S uc u al Basis o Pha macological Chape oning o Human

-Galac osidase
*
□
S
Recei ed o publica ion, Oc obe 23, 2013, and in e ised o m, Ma ch 12, 2014 Published, JBC Pape s in P ess, Ap il 15, 2014, DOI 10.1074/jbc.M113.529529
Hi ono i Suzuki
‡
, Umeha u Oh o
‡
, Ka sumi Higaki
§
, Te esa Mena-Ba agán
¶
, Ma ilde Aguila -Moncayo
¶
,
Ca men O iz Melle
¶
, Eiji Nanba
§
, Jose M. Ga cia Fe nandez
储
, Yoshiyuki Suzuki**
1
, and Toshiyuki Shimizu
‡ ‡‡2
F om he
‡
G adua e School o Pha maceu ical Sciences, The Uni e si y o Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan,
he
§
Di ision o Func ional Genomics, Resea ch Cen e o Bioscience and Technology, To o i Uni e si y, 86 Nishi-cho, Yonago,
To o i 683-8503, Japan, he
¶
Depa men o O ganic Chemis y, Facul y o Chemis y, Uni e si y o Se ille, P o eso Ga cía
González 1, E-41012 Se ille, Spain, he
储
Ins i u e o Chemical Resea ch (IIQ), CSIC, Uni e si y o Se illa, Ame ico Vespucio 49, Isla de
la Ca uja, E-41092 Se illa, Spain, he **In e na ional Uni e si y o Heal h and Wel a e G adua e School, Ki a Kanema u, O awa a,
Tochigi 324-8501, Japan, and
‡‡
CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi,
Sai ama 332-0012, Japan
Backg ound: Pha macological chape one (PC) he apy has been p oposed o lysosomal s o age diseases.
Resul s: Wild ype and mu an

-galac osidases exhibi simila enzymological p ope ies. The ecogni ion mechanism o
glycomime ic PC candida es in ol es bo h suga -like and subs i uen moie ies.
Conclusion: C ys al s uc u es e eal he molecula basis o high binding po ency o PC compounds.
Signi icance: Enzymological p ope ies, binding a ini ies, and ecogni ion modes a e biophysically and s uc u ally
cha ac e ized.
G
M1
gangliosidosis and Mo quio B disease a e au osomal
ecessi e diseases caused by he de ec in he lysosomal

-galac-
osidase (

-Gal), equen ly ela ed o mis olding and subse-
quen endoplasmic e iculum-associa ed deg ada ion. Pha -
macological chape one (PC) he apy is a newly de eloped
molecula he apeu ic app oach by using small molecule ligands
o he mu an enzyme ha a e able o p omo e he co ec old-
ing and p e en endoplasmic e iculum-associa ed deg ada ion
and p omo e a icking o he lysosome. In his epo , we
desc ibe he enzymological p ope ies o pu i ied ecombinan
human

-Gal
WT
and wo ep esen a i e mu a ions in G
M1
gan-
gliosidosis Japanese pa ien s,

-Gal
R201C
and

-Gal
I51T
.We
ha e also e alua ed he PC e ec o wo compe i i e inhibi o s
o

-Gal. Mo eo e , we p o ide a de ailed a omic iew o he
ecogni ion mechanism o hese compounds in compa ison
wi h wo s uc u ally ela ed analogues. All compounds bind o
he ac i e si e o

-Gal wi h he suga -mimicking moie y making
hyd ogen bonds o ac i e si e esidues. Mo eo e , he binding
a ini y, he enzyme selec i i y, and he PC po en ial a e s ongly
a ec ed by he mono- o bicyclic s uc u e o he co e as well as
he o ien a ion, na u e, and leng h o he exocyclic subs i uen .
These esul s p o ide unde s anding on he mechanism o
ac ion o

-Gal selec i e chape oning by newly de eloped PC
compounds.
Human

-D-galac osidase (EC 3.2.1.23,

-Gal)
3
is a lyso-
somal hyd olase ha ca alyzes emo al o e minal

-linked
galac ose in G
M1
ganglioside and ke a an sul a e (1–3). In
humans, de iciency o

-Gal enzyme causes G
M1
gangliosidosis
and Mo quio B disease, wo lysosomal s o age diseases (LSDs)
cha ac e ized by he p og essi e accumula ion o me aboli es
in he cell (4–6). G
M1
gangliosidosis is a se e e neu odegene a-
i e disease ha is classi ied in o h ee ypes, in an ile, ju enile,
and adul , depending on he onse and se e i y (7). Mo quio B
disease is a a e bone disease wi hou cen al ne ous sys em
in ol emen (7). Cu en ly, mo e han 160 mu a ions in he
human

-Gal gene ha e been iden i ied as ac o s causa i e o
i s de iciency (8, 9).
Two p incipal ea men s a egies a e cu en ly app o ed o
in clinical ials o LSDs. The i s is enzyme eplacemen he -
apy, whe e he de icien enzyme is supplied by egula injec ion
o pu i ied ecombinan human enzyme (10–12). Howe e , li -
leo noimp o emen hasbeenobse edin hecen alne ous
sys em a ec a ions in LSD pa ien s because he enzyme canno
*This wo k was suppo ed by G an s-in-Aid om he Minis y o Educa ion,
Cul u e, Science, Spo s, and Technology o Japan ( o U. O. and T. S.) and
G an s 13680918 and 14207106 ( o Y. S.), Minis y o Heal h, Labo and
Wel a e o Japan H20-Koko o-022 and H22-Nanji-Ippan-002, and Japan
Science and Technology Agency G an AS232Z00009G ( o Y. S.), g an s
om he Takeda Science Founda ion and Mochida Memo ial Founda ion
o Medical and Pha maceu ical Resea ch ( o U. O. and T. S.), and Spanish
Minis e io de Economía y Compe i i idad con ac s SAF2010-15670 and
CTQ2010-15848, he Fundación Ramón A eces, he Jun a de Andalucía,
he Eu opean Regional De elopmen Funds (FEDER), he Eu pean Social
Funds (FSE), and he Cen e o Resea ch, Technology and Inno a ion o
he Uni e si y o Se ille (CITIUS).
□
S
This a icle con ains supplemen al da a and Figs. S1–S5.
The a omic coo dina es and s uc u e ac o s (codes 3WEZ, 3WF0, 3WEZ, 3WF0,
3WF1, 3WF2, 3WF3, and 3WF4) ha e been deposi ed in he P o ein Da a Bank
(h p://wwpdb.o g/).
1
P esen add ess: Tokyo Me opoli an Ins i u e o Medical Science, 2-1-6
Kami-Ki azawa, Se agaya-ku, Tokyo 156-8506, Japan.
2
To whom co espondence should be add essed: 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan. Tel.: 81-03-5841-4840; Fax: 81- 03-5841-4891;
E-mail: [email p o ec ed].
3
The abb e ia ions used a e:

-Gal,

-galac osidase; G
M1
, monosialo e a-
hexosylganglioside; LSD, lysosomal s o age disease; DGJ, 1-deoxygalac-
onoji imycin; PC, pha macological chape one; NOEV, N-oc yl-4-epi-

