsin digestion at 25 and 37 °C. At 25 °C, the EGCII−6
complex is more resistant to proteolytic activity, compared to the
protein free state, as shown by SDS−PAGE (Figure 7a). At 37
°C, the protein is still digested into two bands. However, these
two bands persist during the incubation time (Figure 7c),
contrary to what was observed for the free protein. This latter
peptic profile resembles that seen in the free state at 25 °C. The
peptic fragments were analyzed by Western blotting with an anti-
His tag antibody against the C-terminal histidine tag of EGCII.
The blot confirms that the lower band contains the C-terminal
fragment of the protein (Figure 7b). The same peptic pattern is
observed when the protein is labeled with activity-based probe 9.
The lower band is the part of the protein that contains covalently
bound ABP 9, presumably to the nucleophile Glu351 (Figure
7d). To investigate the EGCII−6 complex peptic fragment, we
analyzed the trypsin-cleaved and uncleaved EGCII samples by
SEC. Their chromatograms show the same elution volume for a
single peak, and also single bands are observed via native gel
electrophoresis (Figure 7e,f). These observations support the
conclusion that after digestion the two protein fragments stick
together forming a strong noncovalent complex. On SDS−
PAGE, two bands were observed due to the dissociation of the
complex under the denaturing conditions (Figure 7f). Therefore,
we could speculate that the hydrophobic interaction between the
two nicked fragments is the main force that keeps the complex
together. This hydrophobic interaction is likely enhanced by the
aliphatic tail of the irreversible inhibitors. On the basis of these
observations, we can conclude that in its complexed state EGCII
experiences pronounced conformational changes, as seen from
its 1H−15N HSQC−TROSY spectrum and the measured
accessible hydrophobic solvent. Additionally, an increase in
trypsin resistance was observed, although the trypsin cleavage
site remains accessible. This conformation of the protein may
H
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Figure 7. Rigidification of EGCII by compound 6. EGCII−6 complex peptic fragment pattern at 25 °C (a) and 37 °C (c) on 10% SDS−PAGE. (b) Anti-
histidine tag Western blot membrane of digested EGCII in its free state and bound to compound 6 or ABP 9. (d) Fluorescence image (Cy3) of the anti-
histidine tag blot showing the fluorescence of ABP. (e) Size exclusion chromatograms of trypsin-treated (red) and untreated (blue) EGCII−6complex.
The elution volumes of the peaks are indicated. (f) Untreated EGCII−6 complex and EGCII−6 treated with trypsin analyzed under native and
denaturing electrophoresis conditions.
represent a closed and rigid state that could be stabilized by the
hydrophobic interaction between the aliphatic tail of the
irreversible inhibitor and the hydrophobic part of the substrate
binding site.
protein adopts a more closed and compact conformation. An
exception is observed for compound 3, which could be due to its
bulky aromatic ring that may cause steric hindrance in the narrow
hydrophobic channel of the substrate binding pocket. On the
basis of these results, we can conclude that the shape and length
of the aliphatic tail, and therefore its molecular lipophilic
potential, are key factors for rigidification and stabilization of
EGCII.
Correlation between EGCII Stabilization and the
Lipophilic Potential of Cyclophellitols. A series of cyclo-
phellitol aziridines N-alkylated or N-acylated with different
lipophilic moieties (Figure 8a) were screened and the effects on
EGCII melting temperature monitored by CD. Compounds 1
and 2 have no effect on the EGCII apparent Tm, in contrast to
compounds 4−8, for which an increase in the apparent Tm was
observed with a maximal shift of 14.5 °C obtained with
compound 8, containing an adamantane moiety (Figure 8b). A
correlation between the molecular lipophilic potential of the
compounds and the increase in EGCII melting temperature is
observed. This correlation can be explained by the more compact
and rigid conformation adopted by the protein upon the
interaction with the cyclophellitol aliphatic tags.
