Probing the substrate specificity of golgi α-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant

Article abstract of DOI:10.1021/ja711248y

Inhibition of Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an α(1,3)- or α(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the α(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B_2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the α(1,6)-linked Man of the natural substrate and the β(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the β(1,2)-linked GlcNAc. The structure is consistent with the ～80-fold preference of dGMII for the cleavage of substrates containing a nonreducing β(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.

Full text of DOI:10.1021/ja711248y

Published on Web 06/18/2008
Probing the Substrate Specificity of Golgi r-Mannosidase II
by Use of Synthetic Oligosaccharides and a Catalytic
Nucleophile Mutant
Wei Zhong,† Douglas A. Kuntz,*,‡ Brian Ember,† Harminder Singh,†
Kelley W. Moremen,† David R. Rose,*,‡,§ and Geert-Jan Boons*,†
Complex Carbohydrate Research Center, UniVersity of Georgia, 315 RiVerbend Road, Athens,
Georgia 30602, and Ontario Cancer Institute and Department of Medical Biophysics, UniVersity
of Toronto, 101 College Street, Toronto, Ontario, Canada M5G 1L7
Received January 2, 2008; E-mail: drose@uhnresearch.ca; dkuntz@uhnresearch.ca; gjboons@ccrc.uga.edu
Abstract: Inhibition of Golgi R-mannosidase II (GMII), which acts late in the N-glycan processing pathway,
provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the
substrate requirements of GMII, oligosaccharides were synthesized that contained an R(1,3)- or R(1,6)-linked
1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native
Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed
to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding
site for the R(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational
change of the bound mannoside to a high-energy B2,5 conformation is facilitated by steric hindrance from, and
the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well
tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the R(1,6)-linked
Man of the natural substrate and the ꢀ(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended
binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular
interactions with the ꢀ(1,2)-linked GlcNAc. The structure is consistent with the ∼80-fold preference of dGMII for
the cleavage of substrates containing a nonreducing ꢀ(1,2)-linked GlcNAc. By contrast, the lysosomal
mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates
without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII
could exploit the additional binding specificity of the GlcNAc binding site.
thetic precursor for polylactosamine chains.12 It has been
proposed that these polysaccharides on integrins and cadherins
Introduction
Aberrant glycosylation of glycoproteins and glycolipids is a
molecular change typical of malignant transformations.1–3 For
example, human cancers of the breast, colon, and melanomas
often overexpress the glycosyltransferase N-acetylglucos-
aminyltransferase V (GlcNAc-TV).4–11 This enzyme introduces
a specific branching N-acetylglucosamine moiety at the N-linked
oligosaccharides of glycoproteins, thereby forming the biosyn-
or adhesion receptors facilitate focal adhesion turnover, cell
migration, and tumor metastasis.13
There is evidence that inhibition of GlcNAc-TV may be
useful for the treatment of malignancies. However, it has been
difficult to design and synthesize potent and cell-permeable
inhibitors of glycosyltransferases.14–16 Therefore, efforts to block
the biosynthesis of polylactosamine chains of N-linked glyco-
proteins have focused on inhibitors of glycosidases that act
earlier in the biosynthesis of N-glycans to prevent the formation
of the biosynthetic pathway primer of GlcNAc-TV. Most efforts
in the field have focused on Golgi R-mannosidase II (GMII),17–19
which trims two mannosyl residues from GlcNAcMan5GlcNAc2
to form the core GlcNAcMan3GlcNAc2 moiety.20
† University of Georgia.
‡ Ontario Cancer Institute, University of Toronto.
§ Department of Medical Biophysics, University of Toronto.
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The alkaloids swainsonine and mannostatin are potent inhibi-
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tors of Golgi R-mannosidase II.18 However, these compounds
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9
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Zhong et al.
1
0
also inhibit the GH38 lysosomal R-mannosidases (LM) with
potencies equal to those of the GMII and therefore induce
symptoms similar to those of the lysosomal storage disease
R-mannosidosis.18,21–23 Attempts to develop more selective
inhibitors of GMII by chemically modifying swainsonine or
mannostatin have been unsuccessful to date.17–19,24,25 Under-
standing the molecular basis for the substrate specificities of
these enzymes may provide unique opportunities for the design
of more selective inhibitors.
mannanase adopts a S5 and its covalent intermediate an S2
conformation, strongly implicating a B2,5 transition state for this
enzyme.33 Thus, it appears that these two disparate enzymes
follow similar pseudorotational itineraries but in opposite
directions.
The presence of an extended binding site is common for
glycosidases and may be important for substrate specificity. For
example, GMII is dependent upon the ꢀ(1,2)-GlcNAc moiety
of the natural substrate GlcNAcMan5GlcNAc2 for efficient
catalysis,34 and it has been suggested that this saccharide moiety
makes important interactions with the enzyme.