-
alienamine; 6S-NBI-DGJ, 5N,6S-(N⬘-bu yliminome hylidene)-6- hio-1-de-
oxygalac onoji imycin; 6S-NBI-GJ, 5N,6S-(N⬘-bu yliminome hylidene)-6-
hiogalac onoji imycin; NBT-DGJ, N-(N⬘-bu yl hioca bamoyl)-1-deoxy-
galac onoji imycin.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 21, pp. 14560–14568, May 23, 2014
© 2014 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. Published in he U.S.A.
14560 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014
This is an open access a icle unde he CC BY license.
c oss heblood-b ainba ie .Thesecondissubs a e educ ion
he apy, which uses an o ally a ailable small molecule ha
inhibi s he biosyn hesis o he glycosphingolipid (13). How-
e e , his ea men is nonspeci ic, leading o se ious side
e ec s. An al e na i e ea men , pha macological chape one
(PC) he apy, has been p oposed o G
M1
gangliosidosis,
Mo quio B disease, and o he LSDs (14). This he apy uses a
small molecule ligand ha can bind o he mu an p o ein and
s abilize he co ec con o ma ion o he p o ein a neu al pH
in he endoplasmic e iculum, allowing i o be anspo ed o
he lysosome whe e he ligand dissocia es a acidic pH and in
he p esence o excess subs a e.
Galac ose, he ca aly ic p oduc o
␣
-o

-Gal, he imino-
suga 1-deoxygalac onoji imycin (DGJ; Fig. 1), a mimic o
galac ose, and some de i a i es ha e been well s udied as PCs
o hese LSDs (15, 16). Howe e , DGJ has p omiscui y o a
numbe o galac opy anoside-p ocessing isoenzymes, which
may hampe clinical de elopmen .
We had p e iously epo ed he alienamine de i a i e
N-oc yl-4-epi-

- alienamine (NOEV) and he sp
2
-iminosuga
de i a i e 5N,6S-(N⬘-bu yliminome hylidene)-6- hio-1-deoxy-
galac onoji imycin (6S-NBI-DGJ; Fig. 1), as e ec i e PC com-
pounds ha imp o e human mu an

-Gal ac i i y in cul u ed
cells(8, 17–20).The e ec o NOEV and6S-NBI-DGJ on educ-
ing he accumula ion o G
M1
ganglioside was mu a ion speci ic.
In ac , NOEV educed he accumula ion in

-Gal
R201C
bu no
in

-Gal
I51T
cells, whe eas 6S-NBI-DGJ was e ec i e in bo h
cell lines (8, 20). On he o he hand, he inhibi o y ac i i y o
NOEV o e came he chape one ac i i y a mic omola concen-
a ions, whe eas he chape one e ec o 6S-NBI-DGJ s eadily
inc easedin adose-dependen manne up omilimola concen-
a ions (20). The p esence o an ex a pseudoanome ic
hyd oxyl in 5N,6S-(N⬘-bu yliminome hylidene)-6- hiogalac-
onoji imycin (6S-NBI-GJ; Fig. 1) esul s in a selec i i y shi
owa d

-glucosidase (

-glucoce eb osidase), which has been
exploi ed o he de elopmen o e icien pha macological
chape ones o Gauche disease (21–23). The monocyclic
N-(N⬘-bu yl hioca bamoyl)-1-deoxygalac onoji imycin (NBT-
DGJ;Fig. 1) de i a i e u ned ou o be ins ead a selec i e inhib-
i o o lysosomal
␣
-Gal wi h po en ial as a pha macological
chape one o Fab y disease (17).
We epo ed he c ys al s uc u e o human

-Gal (24). Ge -
ing mo e s uc u al in o ma ion on how his se o compounds
in e ac wi h human

-Gal and es ablishing PC䡠enzyme com-
plex s uc u e-ac i i y ela ionships p o ide use ul in o ma ion
abou he ea u esgo e ningchape one-enzymein e ac ionsa
he molecula le el and how hey could be implemen ed in he
design o new gene a ions o PC d ug candida es o G
M1
gangliosidosis.
In his s udy, we examined he enzymological p ope ies o
ecombinan human

-Gal
WT
and wo ep esen a i e mu a-
ions in Japanese pa ien s,

-Gal
R201C
and

-Gal
I51T
. To da e,
he chape one abili y o candida e compounds was e alua ed
agains lysa es om cul u ed human ib oblas s o ansien ly
ans ec ed cells. In his epo , all assays we e pe o med using
pu i ied ecombinan human

-Gal, so ha he chape one
e ec canbe es ima edwi hou anyin e e ence. Wealsode e -
mined c ys al s uc u es o he complexes o he s ong

-Gal
inhibi o s, NOEV and 6S-NBI-DGJ, and o he much weake
ligands 6S-NBI-GJ and NBT-DGJ bound o human

-Gal
WT
.

-Gal
I51T
mu an s uc u es complexed wi h galac ose o
6S-NBI-DGJ we e also de e mined.
EXPERIMENTAL PROCEDURES

-Gal Inhibi o s/Chape ones—The alienamine de i a i e
NOEV, and he h ee sp
2
-iminosuga de i a i es 6S-NBI-DGJ,
6S-NBI-GJ, and NBT-DGJ, we e syn hesized ollowing p e i-
ously epo ed p ocedu es (Fig. 1) (17, 20, 25, 26). All com-
pounds a e s able a oom empe a u e and soluble in wa e up
o 10 mM(NOEV and 6S-NBI-DGJ) o 20 mM(6S-NBI-GJ and
NBT-DGJ). The molecula weigh s a e 287.4, 260.4, 278.4, and
276.4, espec i ely. S ock solu ion was p epa ed in s e ile H
2
O
and s o ed a ⫺20 °C. NOEV and 6S-NBI-DGJ inhibi human

-Gal in i o wi h high selec i i y, whe eas 6S-NBI-GJ and
NBT-DGJ a e e y weak inhibi o s o

-Gal bu low mic omo-
la inhibi o s o

-glucoce eb osidase o
␣
-Gal, espec i ely.
Cloning, Exp ession, and Pu i ica ion o Human

-Gal—The
de ails o cloning, exp ession, and pu i ica ion o

-Gal we e
epo ed p e iously (27). Mu a ions we e in oduced by P ime-
STAR Max DNA polyme ase (Taka a) using oligonucleo ide
p ime s ollowing he manu ac u e ’s p o ocol. B ie ly,

-Gal
WT
and wo mu an p o eins,

-Gal
R201C
and

-Gal
I51T
( esidues 24–677), used o he N- e minal hexahis idine and
FLAG ag, we e exp essed in yeas Pichia pas o is KM71 and
pu i ied o homogenei y in a nickel-Sepha ose column. In he
cou se o pu i ica ion, polysaccha ide moie ies a ached o he
p o ein we e immed o by endoglycosidase H ea men . Fo
c ys alliza ion,

-Gals we e subjec ed o limi ed p o eolysis
wi h bo ine ypsin and u he pu i ied by ca ion-exchange
column ch oma og aphy, and Supe dex 200 size exclusion col-
umn (GE Heal hca e) equilib a ed wi h bu e A (0.01 MMES,
pH 6.0, 0.1 MNaCl). Finally, pu i ied

-Gals we e concen a ed
o 10 mg/ml in bu e A wi h o wi hou 10 mMgalac ose.
FIGURE 1. S uc u es o DGJ de i a i es and PC compounds discussed in
his s udy.
S uc u al Insigh s in o Pha macological Chape ones
MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14561
Kine ics o Pu i ied

-Gals and E alua ion o Inhibi o s—

-Gal ac i i y was measu ed by using 4-me hylumbelli e yll-

-
D-galac opy anoside in bu e B (0.15 Msodium ci a e, pH 4.5,
and 0.2 MNaCl) as a subs a e, and incuba ing a 37 °C o 6
min. The eac ion was e mina ed by adding 0.2 Mglycine-
NaOH bu e (pH 10.7). The libe a ed 4-me hylumbelli e yll
was measu ed wi h a luo escence pla e eade (exci a ion 355
nm; emission 460 nm; luo oskan ascen , The mo elec on
Co p.).
A kine ic expe imen on pu i ied

-Gal was pe o med using
a ious concen a ions (75–375 mM) o 4-me hylumbelli e yll-