Next, the solvent-accessible hydrophobic surfaces (SAHSs) of
EGCII−cyclophellitol/cyclophellitol aziridine complexes were
measured by ANSA (Figure 8c), and a decrease in EGCII SAHS
was observed with an increase in inactivator lipophilicity. To
highlight this correlation, the SAHS of the complexes is plotted
against the theoretical “log P” of the compounds (Figure 8d).
The plot suggests that with an increasing tag hydrophobicity, the
DISCUSSION
■
The interaction of a ligand binding protein with its cognate
substrate usually induces changes in the protein thermal stability,
resulting in a shift of the observed melting temperature (Tm).
This phenomenon is widely exploited as a tool to identify
potential binders for scientific and medicinal applications.32,33
For instance, this approach found successful application in the
identification of target inhibitors in drug screening and for
decrypting unknown protein functions. As an illustrative
example, ligand-induced protein stabilization was used to identify
inhibitors against human serine/threonine kinases. It was found
that compounds that enhanced protein thermostability have a
higher inhibition activity.34 Another example is the elucidation of
a protein function of an essential gene from Streptococcus
pneumoniae using a thermofluor screening assay.35 A library of
3000 compounds was screened against the unknown functional
I
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Figure 8. EGCII−cyclophellitol−aziridine derivative complexes. (a) Molecular lipophilic potential of cyclophellitol−aziridine derivatives as visualized
using MLP Tools, a PyMOL plugin.60(b) Fits of thermal denaturation curves of the different EGCII compound complexes, monitored by circular
dichroism at pH 5.5 (the colors agree with those in panels c and d, and experimental data points were omitted for the sake of clarity). (c) Effect of the
tested compounds on the relative EGCII solvent-accessible hydrophobic surface monitored by ANSA fluorescence. (d) Correlation between the
calculated log P of the different tested compounds and the relative solvent-accessible hydrophobic surface of EGCII compound complexes. The free
EGCII solvent-accessible hydrophobic surface was set to 100%.
protein, and two hits were found to increase the protein thermal
stability by 25 and 5 °C. The shifts of the protein melting
temperature correlated with the ligand dissociation constant (Kd
values of ≈50 pM and ≈2.5 μM, respectively). The protein was
identified as a nucleoside diphospho-keto-sugar aminotransfer-
ase.35
levels in Gaucher patient tissues, including brain.46 Additionally,
oral administration of IFG increased GBA L444P mutant activity
in mice up to 5-fold.7 Several lipophilic IFG derivatives proved to
have a higher affinity and chaperoning activity toward GBA
mutants.47−49 In connection to these findings, irreversible
inhibitor cyclophellitol and its aziridine derivatives were also
found to be effective mechanism-based ligands against retaining
β-glucosidases;50−52 therefore, they were used to study the
kinetics and structural basis of β-glucosidase inhibition.53
In the work presented here, on the basis of the similarity of the
structural topology between β-glucosidases GBA and EGCII as
well as of their target of catalysis, the β-glucosidic linkage in
glucosylceramide, we speculated that cyclophellitol derivatives
binding covalently to the catalytic nucleophile of GBA might also
react with EGCII. Our observation that EGCII is indeed
irreversibly inhibited by cyclophellitol derivatives opened new
possibilities for research on the enzyme. We investigated with
several biophysical and biochemical techniques the factors
influencing the conformational stability of EGCII, with emphasis
on the effect of occupancy of the active site by cyclophellitols.
Our investigation led to a number of findings warranting further
discussion.