To probe the substrate requirements of GMII, we report here
the chemical synthesis of a number of oligosaccharides (1-5)
that have been employed in cocrystallographic studies with wild-
type and mutant (D204A) Drosophila GMII (dGMII). These
studies uncovered the molecular interactions of the enzyme with
the R(1,3)-linked mannoside and ꢀ(1,2)-linked GlcNAc moiety
of the natural substrate. The importance of the GlcNAc binding
site is consistent with a ∼80-fold preference of dGMII for
cleavage of substrates containing the ꢀ(1,2)GlcNAc residue.
GMII is a retaining glycosylhydrolase of family GH38.
Retaining glycosyl hydrolases employ a two-step mechanism
involving the formation of a covalent glycosyl enzyme complex.
Two carboxylic acids positioned within the active site act in
concert: one as a catalytic nucleophile and the other as a general
acid/base catalyst.26–28 Studies with 5-fluoro pseudosubstrates
and deuterium-labeled substrates have shown that the transition
states on either side of the covalent intermediate have a marked
oxacarbenium ion character, involving electron delocalization
across the C-1-O-5 bond. This demands planarity of C-2, C-1,
O-5, and C-5 at or near the transition state, which for the
pyranose ring can be accomplished by several boat and half-
chair conformations, namely, 2,5B, B2,5, 4H3, and 3H4.29–31 X-ray
crystal structures of wild-type and mutant (D341N) Drosophila
melanogaster GMII (in which the D204 side chain is present)
with fluorinated mannose analogues have revealed that the
covalent mannosyl-enzyme intermediate adopts a distorted 1S5
skew boat conformation.32 In this conformation, the leaving
group is placed antiperiplanar to the lone pair of the ring oxygen,
which is required for the departure of the leaving group
according to Deslongchamp’s antiperiplanar lone-pair hypoth-
esis. Furthermore, steric clashes between the syn-hydrogens at
C-3 and C-5 and the attacking water molecule are minimal in
the 1S5 skew boat conformation. There are only a limited number
of pseudorotational itineraries that glycosidase can take,29–31
and hence it has been suggested that retaining R-mannosidases
follow a 1S5 f B2,5 f 0S2 itinerary.32 Interestingly, it has been
shown that the Michaelis complex of a retaining endo-ꢀ-
Results
Oligosaccharide Synthesis. Oligosaccharides 1-5, derived
from the natural substrate of GMII, were synthesized for
cocrystallization studies. Compounds 1-4 contain an R(1,6)-
or R(1,3)-linked 1-thio-R-mannoside. Such compounds are
resistant toward hydrolysis by mannosidases and were expected
to allow cocrystallographic studies with wild-type dGMII.35–37
Furthermore, the C-S bond is significantly longer than a C-O
linkage, which was expected to make the compounds better
ligands because the longer C-S bond mimics lengthening of
the exocyclic C-O bond in the transition state of the cleaved
R-mannoside. Thus, compound 1 is an R(1,3)-linked dimanno-
side in which the exocyclic oxygen of the nonreducing man-
noside is replaced by sulfur (Figure 1). Compound 2 is derived
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Asymmetry 2000, 11, 1645–1680.
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Figure 1. Structures of synthetic targets.
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Royal Society of Chemistry: London, 1987; pp 259-297.
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Szabo, L.; Stoll, D.; Withers, S. G.; Davies, G. J. Angew. Chem., Int.
Ed. 2002, 41, 2824–2827.
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Substrate Specificity of Golgi R-Mannosidase II
A R T I C L E S
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) NIS, TfOH, DCM, 0 °C (80%); (ii)
NaOMe, MeOH (89%); (iii) NIS, TfOH, DCM, 0 °C (76%); (iv) BH3 in
THF, Bu2BOTf in DCM, 0 °C (67%).
from 1 but contains an additional R(1,6)-mannoside. Thus, this
derivative contains both mannosides that can be cleaved by
GMII and was initially designed to determine whether the 1-thio-
linked mannoside would be preferentially recognized by GMII.
However, studies described in the next section established that
thio-linked mannosides are not well tolerated by the catalytic
site of dGMII. On the other hand, a crystal structure of
compound 2 with a mutant enzyme revealed the binding site
for the R(1,3)-linked mannoside of the natural substrates.
Compounds 3 and 4 are more complex oligosaccharides and
contain an R(1,6)-linked 1-thiomannoside. Finally, the observa-
tion that the 1-thio-linked mannosides are not well tolerated by
the catalytic site of wild-type dGMII led to the synthesis of
pentasaccharide 5, which is derived from the natural substrate
of GMII and contains the R(1,6)-linked Man that is cleaved by
the enzyme and the ꢀ(1,2)-linked GlcNAc moiety, which is
proposed to be accommodated by the extended binding site of
the enzyme.