-Gal as a subs a e o de e mine he maximum eloci y (V
max
)
and Michaelis cons an (K
m
) alues. Fo inhibi ion assays, he
ligand compounds (galac ose, 25 mM; DGJ, 250
M; NOEV, 2.5
M; 6S-NBI-DGJ, 250
M; 6S-NBI-GJ, 2.5 mM; and NBT-DGJ,
2.5 mM) we e added o he pu i ied

-Gal, and hen he ac i i y
o

-Gal was measu ed as desc ibed abo e. The inhibi ion con-
s an (K
i
) alues (Table 1) we e calcula ed om he plo s o
slope e sus inhibi o concen a ion.
E ec s o Pha macological Chape ones on he S abili y o he

-Gals—To compa e he s abili y o he

-Gal
WT
and ha o
mu an p o eins unde a ious pH condi ions in a bu e , 10
g/ml o

-Gal was incuba ed in ei he 0.1 Msodium ci a e
bu e (pH 3.5–7.0) o 0.1 MHepes bu e , pH 7.5–8.0, wi h 0.1
MNaCl a 48 °C o 40 min. A e incuba ion,

-Gal ac i i y was
assayed immedia ely unde pH 4.5. To assess he e ec s o PC
compounds on he s abili y o WT and mu an

-Gal, a ious
concen a ions(galac ose,0.2,2,and20mM;DGJ,2, 20,and 200
M; NOEV, 0.02, 0.2, and 2
M;6S-NBI-DGJ 2, 20, and 200
M)
o PC compounds we e added o 10
g/ml o

-Gal in bu e C
(0.1 Msodium ci a e, pH 7.0, and 0.1 MNaCl), ollowed by
incuba ion a 48 °C o a ious imes (0, 10, 20, and 40 min). As
a con ol, he same expe imen was ca ied ou wi h o wi hou
PCs (galac ose, 20 mM; DGJ, 200
M; NOEV, 2
M;6S-NBI-
DGJ, 200
M) unde he acidic condi ion (0.1 Msodium ci a e,
pH 4.8, and 0.1 MNaCl). Values a e a e ages o h ee indi idual
expe imen s.
CD Spec um—Ci cula dich oism (CD) spec a we e
eco ded a 20 °C on a Jasco J-720W spec opola ime e
equipped wi h a Julabo F25-ED empe a u e con olle . The
wild ype and wo mu an p o eins o

-Gal we e dilu ed o 0.1
mg/ml in 20 mMsodium ace a e, pH 5.0, wi h o wi hou galac-
ose (50 mM). CD spec a we e collec ed o e he 200–280 nm
wa eleng h ange and wi h a esolu ion o 0.1 nm, a bandwid h
o 1 nm, and a esponse ime o 1 s. Final spec a we e he sum
o 16 scans accumula ed a a speed o 50 nm/min.
Iso he mal Ti a ion Calo ime y—Iso he mal i a ion cal-
o ime e Mic oCal iTC200 was employed o de e mine he
a ini ies be ween

-Gal and he wo PCs, NOEV o 6S-NBI-
DGJ, a 25 °C. The calo ime ic cell was illed wi h 50 ( o
NOEV) o 100
M( o 6S-NBI-DGJ)

-Gal, and PCs (0.5 mM
NOEV and 1 mM6S-NBI-DGJ) we e injec ed in o he cell wi h
a 60-
l sy inge. The eleased hea was measu ed by in eg a ing
he calo ime ic ou pu cu es. Da a we e i wi h he model o
one se o binding si es in O igin (O iginLab).
C ys alliza ion and Da a Collec ion—C ys alliza ion expe i-
men s we e pe o med by si ing-d op apo di usion me hods
a 4 °C. C ys als o

-Gal
WT
䡠6S-NBI-DGJ complex we e g own
om he equi olume o 10 mg/ml o

-Gal
WT
in bu e A con-
aining1mM6S-NBI-DGJ and he ese oi solu ioncon aining
20–26% PEG3350, 0.2 Mammonium sul a e, and 0.1 MHepes,
pH 7.5. The o he s we e i s p epa ed as

-Gal
WT
o

-Gal
I51T
䡠galac ose complex c ys als, and hen he c ys als
we e soaked in o he solu ion con aining each PC compound.
C ys als o

-Gal
WT
o

-Gal
I51T
䡠galac ose complex we e
g own om he equi olume o 10 mg/ml o

-Gal
WT
o

-Gal
I51T
in bu e A con aining 10 mMgalac ose and he es-
e oi solu ion con aining 20–26% PEG3350, 0.2 Mammonium
sul a e, and 0.1 MHepes, pH 7.5. Then,

-Gal
WT
o

-Gal
I51T
-
galac ose c ys als we e ans e ed in o mo he liquo (25%
PEG3350, 0.2 Mammonium sul a e, and 0.1 MHepes, pH 7.5)
supplemen ed wi h PC compounds (NOEV o 6S-NBI-DGJ, 1
mM;6S-NBI-GJ o NBT-DGJ, 10 mM) and incuba ed o 3–55
min (NOEV, 55 min; 6S-NBI-DGJ, 40 min; 6S-NBI-GJ, 15 min;
NBT-DGJ, 3 min) a 4 °C.
Di ac ion da ase s we e collec ed a beamline BL-17A and
AR-NE3Aa he Pho on Fac o y (Tsukuba,Japan).P io o da a
collec ion, he c ys als o he each PC compound䡠

-Gal com-
plex we e soaked o a ew seconds in he ese oi solu ion
supplemen ed wi h he co esponding PC compound (galac-
ose 10 mM; NOEV and 6S-NBI-DGJ, 1 mM;6S-NBI-GJ and
NBT-DGJ, 2 mM) and 15% e hylene glycol, and lash-cooled o
95 K. The da ase s we e p ocessed wi h he HKL2000 package
(Table 2) (28).
S uc u e De e mina ion and C ys allog aphic Re inemen —
S uc u es o

-Gal
WT
complexed wi h PC compounds
(NOEV, 6S-NBI-DGJ, 6S-NBI-GJ, o NBT-DGJ) and

-Gal
I51T
complexed wi h galac ose o 6S-NBI-DGJ we e de e mined by
he molecula eplacemen me hod using he

-Gal
WT
-galac-
ose complex s uc u e (PDB code 3THC) as a sea ch model
and he p og am Mol ep implemen ed in he CCP4 sui e (29).
Modelbuilding andadjus men we eca iedou usingp og am
COOT (30). C ys allog aphic e inemen was pe o med using
he p og am REFMAC (31) implemen ed in he CCP4 package
un il he R ac o was con e ged (Table 2). The a omic coo di-
na es and s uc u e ac o s ha e been deposi ed in he P o ein
TABLE 1
Enzymological pa ame e s o he wild- ype and mu an

-Gals, and inhibi o y cons an alues o PC compounds
The enzyme ac i i y o

-Gal was luo ome ically de e mined wi h 4-me hylumbelli e yll-

-D-galac opy anoside as a subs a e.
K
i
Speci ic
ac i i y V
max
K
i
Galac ose DGJ NOEV 6S-NBI-DGJ 6S-NBI-GJ NBT-DGJ
mol/min/mg
M/min mMmM
M
WT 15.6 1.78 0.49 4.14 61.8 1.1 68.3 253.1 372.7
R201C 13.1 1.74 0.44 5.61 49.0 0.94 53.3 320.4 388.3
I51T 16.3 1.82 0.55 4.91 44.7 1.2 68.8 319.6 329.0
S uc u al Insigh s in o Pha macological Chape ones
14562 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014
Da a Bank (3WEZ (