Ligand-induced protein stabilization can also be attained by
changes of the protein conformational flexibility.36−38 By
studying the interaction between ANSA derivatives and BSA,
Celej and co-workers found that binding of ligand to the protein
induces an increase in protein conformational rigidity that
correlates with thermostability.38 Rigidification upon ligand
binding is used to screen for small compounds that assist protein
refolding and prevent their aggregation39 or for specific active site
binders that stabilize the folded state of a protein and improve its
functionality.40 The latter compounds are called pharmacological
chaperones and have potential application in drug discovery for
rescuing protein activity in several misfolding diseases.41−44 As a
representative example, a deficiency of GBA activity represents
the etiology of Gaucher disease, the most common lysosomal
storage disorder.45 This deficiency can be due to various point
mutations, destabilizing the protein fold and disrupting cellular
trafficking. It has been reported that active site binders restore to
some extent the activity of lysosomal retaining β-glucosidase
GBA mutants by stabilizing the folded conformation of the
protein. For instance, IFG increased GBA thermal stability at
neutral pH by 8.7 °C and improved protein trafficking of the
N370S GBA mutant, resulting in an increase in GBA cellular
The thermostability study of EGCII by circular dichroism
indicated that the protein has an apparent midpoint of the
unfolding transition of 42 °C with an irreversible unfolding
process, forming an aggregate as a final state. The isothermal
denaturation of EGCII at 37 °C, which represents its optimal
catalytic temperature, shows that the protein has a short half-life
J
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of 10 min, unlike what was observed at 25 °C where the protein
shows a remarkable kinetic stability. These findings differ from
those reported by Ito and co-workers. They found that the
protein has a thermal stability of 60 min at 37 °C at the same pH.1
An explanation for this discrepancy is presently not available; the
buffer conditions were not reported, so they may have deviated
from the ones used here. The noted thermolability of EGCII
could stem from its 3D structure containing several flexible
regions without a well-packed hydrophobic core.54 Analysis of
EGCII flexibility in solution by proteolysis shows that the protein
is susceptible to trypsin and has a major cleavage site localized in
an α-helix in the crystal structure. It has been reported that
protease cleavage sites for a variety of proteins of known 3D
structure never occur in α-helices, but largely in loops.55
Therefore, we speculate that this region in solution is either
highly flexible or locally disordered. In the presence of
detergents, this cleavage site becomes inaccessible to the trypsin
and adopts a more structured conformation, as observed in the
crystal structure of the protein. This local conformational
flexibility, in combination with the high positive charge in this
region, could play a crucial role in membrane association of
EGCII, required to exert its enzymatic activity toward
glycosphingolipids. The fact that EGCII processes a wide range
of substrates,24 which requires a high flexibility and plasticity of
the substrate binding site, may also explain its flexible behavior in
solution. Protein flexibility may also be important in protein−
protein complex formation. EGCII has an activator of 69 kDa
that increases the catalytic activity of the enzyme against
gangliosides in the absence of detergents under in vitro
conditions.56 It could be that protein flexibility is necessary for
protein−activator complex formation via an induced-fit mech-
anism. EGCII has a typical TIM (triose-phosphate isomerase)
barrel folding pattern with a (β/α)8 N-terminal domain and a β-
sheet C-terminal domain.27 The function of the β-sheet domain
is unclear, even though it is highly conserved in different families
of retaining β-glucosidases. We attempted to express only the
catalytic domain to no avail, and all of the produced protein was
found in inclusion bodies. This observation, combined with the
high flexibility of the catalytic domain, supports the hypothesis
that this domain not only is necessary for the stability of the
catalytic domain but also may play a crucial role as an internal
chaperone to guide the folding of the catalytic domain.