Compounds 4 and 5 were prepared by a convergent 3 + 2
glycosylation strategy using glycosyl acceptor 12 and glycosyl
donors 18 and 22, respectively (Schemes 1 and 2). The
trisaccharide acceptor 12 could easily be prepared from known
monosaccharides 6, 7, and 8.38–40 The thio-linked mannosyl
donor 22 was obtained by a nucleophilic displacement of triflate
19 with a thioaldose prepared by in situ deacetylation of 2036,41
followed by a two-step conversion of the trimethylsilylethyl (SE)
glycoside of the resulting compound into a trichloroacetimidate.
Thus, NIS/TfOH-mediated coupling of thioglycoside 640 with
glycosyl acceptor 739 (Scheme 1) gave disaccharide 9 in an
excellent yield of 80%. Neighboring group participation by the
C-2 acetyl ester of 6 ensured exclusive formation of the
R-anomer. Next, the acetyl ester of 9 was removed by treatment
with NaOMe in methanol to give glycosyl acceptor 10, which
was employed in a NIS/TfOH-mediated coupling with thio-
glycosyl donor 838 to give trisaccharide 11in a yield of 76%.
The bulky C-2 phthalidene of 8 ensured that only the ꢀ-anomer
was formed. The benzylidene ring of 11 was selectively opened
a Reagents and conditions: (i) TrCl, pyridine, 80 °C, and then Ac2O,
pyridine (96%); (ii) FeCl3 ·6H2O, DCM (82%); (iii) Tf2O, 2,6-lutidine,
DCM, -40 °C; (iv) NIS, TfOH, DCM, 0 °C (76% for 17, 86% for 22); (v)
diethylamine, DMF, 0 °C (73%); (vi) TFA, DCM, and then trichloro-
acetonitrile, DBU, DCM; (vii) TMSOTf, DCM, (80% for 23, 83% for 25);
(viii) H2NNH2 ·H2O, EtOH, 90 °C, and then Ac2O, pyridine; (ix) NaOMe,
MeOH, and then Na (s), NH3 (l), THF, -78 °C (86% for 4, 87% for 5).
by treatment with borane in THF and Bu2BOTf in DCM,42
resulting in the formation of glycosyl acceptor 12 having a C-4
benzyl ether and a C-6 hydroxyl.
Next, attention was focused on the preparation of glycosyl
donors 18 and 22 (Scheme 2). Thus, glycosyl acceptor 15 was
obtained by regioselective tritylation of the C-6 hydroxyl of
mannoside 1343 with trityl chloride and pyridine, followed by
acetylation with acetic anhydride and pyridine and removal of
the trityl ether by employing FeCl3 ·6H2O in DCM. Glycosyl-
ation of 15 with 1644 with NIS/TfOH as the promoter45 gave
disaccharide 17 in an excellent yield of 76% as only the
R-anomer. The 1-thio-R-mannoside 21 was prepared by treat-
ment of 15 with triflic anhydride in the presence of 2,6-lutidine
in DCM, followed by displacement of the triflate of the resulting
compound 19 with 2,3,4,6-tetra-O-acetyl-1-thio-R-D-mannose,
which was prepared by in situ S-deacetylation of 2046 by
diethylamine in DMF.
The requisite trichloroacetimidates 18 and 22 were obtained
by hydrolysis of the trimethylsilyl ethyl glycosides of 17 and
(42) Jiang, L.; Chan, T. H. Tetrahedron Lett. 1998, 39, 355–358.
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(44) Weingart, R.; Schmidt, R. R. Tetrahedron Lett. 2000, 41, 8753–8758.
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Lett. 1990, 31, 1331–1334.
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A R T I C L E S
Zhong et al.
Scheme 3a
Initially, we attempted to determine the structure of non-
cleavable 1-thiomannosides 1-4 bound in the active side of
native dGMII. X-ray crystallographic studies were carried out
on crystals grown in the presence of the analogues (cocrystals)
or soaked into phosphate-washed crystals. The washing step
removes bound Tris from the active site49 and previously led
to improvements in the visualization of weakly binding
compounds.49,50 However, after analysis of electron densities
from more than 10 different crystals prepared under various
conditions, no evidence was found of an oligosaccharide in
complex with the native enzyme.
a Reagents and conditions: (i) DMSO, 1:2 Ac2O/DMSO, and then NaBH4,
1:1 DCM/MeOH (75%); (ii) Tf2O, 1:2 pyridine/DCM, and then 20, DMF,
diethylamine, 0 °C (61%); (iii) BH3 in THF, Bu2BOTf (63%); (iv) 16, NIS,
TfOH, DCM, 0 °C (77%); (v) NaOMe, MeOH, and then Na/NH3(l), -78
°C (70%).