-Gal
WT
-NOEV), 3WF0 (

-Gal
WT
-6S-
NBI-DGJ), 3WF1 (

-Gal
WT
-6S-NBI-GJ), 3WF2 (

-Gal
WT
-
NBT-DGJ), 3WF3, (

-Gal
I51T
-galac ose) and 3WF4 (

-Gal
I51T
-
6S-NBI-DGJ)) (supplemen al da a).
RESULTS
Enzymological P ope ies o

-Gal—The Michaelis-Men en
pa ame e so hepu i ied ecombinan

-Galwe ede e mined
(Table 1). The V
max
alues o

-Gal
WT
,

-Gal
R201C
, and

-Gal
I51T
we e1.8, 1.7, and 1.8
M/min,and he K
m
alueswe e
0.5, 0.4, and 0.6 mM, espec i ely. The esul s sugges ha he
wild ypeand mu an p o eins showsimila enzymeac i i yand
subs a e a ini y. These obse a ions u he suppo he
p omise o PCs capable o escuing he mu an p o ein om
endoplasmic e iculum-associa ed deg ada ion and p omo ing
a icking o he lysosome o he ea men o G
M1
gangli-
osidosis. The co esponding inhibi ion cons an (K
i
) alues a e
p esen ed in Table 1. Da a o D-galac ose and DGJ ha e been
collec ed o compa a i e pu poses. Galac ose is a e y weak
inhibi o , al hough DGJ shows 100- old s onge inhibi ion
han galac ose. NOEV exhibi ed he mos po en inhibi ion o

-Gal among he se ies, wi h K
i
close o 1
M.6S-NBI-DGJ
inhibi s

-Gal o he same deg ee as DGJ, whe eas he s uc u -
ally ela ed de i a i es 6S-NBI-GJ and NBT-DGJ showed much
weake inhibi o y p ope ies. Consis en wi h he deg ee o he
inhibi ion, a iso he mal i a ion calo ime y expe imen dem-
ons a ed ha NOEV exhibi ed a s ong binding a ini y wi h
he dissocia ion cons an o submic omola , whe eas 6S-NBI-
DGJ exhibi ed a ⬃100- old educed a ini y (supplemen al Fig.
S1). I should be no ed ha all he compounds inhibi ed WT
and mu an

-Gal wi h i ually iden ical po encies.
The S abili y o WT and Mu an

-Gal unde Va ious pH
Condi ions—The ac i i y o WT and mu an

-Gal a e hea
ea men (48 °C, 40 min) was examined unde a ious pH con-
di ions (Fig. 2). As p e iously epo ed (32), each

-Gal was
mo eac i e a acidic pH (4.5–5.5), whe eas he ca aly ic ac i i y
was g ea ly educed unde s ong acidic (pH ⬍3.5) and basic
(pH ⬎8.0) condi ions. When compa ing wild ype and mu an
enzymes, i was ound ha

-Gal
I51T
was only sligh ly mo e
sensi i e o hea -induced inac i a ion in he whole ange o pH
alues om 3.5 o 8.0, whe eas he ac i i y o

-Gal
R201C
was
signi ican ly educed unde a ious pH condi ions compa ed
wi h

-Gal
WT
. As a conclusion om hese esul s,

-Gal mu a-
ions associa ed wi h G
M1
gangliosidosis lead o he educ ion
o enzyme s abili ies bu do no lose he ca aly ic ac i i y a all,
sugges ing ha bo h

-Gal
R201C
and

-Gal
I51T
could p ocess
G
M1
ganglioside and p e en / educe i s accumula ion p o ided
hey a e p ope ly ans e ed o he lysosome wi h he help o
PCs.
TABLE 2
Da a collec ion and c ys allog aphic e inemen s a is ics

-Gal
WT WT WT WT I51T I51T
PC compound NOEV 6S-NBI-DGJ 6S-NBI-GJ NBT-DGJ Galac ose 6S-NBI-DGJ
Da a collec ion
X- ay sou ce PF-BL17A PF-BL17A PF-AR NE3A PF-AR NE3A PF-AR NE3A PF-AR NE3A
Wa eleng h (Å) 0.98000 0.98000 1.00000 1.00000 1.00000 1.00000
Resolu ion ange (Å) 50–2.1 (2.14–2.10) 50–2.2 (2.24–2.20) 50–2.0 (2.03–2.00) 50–2.3 (2.34–2.30) 50–2.2 (2.19–2.15) 50–2.3 (2.34–2.30)
Space g oup P2
1
P2
1
P2
1
P2
1
P2
1
P2
1
Uni cell pa ame e s
a(Å) 94.8 95.0 95.0 95.1 95.0 95.0
b(Å) 115.6 116.5 116.0 116.3 115.9 116.2
c(Å) 140.3 140.4 140.6 141.8 140.3 140.7

(°) 92.3 92.2 92.3 92.4 92.2 92.3
No. o obse ed e lec ions 709,280 367,282 743,452 597,215 515,585 505,251
No. o unique e lec ions 172,353 139,532 199,457 133,461 160,267 134,623
Redundancy 4.1 (3.8) 2.6 (2.5) 3.7 (3.6) 4.5 (4.5) 3.2 (3.3) 3.8 (3.7)
Comple eness (%) 98.6 (96.4) 90.4 (93.7) 97.5 (96.4) 98.3 (98.1) 96.4 (98.6) 99.1 (100.0)
A e age I/
(I) 16.7 (3.2) 9.0 (2.0) 15.8 (2.2) 13.5 (2.5) 15.5 (3.5) 11.5 (2.3)
R
me gea
0.136 (0.602) 0.148 (0.571) 0.128 (0.605) 0.171 (0.763) 0.154 (0.478) 0.173 (0.677)
Re inemen
Resolu ion ange (Å) 47.4–2.1 43.4–2.2 25.9–2.0 26.0–2.3 27.3–2.2 25.3–2.3
P o ein esidues 2,415 2,416 2,416 2,416 2,416 2,416
No. o a oms 19,291 19,302 19,305 19,243 19,280 19,263
Ligand 80 68 72 72 48 68
N-Ace ylglucosamine 224 224 224 224 224 224
Wa e molecules 1,513 1,248 1,596 1,093 1,429 1,241
SO
4
2⫺
/Cl
⫺
/EG
b
40/4/32 40/4/32 40/4/32 40/4/32 40/4/32 40/4/32
A e age B ac o (Å
2
) 22.9 21.9 28.8 32.0 28.9 29.0
R
wo kc
/R
eed
(%) 17.9/22.4 17.7/23.0 18.6/23.3 17.4/24.0 19.6/24.2 17.9/24.0
Roo mean squa e de ia ion
Bond leng h (Å) 0.012 0.013 0.014 0.016 0.014 0.016
Bond angles (°) 1.54 1.59 1.69 1.84 1.70 1.86
Ramachand an plo (%)
Fa o ed 96.6 96.6 97.3 96.0 96.5 96.1
Allowed 3.4 3.3 2.6 3.8 3.4 3.8
Disallowed 0.0 0.1 0.1 0.2 0.1 0.1
Values in pa en heses a e o he shell wi h he highes esolu ion.
a
R
me ge
(I)⫽⌺兩I⫺具I典兩/⌺I,whe e Iis he di ac ion in ensi y.
b
EG indica es e hylene glycole.
c
R⫽⌺兩F
o
⫺F
c
兩/⌺F
o
, whe e F
o
and F
c
a e he obse ed and calcula ed s uc u e ampli udes, espec i ely.
d
R
ee
is an R alue o a 5% subse o all e lec ions, bu was no used in he e inemen .
S uc u al Insigh s in o Pha macological Chape ones
MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14563
E ec s o he Pha macological Chape ones on he S abili y o
he

-Gal—Theabili yo aligand o p e en hea -induced inac-
i a ion o a gi en glycosidase has been p e iously used as an
indica ion o i s pha macological chape one po en ial (33). In
ou case, galac ose, DGJ, NOEV, and 6S-NBI-DGJ we e added
o

-Gal a neu al pH in bu e s, and hen he emaining
enzyme ac i i ies we e examined a e hea ea men (Fig. 3).
All compounds we e able o inc ease he esidual ac i i ies o
he

-Gal in a dose-dependen manne .