The dramatic stabilization and rigidification of EGCII upon
complex formation with the irreversible inhibitors are caused by
the hydrophobic interaction with their aliphatic tail. A correlation
between the molecular lipophilic potential of the tested
compounds and the decrease in the solvent-accessible hydro-
phobic surface was observed. When accommodated into the
enzyme binding pocket, the hydrophobic moieties of the
compound appear to function as a “hydrophobic zipper”. This
interaction makes the protein more rigid but with the
preservation of the trypsin cleavage site. After cleavage, the two
fragments were shown to stay together in a noncovalent complex,
mediated by the hydrophobic interactions with the aliphatic tail
of the irreversible inhibitor. A similar mechanism of binding
pocket occupancy effects was observed for the complex between
renin, an aspartic protease playing a key physiological role in the
regulation of blood pressure, and a nonpeptidomimetic inhibitor,
showing dramatic structural changes in renin. In this case, a
movement of protein flexible loops of the binding pocket was
observed and further changes in the protein around the bulky
lipophilic substitutes of the inhibitors were seen. The lipophilic
substitutes and hydrophobic side chains in renin act to form a
new compact hydrophobic region within the protein.57
A
stabilization and rigidification effect was also observed in the
case of the covalent modification of the β-(1−4)-glycosidase Cex
catalytic domain, from the soil bacterium Cellulomonas fimi, with
the mechanism-based inhibitors 2,4-dinitrophenyl 2-deoxy-2-
fluoro-β-cellobioside. The free state of the Cex catalytic domain
was markedly stabilized upon formation of the glycosyl−enzyme
intermediate with an increase in its Tm of 10.5 °C. This protein
stabilization was accompanied by a distinct thermolysin
proteolytic resistance, with no proteolysis of the glycosyl−
enzyme complex occurring, even after incubation for 50 h at 50
°C.58
In conclusion, we used EGCII as a research model to
investigate the effects of occupancy of the active site by
cyclophellitol analogues with variable lipophilic potential. The
hydrophobic interaction plays a decisive role in the protein−
inhibitor complex, promoting rigidification of the protein and
conformational stability. In theory, endoglycoceramidases could
be employed to treat a number of inherited glycosphingoli-
pidoses caused by deficiency in a lysosomal exoglycosidase.
Insight into factors influencing the conformational stability of
endoglycoceramidases is one important prerequisite for their
design as successful therapeutic agents.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.6b00363.
EGCII and GBA primary sequence alignment, readouts of
active EGCII molecules, tryptic digestion of EGCII at
different ratios, SEC−MALLS analysis of EGCII in
1
solution, and H and 13C NMR and LC−MS spectra of
compound 8 (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: m.ubbink@chem.leidenuniv.nl. Phone: +31 71
5274628.
■
Author Contributions
F.B.B., H.S.O., J.M.A., and M.U. conceived the study. F.B.B., J.J.,
W.K., A.d.H., B.I.F., B.B., R.B., H.S.O., and M.U. designed and
performed the experiments. F.B.B., J.M.A., and M.U. wrote the
manuscript. All authors reviewed the results and approved the
final version of the manuscript.
Funding
This work was supported by The Netherlands Organization for
Scientific Research (NWO) Grant 022.004.027 to H.S.O. J.J. was
supported by a China Scholarship Council fellowship.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Dr. E. R. Newman for technical assistance with the
SEC−MALLS experiment.
■
ABBREVIATIONS
■
EGCII, endoglyoceramidase II; GBA, glucocerebrosidase;
ANSA, 8-anilino-1-naphthalenesulfonic acid; IFG, isofagomine;
NN-DNJ, N-nonyldeoxynojirimycin; ABPs, activity-based
probes; TROSY, transverse relaxation optimized spectroscopy;
K
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catalytically active normal and Gaucher disease enzymes. J. Biol. Chem.
261, 8263−8269.
COSY, correlation spectroscopy; HSQC, heteronuclear single-
quantum correlation spectroscopy; 4MU-Lac, β-D-lactopyrano-
side; LC−MS, liquid chromatography−mass spectrometry; CD,
circular dichroism; SEC−MALLS, size exclusion chromatog-
raphy and multiangle laser light scattering; TLC, thin-layer
chromatography; MeCN, acetonitrile; TMS, tetramethylsilane;
SAHS, solvent-accessible hydrophobic surface.
(15) Brumshtein, B., Greenblatt, H. M., Butters, T. D., Shaaltiel, Y.,
Aviezer, D., Silman, I., Futerman, A. H., and Sussman, J. L. (2007)
Crystal Structures of Complexes of N-Butyl- and N-Nonyl-Deoxynojir-
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chemical chaperone action in gaucher disease. J. Biol. Chem. 282,
29052−29058.
(16) Jiang, J., Beenakker, T. J. M., Kallemeijn, W. W., van der Marel, G.
́
A., van den Elst, H., Codee, J. D. C., Aerts, J. M. F. G., and Overkleeft, H.
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