-Gal
WT
ac i i y was
dec eased o ⬃20% a e 40 min a 48 °C incuba ion, bu galac-
ose- ea ed

-Gal e ained ⬃70% ac i i y. In acco dance wi h
he inhibi o y ac i i y, NOEV- ea ed

-Gal, and DGJ and
6S-NBI-DGJ- ea ed

-Gal eached a simila ac i i y imp o e-
men a ⬃10,000- and ⬃100- old lowe concen a ions, espec-
i ely, compa ed wi h galac ose, sugges ing ha hese com-
pounds bea po en ial as PCs. Li le chape one e ec s we e
obse ed unde he acidic condi ion (supplemen al Fig. S2).
S uc u al Basis o Pha macological Chape one In e ac ion
wi h Human

-Gal—To gain a omic insigh in o he PC com-
pounds binding o

-Gal, we de e mined c ys al s uc u es o

-Gal
WT
complexed wi h NOEV, 6S-NBI-DGJ, 6S-NBI-GJ,
and NBT-DGJ. The o e all s uc u es o

-Gal emained
la gely unchanged among hese ou complexes and all s uc-
u es showed a s ong o e all ag eemen wi h galac ose-bound

-Gal
WT
(PDB code 3THC) (24) wi h oo mean squa e de ia-
ions anging om 0.17 o 0.26 Å.

-Galwas oldedin o h eedomains: he TIMba el domain,

-domain 1, and

-domain 2 (supplemen al Fig. S3). The galac-
ose mime ics we e embedded in he same ligand binding
pocke o he TIM ba el domain in

-Gal, wi h he alkyl chain
a he exocyclic ni ogen a om o ien ed owa d he en ance o
he ac i e si e pocke (Fig. 4, A–D). In he case o NOEV, all
hyd oxyl g oups made di ec hyd ogen bonds wi h

-Gal in a
simila manne o galac ose. In addi ion, a hyd ogen bond was
o med be ween he exocyclic N a om o NOEV and Glu-188 o

-Gal, s abilizing he o ien a ion o he oc yl chain. The hyd o-
phobic ail ex ended along he p o ein su ace consis ing o
Ty -485, T p-273, Leu-274, His-276, and Asn-321 (Fig. 4A).
The e minal me hyl g oup (C16 ca bon a om) was in con ac
wi h he CE1 a om o His-276, and CB and CG a oms o Asn-
321 (Fig. 5A).
Simila o NOEV, hyd oxyls OH2, OH3, and OH4 o he
sp
2
-iminosuga 6S-NBI-DGJ made di ec hyd ogen bonds wi h

-Gal (Fig. 4B). The exocyclic N a om o 6S-NBI-DGJ also
made a hyd ogen bond wi h Glu-188 o

-Gal, which o ien s
he bu yl chain owa d he hyd ophobic pocke lanked by Ty -
485 and T p-273 (Fig. 5B). Con a y o NOEV, o which he
endocyclic double-bond imposes a hal -chai con o ma ion a
he alienamine ing, he 6-membe ed ing o 6S-NBI-DGJ
exhibi s an almos ideal chai con o ma ion close o ha
encoun e ed o galac ose in he galac ose䡠

-Gal complex. The
5-membe ed ing is used o he 6-membe ed ing a an angle o
110° (supplemen al Fig. S4). Because 6S-NBI-DGJ lacks he p i-
ma y hyd oxyl equi alen o he OH6 g oup in galac ose, he
side chain o ien a ion o Ty -333 is shi ed so as o ill he co -
esponding space. This ea u e is unique o he 6S-NBI-DGJ䡠

-
Gal complex s uc u e.
The p esence o a pseudoanome ic hyd oxyl g oup, OH1, in
he galac onoji imycin analogue 6S-NBI-GJ led o a e y signi -
ican dec easein hebindinga ini y owa d

-Galascompa ed
wi h he DGJ congene 6S-NBI-DGJ. Ac ually his compound is
a a he selec i e inhibi o o

-glucosidase (21). Whe eas
6S-NBI-GJ has been shown o exis exclusi ely in he
4
C
1
chai
con o ma ion in wa e solu ion, wi h OH1 axially o ien ed in
he
␣
-con igu a ion (34, 35), in he co esponding complex
wi h

-Gal he opposi e

-con igu a ionwas encoun e ed, wi h
he 6-membe ed ing and he 5-membe ed ing in he same
plane(supplemen alFig.S4).The

-o ien edOH1g oupisnow
in ol ed in hyd ogen bonding wi h Glu-188 (Fig. 4C), wi h he
bu yl chain ex ended o Ty -485 and T p-273 (Fig. 5B).
The monocyclic de i a i e NBT-DGJ eco e ed he hyd o-
gen bonding in e ac ion be ween he p ima y hyd oxyl OH6
and Ty -333 (Fig. 4D). Ac ually, all hyd oxyl g oups o NBT-
DGJ made di ec hyd ogen bonds wi h

-Gal as in he
NOEV䡠

-Gal complex. Howe e , he exocyclic N and S a oms
had no in e ac ions wi h

-Gal, which is in ag eemen wi h he
weak binding a ini y obse ed in he kine ic inhibi ion s udies.
We also de e mined c ys al s uc u es o

-Gal
I51T
com-
plexed wi h galac ose o 6S-NBI-DGJ (Fig. 6). The quali y o he
elec on densi y maps is su icien ly high o allow modeling o
he SNP esidue (Fig. 6B). These o e all s uc u es a e essen-
ially iden ical o hose o

-Gal
WT
bound o he co esponding
ligand. In ac , he main chain a oms in each complex could be
supe imposed on hose o he galac ose o 6S-NBI-DGJ com-
plex wi h oo mean squa e de ia ions o 0.16 and 0.17 Å,
espec i ely. CD spec um demons a ed ha s uc u es o he
wild ype and mu an p o eins a e essen ially he same ega d-
less o he p esence o absence o he PC compounds (supple-
men al Fig. S5).
DISCUSSION
In his s udy, we de e mined he enzymological p ope ies o
pu i ied ecombinan human

-Gal (

-Gal
WT
) and wo ep e-
sen a i e mu an p o eins,

-Gal
R201C
and

-Gal
I51T
. Mo e-
o e , we de e mined he c ys al s uc u es o

-Gal
WT
com-
plexed wi h ou ligand compounds, wo o which, NOEV and
6S-NBI-DGJ, ha e shown pha macological chape one ac i i y
FIGURE 2. Enzyma ic ac i i y o he WT and wo mu an p o eins o

-Gal
unde a ious pH condi ions. The pu i ied WT and wo mu an p o eins o

-Gal we e incuba ed unde he indica ed pH condi ions in a bu e a 48 °C
o 40 min. Then, enzyme assay was pe o med unde pH 4.5 condi ions. The
esidual enzyme ac i i y is shown as a pe cen age o he alues a he ime 0.
Values a e a e ages o h ee indi idual expe imen s and exp essed as he
pe cen a io o he ac i i y a a pa icula pH condi ion o he alue a he
ime 0.
S uc u al Insigh s in o Pha macological Chape ones
14564 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014
o se e al G
M1
gangliosidosis-associa ed mu a ions, whe eas
he o he wo, 6S-NBI-GJ and NBT-DGJ, showed only weak
a ini y o he enzyme despi eha ing a s uc u e closely ela ed
o ha o 6S-NBI-DGJ. C ys al s uc u es o

-Gal
I51T
com-
plexed wi h galac ose o 6S-NBI-DGJ ha e also been de e -
mined. Al hough NOEV and 6S-NBI-DGJ exhibi mic omola
and ens o mic omola K
i
alues, hese compounds we e
shown o signi ican ly enhance he

-Gal ac i i ies (up o
6- old) in G
M1
ib oblas s, demons a ing hei e icacy in i o.
I was also demons a ed ha he PC compounds amelio a ed
accumula ion o G
M1
ganglioside in a mouse model (human

-Gal
R201C
), including he b ain, a e o al adminis a ion (8,
18, 20).
The Ile-51 mu a ion is loca ed in he inne egion o

-Gal,
whe eas he A g-201 mu a ion is loca ed ins ead on he la e al
ace o he TIM ba el domain, being exposed o he sol en . In
bo h he A g-201 and Ile-51 mu an p o eins, he mu a ed
amino acid is a om he ac i e si e, indica ing ha he mu a-
ions a e unlikely o a ec he ac i e si e di ec ly. Acco dingly,

-Gal
I51T
and

-Gal
R201C
showed simila enzymological
pa ame e s as

-Gal
WT
(Table 1). This coincides wi h he ac
ha no signi ican con o ma ional change in

-Gal
I51T
was
obse ed in he c ys al s uc u e o he mu an p o ein as com-
pa ed wi h he wild ype enzyme (Fig. 6A). By con as ,

-Gal
R201C
was mo e uns able han

-Gal
WT
and

-Gal
I51T
unde a ious pH condi ions (Fig. 2). A g-201 o ms a sal
b idge o Asp-198 (24) and he loss o his sal b idge p obably
a ec s he s abili y o he p o ein, inc easing dena u a ion p o-
pensi y. These enzymological p ope ies, ob ained using
ecombinan

-Gal, we e consis en wi h p e iously epo ed
da a (32).
The s uc u al s udy p o ides an a omic basis o he binding
mechanism o ac i e si e-di ec ed pha macological chape ones
o

-Gal, by highligh ing he impo ance o he suga -like agly-
con moie y, he na u e o he exocyclic subs i uen and he con-
o ma ional p ope ies o he ligand. NOEV and 6S-NBI-DGJ
we e ecognized in a simila manne ; howe e , NOEV was a
igh binding inhibi o , whe eas 6S-NBI-DGJ binds ⬎60- old
weake (Table 1). This high a ini y o NOEV esul s om he
exocyclic N a om ecogni ion, he leng h and he o ien a ion o
he ex ended hyd ophobic ail and hal -chai con o ma ion
imposedby hedouble bond.The sp
2
-iminosuga ype inhibi o
6S-NBI-DGJ is cha ac e ized by a igid bicyclic co e de i ed
om DGJ. 6S-NBI-DGJ lacks OH6, exhibi s a di e en leng h
and o ien a ion o he hyd ophobic ail, and adop s he chai
con o ma ion, esul ing in he weake a ini y. As a conse-
FIGURE 3. E ec s o PC compounds on he s abili y o he WT and wo mu an p o eins o

-Gals a pH 7.0. The pu i ied WT and wo mu an p o eins o

-Gal we e incuba ed in 0.1 Msodium ci a e, pH 7.0, wi h o wi hou galac ose, DGJ, NOEV, and 6S-NBI-DGJ a 48 °C o he incuba ed imes. Then, he enzyme
assay was pe o med a pH 4.5. The esidual enzyme ac i i y is shown as a pe cen age o he alues a he ime 0. Values a e a e ages o h ee indi idual
expe imen s and a e exp essed as he pe cen a io o he ac i i y a a pa icula pH condi ion o he alue a he ime 0.
S uc u al Insigh s in o Pha macological Chape ones
MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14565
quence, he co esponding hyd ogen bond in e ac ion wi h
Ty -333 is missing, which is expec ed o ha e a de imen al
impac in complex s abili y. No ewo hy, his scena io leads o
a shi o Ty -333 in he 6S-NBI-DGJ䡠

-Gal complex, occupy-
ing he space whe e OH6 is loca ed in he co esponding com-
plex wi h NOEV, e ealing a ce ain deg ee o lexibili y a his
egion o

-Gal. The sho ened alkyl subs i uen in 6S-NBI-
DGJ as compa ed wi h NOEV, bu yl ins ead o oc yl, is also
expec ed o a ec he binding a ini y. In ac , N-alkyl-4-epi-

-
alienamine de i a i es wi h longe alkyl chains han NOEV
ha e been shown o exhibi highe a ini y o bo ine

-Gal (36),
whe eas N-hexyl-4-epi-

- alienamine had 3- old weake a in-
i y han NOEV (14). Consis en ly, he N⬘-oc yl analogue o
6S-NBI-DGJ was also ound o be a 4- old s onge inhibi o o
bo ine

-Gal han 6S-NBI-DGJ, bu i was disca ded o PC
chape one s udies wi h he human lysosomal enzyme due o
oxici y issues (17). P obably he longe alkyl chain in e ac s
wi h he neu al g oo e, o med by His-276, Asn-321, P o-323,
and Ala-325, in a wide su ace a ea han he bu yl chain in
6S-NBI-DGJ, he eby inc easing he binding a ini y (Fig. 5).
Unlike he oc yl chain in NOEV, he bu yl chain does no
ex end o e he neu al g oo e a he en ance o he ac i e si e
FIGURE 4. Binding o PC compounds o he ac i e si e o human

-Gal. A–D,le , PC compounds bound o

-Gal iewed om he en ance o ac i e si e
pocke : A, NOEV; B, 6S-NBI-DGJ; C, 6S-NBI-GJ; D, NBT-DGJ. Hyd ogen bonds de ined by ccp4 mg (37) a e shown by dashed lines.InB, a unique side chain
a angemen o Ty -333 in he 6S-NBI-DGJ complex is shown in c imson. The F
o
⫺F
c
elec on densi y maps o PC compounds a e shown in g ay.Aand Ba e
con ou ed a 2.0
,Ca 1.0
, and Da 1.5
.Righ , schema ic ep esen a ion o he in e ac ion be ween

-Gal and each PC compound: A, NOEV; B, 6S-NBI-DGJ;
C, 6S-NBI-GJ; and D, NBT-DGJ. Hyd ogen bonds a e shown as do ed lines, and an de Waals in e ac ions a e shown by a cs.
FIGURE 5. Compa ison o he binding o he PC compounds. Elec os a ic po en ial su ace o he su ounding o he ac i e si e pocke . A, NOEV (o ange)
complex s uc u e is supe imposed on NBT-DGJ (g ay) complex. B, 6S-NBI-DGJ (g een) and 6S-NBI-GJ (cyan) a e d awn in a simila manne as in A.
S uc u al Insigh s in o Pha macological Chape ones
14566 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014
in

-Gal (Fig. 5B), which in pa is esponsible o i s mode a e
binding a ini y o

-Gal.
The p esen s udy e ealed ha he s abili y o he mu an
p o eins (R201C, I51T) coincides wi h he se e i y o he dis-
ease and he PC e ec owa d

-Gal
WT
and

-Gal
R201C
a e well
consis en wi h he p e ious da a (8, 20). Howe e , NOEV has
been shown o exhibi di e ences in hei PC e ec s owa d
some

-Gal mu an p o eins in i o (20). This beha io does
no co ela e, howe e , wi h he p esen da a on he s abiliza-
ion e ec owa d hea -induced inac i a ion o pu i ied ecom-
binan

-Gal,whe e NOEV p o ed mo ee icien han 6S-NBI-
DGJ i espec i e o he mu a ion. Mu an enzyme escued by
PCs is a complex p ocess ha also in ol es s abiliza ion o he
al eady olded enzyme o p omo e a icking and ma u a ion.
How close a e he olded s a es is p obably s ongly mu a ion-
dependen , which may explain he abo e disc epancy be ween
in i o and cellulo esul s. Fu he in es iga ion will be
equi ed.
The hyd ogen bond in e ac ions in ol ing OH2, OH3, and
OH4 o he suga -like moie y and wo glu amic acid esidues
(Glu-129 and Glu-268) a he ac i e si e o

-Gal a e c i ical o
binding in bo h he NOEV and 6S-NBI-DGJ complexes and
likely de ine he D-galac o con igu a ional selec i i y o he
enzyme. A hi d glu amic acid, Glu-188, in e ac s wi h he exo-
cyclic basic ni ogen a om. This hyd ogen bond con ibu es
subs an ially o

-Galbinding a ini y and i s absence is likely o
be a he o igin o he much lowe inhibi o y po encies o he
s uc u ally ela ed sp
2
-iminosuga s 6S-NBI-GJ and NBT-DGJ
as well as o he monosaccha ide galac ose. The weakening o
he

-Gal binding a ini y o NOEV and 6S-NBI-DGJ obse ed
a acidic pH (8, 17) can be a ionalized in e ms o he expec ed
dec ease in he s eng h o he key hyd ogen bonds a e p o o-
na ion o he glu amic acid esidues in he p o ein and he basic
ni ogen unc ionali y in he chape one.
The sha p dec ease in

-Gal binding a ini y o 6S-NBI-GJ
and NBT-DGJ as compa ed wi h 6S-NBI-DGJ u he illus-
a es he s ong dependenceo he inhibi o y/chape oneac i -
i y o sp
2
-iminosuga glycomime ics on sub le chemical modi-
ica ions. The indings p esen ed he e may be pa icula ly
use ul o he a ional design o second gene a ion PCs o he
ea men o he mu an

-Gal-associa ed LSDs G
M1
gangli-
osidosis and Mo quio B disease. Thus, acco ding o he x- ay
da a, s uc u al changes a he exocyclic subs i uen in he
6S-NBI-GJ sca old a e expec ed o be ole a ed by he enzyme,
o e ing po en ial o d ug op imiza ion. In addi ion, he inco -
po a ion o an app op ia e subs i uen a he i e-membe ed
ing me hylene ca bon migh lead o a o able in e ac ions
wi h Ty -333, which could be exploi ed in he design o highe
a ini y ligands. Resea ch in ha di ec ion is cu en ly sough in
ou labo a o ies.
Acknowledgmen —We hank he beamline s a s a he Pho on Fac-
o y o assis ance wi h da a collec ion.
REFERENCES
1. Alpe s, D. H. (1969) On

-galac osidase ac i i y. Gas oen e ology 56,
985–986
2. Asp, N. G., and Dahlq is , A. (1972) Human small in es ine-galac osi-
dases: speci ic assay o h ee di e en enzymes. Anal. Biochem. 47,
527–538
3. Dis le , J. J., and Jou dian, G. W. (1973) The pu i ica ion and p ope ies o

-galac osidase om bo ine es es. J. Biol. Chem. 248, 6772–6780
4. Callahan, J. W. (1999) Molecula basis o G
M1
gangliosidosis and Mo quio
disease, ype B: s uc u e- unc ion s udies o lysosomal

-galac osidase
and he non-lysosomal

-galac osidase-like p o ein. Biochim. Biophys.
Ac a 1455, 85–103
5. Oshima, A., Yoshida, K., Shimmo o, M., Fukuha a, Y., Saku aba, H., and
Suzuki, Y. (1991) Human

-galac osidase gene mu a ions in Mo quio B
disease. Am. J. Hum. Gene . 49, 1091–1093
6. Yoshida, K., Oshima, A., Shimmo o, M., Fukuha a, Y., Saku aba, H.,
Yanagisawa, N., and Suzuki, Y. (1991) Human

-galac osidase gene mu-
a ions in G
M1
-gangliosidosis: a common mu a ion among Japanese adul /
ch onic cases. Am. J. Hum. Gene . 49, 435–442
7. Suzuki, Y., Nanba, E., Ma suda, J., Higaki, K., and Oshima, A. (2013)

-Ga-
lac osidase de iciency (

-galac osidosis): G
M1
-gangliosidosis and
Mo quio B disease in The Online Me abolic and Molecula Bases o Inhe -
i ed Disease (Valle, D., Beaude , A. L., Vogels ein, B., Kinzle , K. W., An-
ona akis, S. F., and Ballabio, A., eds) Chap e 151, McG aw-Hill, New
Yo k
8. Higaki, K., Li, L., Bah udin, U., Okuzawa, S., Takamu am, A., Yamamo o,
K., Adachi, K., Pa aguison, R. C., Takai, T., Ikeha a, H., Tominaga, L.,
Hisa ome, I., Iida, M., Ogawa, S., Ma suda, J., Ninomiya, H., Sakakiba a, Y.,
Ohno, K., Suzuki, Y., and Nanba, E. (2011) Chemical chape one he apy:
FIGURE 6. S uc u es o

-Gal
I51T
complexed wi h galac ose o 6S-NBI-DGJ. A, supe imposi ion o he o e all s uc u es o he

-Gal
WT
-galac ose (magen a),

-Gal
WT
-6S-NBI-DGJ (g een),

-Gal
I51T
-galac ose ( ed), and

-Gal
I51T
-6S-NBI-DGJ (cyan). Mu a ed esidue I51T is shown in space- illing ep esen a ion. B, he F
o
⫺F
c
elec on densi y map (g ay) co esponding o mu a ed esidue Th -51 o

-Gal
I51T
-6S-NBI-DGJ is con ou ed a 3.0
. Shown is he Ile-51 esidue o

-Gal
WT
-6S-NBI-DGJ (g een) and he Th -51 esidue o

-Gal
I51T
-6S-NBI-DGJ (cyan).
S uc u al Insigh s in o Pha macological Chape ones
MAY 23, 2014•VOLUME 289•NUMBER 21 JOURNAL OF BIOLOGICAL CHEMISTRY 14567
chape one e ec on mu an enzyme and cellula pa hophysiology in

-ga-
lac osidase de iciency. Hum. Mu a . 32, 843–852
9. Ho e , D., Paul, K., Fan u , K., Beck, M., Bü ge , F., Caillaud, C., Fumic, K.,
Led ino a, J., Lugowska, A., Michelakakis, H., Rade a, B., Ramaswami, U.,
Plecko, B., and Paschke, E. (2009) G
M1
gangliosidosis and Mo quio B dis-
ease: exp ession analysis o missense mu a ions a ec ing he ca aly ic si e
o acid

-galac osidase. Hum. Mu a . 30, 1214–1221
10. B ady, R. O. (2006) Enzyme eplacemen o lysosomal diseases. Annu.
Re . Med. 57, 283–296
11. Desnick, R. J., and Schuchman, E. H. (2012) Enzyme eplacemen he apy
o lysosomal diseases: lessons om 20 yea s o expe ience and emaining
challenges. Annu. Re . Genomics Hum. Gene . 13, 307–335
12. Pa en i, G. (2009) T ea ing lysosomal s o age diseases wi h pha macolog-
ical chape ones: om concep o clinics. EMBO Mol. Med. 1, 268–279
13. Pla , F. M., and Jeyakuma , M. (2008) Subs a e educ ion he apy. Ac a
Paedia . 97, 88–93
14. Fan, J. Q. (2008) A coun e in ui i e app oach o ea enzyme de iciencies:
use o enzyme inhibi o s o es o ing mu an enzyme ac i i y. Biol. Chem.
389, 1–11
15. Guce, A. I., Cla k, N. E., Rogich, J. J., and Ga man, S. C. (2011) The mo-
lecula basis o pha macological chape oning in human
␣
-galac osidase.
Chem. Biol. 18, 1521–1526
16. Suzuki, Y. (2013) Chape one he apy upda e: Fab y disease, G
M1
-gangli-
osidosis and Gauche disease. B ain De . 35, 515–523
17. Aguila -Moncayo, M., Takai, T., Higaki, K., Mena-Ba agán, T., Hi ano,
Y., Yu a, K., Li, L., Yu, Y., Ninomiya, H., Ga cía-Mo eno, M. I., Ishii, S.,
Sakakiba a, Y., Ohno, K., Nanba, E., O iz Melle , C., Ga cía Fe nandez,
J. M., and Suzuki, Y. (2012) Tuning glycosidase inhibi ion h ough agly-
cone in e ac ions: pha macological chape ones o Fab y disease and G
M1
gangliosidosis. Chem. Commun. 48, 6514–6516
18. Ma suda, J., Suzuki, O., Oshima, A., Yamamo o, Y., Noguchi, A., Ta-
kimo o, K., I oh, M., Ma suzaki, Y., Yasuda, Y., Ogawa, S., Saka a, Y.,
Nanba, E., Higaki, K., Ogawa, Y., Tominaga, L., Ohno, K., Iwasaki, H.,
Wa anabe, H., B ady, R. O., and Suzuki, Y. (2003) Chemical chape one
he apy o b ain pa hology in G
M1
-gangliosidosis. P oc. Na l. Acad. Sci.
U.S.A. 100, 15912–15917
19. Ogawa, S., Ma sunaga, Y. K., and Suzuki, Y. (2002) Chemical modi ica ion
o he

-glucoce eb osidase inhibi o N-oc yl-

- alienamine: syn hesis
and biological e alua ion o 4-epime ic and 4-O-(

-D-galac opy anosyl)
de i a i es. Bioo g. Med. Chem. 10, 1967–1972
20. Takai, T., Higaki, K., Aguila -Moncayo, M., Mena-Ba agán, T., Hi ano,
Y., Yu a, K., Yu, L., Ninomiya, H., Ga cía-Mo eno, M. I., Sakakiba a, Y.,
Ohno, K., Nanba, E., O iz Melle , C., Ga cía Fe nández, J. M., and Suzuki,
Y. (2013) A bicyclic 1-deoxygalac onoji imycin de i a i e as a no el pha -
macological chape one o G
M1
gangliosidosis. Mol. The . 21, 526–532
21. Aguila -Moncayo, M., Ga cía-Mo eno, M. I., T ape o, A., Egido-Gabás,
M., Lleba ia, A., Ga cı´a Fe na´ndez, J. M., and O iz Melle , C. (2011) Bi-
cyclic (galac o)noji imycin analogues as glycosidase inhibi o s: e ec o
s uc u al modi ica ions in hei pha macological chape one po en ial o-
wa ds

-glucoce eb osidase. O g. Biomol. Chem. 9, 3698–3713
22. Beni o, J. M., Ga cía Fe nández, J. M., and O iz Melle , C. (2011) Pha -
macological chape one he apy o Gauche disease: a pa en e iew. Ex-
pe Opin. The . Pa . 21, 885–903
23. Luan, Z., Higaki, K., Aguila -Moncayo, M., Ninomiya, H., Ohno, K.,
Ga cía-Mo eno, M. I., O iz Melle , C., Ga cía Fe nández, J. M., and Su-
zuki, Y. (2009) Chape one ac i i y o bicyclic noji imycin analogues o
Gauche mu a ions in compa ison wi h N-(n-nonyl)deoxynoji imycin.
ChemBioChem 10, 2780–2792
24. Oh o, U., Usui, K., Ochi, T., Yuki, K., Sa ow, Y., and Shimizu, T. (2012)
C ys al s uc u e o human

-galac osidase: s uc u al basis o G
M1
gan-
gliosidosis and mo quio B diseases. J. Biol. Chem. 287, 1801–1812
25. Aguila , M., Glos e , T. M., Ga cía-Mo eno, M. I., O iz Melle , C., Da ies,
G. J., Lleba ia, A., Casas, J., Egido-Gabás, M., and Ga cía Fe nandez, J. M.
(2008) Molecula basis o

-glucosidase inhibi ion by ing-modi ied ca-
lys egine analoges. ChemBioChem 9, 2612–2618
26. Kuno, S., Takahashi, A., and Ogawa, S. (2013) Concise syn heses o po en
chape one d ug candida es, N-oc yl-4-epi-

- alinenamine (NOEV) and
i s 6-deoxy de i a i e, om (⫹)-p o o-que ci ol. Ca bohyd . Res. 368,
8–15
27. Usui, K., Oh o, U., Ochi, T., Shimizu, T., and Sa ow, Y. (2012) Exp ession,
pu i ica ion, c ys alliza ion and p elimina y X- ay c ys allog aphic analy-
sis o human

-galac osidase. Ac a C ys allog . Sec . F S uc . Biol. C ys .
Commun. 68, 73–77
28. O winowski, Z., and Mino , W. (1997) P ocessing o X- ay di ac ion da a
collec ed in oscilla ion mode. Me hods Enzymol. 276, 307–326
29. Collabo a i e Compu a ional P ojec , Numbe 4. (1994) The CCP4 sui e:
p og ams o p o ein c ys allog aphy. Ac a C ys allog . D Biol. C ys allog .
50, 760–763
30. Emsley, P., and Cow an, K. (2004) Coo : model-building ools o molec-
ula g aphics. Ac a C ys allog . D Biol. C ys allog . 60, 2126–2132
31. Mu shudo , G. N., Vagin, A. A., and Dodson, E. J. (1997) Re inemen o
mac omolecula s uc u es by he maximum-likelihood me hod. Ac a
C ys allog . D Biol. C ys allog . 53, 240–255
32. Coelho, C. J., Spopelsa, A. M., Tobo, P. R., Se e ini, M. H., Sil a, C. D., and
Giugliani, R. (1999) Biochemical s udies on leukocy e and ib oblas hu-
man

-galac osidase. Clin. Biochem. 32, 1801–1812
33. Sawka , A. R., Cheng, W. C., Beu le , E., Wong, C. H., Balch, W. E., and
Kelly, J. W. (2002) Chemical chape ones inc ease he cellula ac i i y o
N370S

-glucosidase: a he apeu ic s a egy o Gauche disease. P oc.
Na l. Acad. Sci. U.S.A. 99, 15428–15433
34. Díaz Pé ez, V. M., Ga cía Mo eno, M. I., O iz Melle , C., Fuen es, J., Díaz
A ibas, J. C., Cañada, F. J., and Ga cía Fe nández, J. M. (2000) Gene alized
anome ic e ec in ac ion: syn hesis and e alua ion o s able educing in-
dolizidine glycomime ics as glycosidase inhibi o s. J. O g. Chem. 65,
136–143
35. Sánchez-Fe nández, E. M., Rísquez-Cuad o, R., O iz Melle , C., Ga cía
Fe nández, J. M., Nie o, P. M., and Angulo, J. (2012) sp
2
-Iminosuga O-, S-,
and N-glycosides as con o ma ional mimics o
␣
-linked disaccha ides:
implica ions o glycosidase inhibi ion. Chem. Eu . J. 18, 8527–8539
36. Suzuki, Y., Ogawa, S., and Sakakiba a, Y. (2009) Chape one he apy o
neu onopa hic lysosomal diseases: compe i i e inhibi o s as chemical
chape ones o enhancemen o mu an enzyme ac i i ies. Pe spec . Med.
Chem. 3, 7–19
37. McNicholas, S., Po e on, E., Wilson, K. S., and Noble, M. E. (2011) P e-
sen ing you s uc u es: he CCP4mg molecula -g aphics so wa e. Ac a
C ys allog . D Biol. C ys allog . 67, 386–394
S uc u al Insigh s in o Pha macological Chape ones
14568 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289•NUMBER 21•MAY 23, 2014