Preparation and Crystallographic Analysis of D204A Mutant.
The catalytic nucleophile of Drosophila GMII was previously
identified by trapping of a covalently linked intermediate at
aspartate 204 (in the residue numbering of the Drosophila
construct used for crystal studies).32 Therefore, a mutant in the
catalytic nucleophile Asp204 in which the aspartate was changed
to an alanine (D204A) was prepared as an alternative tool for
visualizing bound substrates. The D204A protein could be
purified in an identical manner to the wild-type enzyme, in
similar yields and with nearly identical crystallization properties.
The crystals formed by D204A gave the same space group
(P212121) and crystal cell dimensions as the native dGMII,
yielding comparable high-resolution diffraction (to 1.1 Å)
(Supporting Information, Table 1S).
21, respectively, by use of trifluoracetic acid in DCM followed
by treatment of the resulting lactols with trichloroacetonitrile
in the presence of DBU.47,48
A TMSOTf-promoted glycosylation of glycosyl acceptor 12
with glycosyl donors 18 and 22 in DCM gave pentasaccharides
23 and 25, respectively, in excellent yields. Cleavage of the
phthalimido group of 23 and 25 was accomplished by treatment
with hydrazine in ethanol, which was followed by N-acetylation
with acetic anhydride in methanol to afford compounds 24 and
26, respectively. Finally, deprotection of 24 and 26 to give target
compounds 4 and 5, respectively, was accomplished by a two-
step procedure entailing treatment with NaOMe in methanol to
hydrolyze the acetyl esters and Birch reduction to remove the
benzyl ethers.
The chemical synthesis of trimannoside 2 was more chal-
lenging due to the presence of a 1-thiomannoside linked to a
secondary C-3 hydroxyl. Thus, the configuration of the C-3
hydroxyl of 27 was inverted by a two-step procedure involving
Swern oxidation to give a ketone, which was immediately
reduced with NaBH4 in a mixture of dichloromethane to give
taloside 28 as the only diastereoisomer in good yield (Scheme
3). Triflation of the C-3 hydroxyl of 28 with triflic anhydride
in the presence of 2,6-lutidine in DCM followed by displacement
of the resulting compound with 2,3,4,6-tetra-O-acetyl-1-thio-
R-D-mannose, which was prepared by in situ S-deacetylation
of 2046 by use of diethylamine in DMF, gave 1-thio-linked 29.
The benzylidene ring of 29 was selectively opened by treatment
with borane in THF and Bu2BOTf in DCM,42 and the C-6
hydroxyl of the resulting compound 30 was glycosylated with
thioglycoside 16 by use of NIS/TfOH as the promoter system
to yield trisaccharide 31. Deprotection of 31 to give target
compound 2 was easily accomplished by the standard two-step
procedure. Compound 1 was easily obtained by a similar
deprotection of 29. Finally, tetrasaccharide 3 was prepared by
regioselective opening of the benzylidene acetal of 9 followed
by glycosylation with trichloroacetimidate 22 and deprotection
by standard procedures (for details see Supporting Information).
The absence of the catalytic aspartate changed the environ-
ment of the active site. One of the principal changes is that the
active-site zinc, which normally coordinates with four amino
acid side chains (Asp204, His90, Asp92, and His471), now only
coordinates with three side chains and shifts approximately 0.3
Å toward Asp92. Second, Arg228, which normally interacts with
Asp204, shows more freedom of movement and a new alternate
conformation becomes visible. Third, Trp95, which forms the
cap of the cleavage pocket, moves upward, generating a slightly
more open pocket. The arrangement of solvent waters within
the empty active-site pocket differs between the native and
mutant GMII, and Tris also binds differently in the active site
of the mutant enzyme (Supporting Information).
Crystallography of dGMII Mutants with Mannose. Initially,
the D204A nucleophile mutant as well as the previously
prepared acid-base catalyst mutant D341N32 was employed to
trap the artificial substrate 2,4-dinitrophenyl R-D-manno-
pyranoside. There is a slight amount of residual mannosidase
activity in these mutants, which was evident as the crystals
turned yellow, indicating substrate cleavage. As a result the
uncleaved substrate could not be visualized; however, the
electron density for mannose was visible in the active site (-1
site) of each mutant (Figure 2).
In the acid-base mutant (D341N) containing natural Asp204,
the ring was found in what is best modeled as a distorted high-
energy B2,5 conformation (Figure 2A), which is necessary for
it to fit in the confined space of the binding pocket. It should
be noted, however, that the fitting is not unambiguous, and a
0
0
Crystallography. The crystallographic results of 10 complexes
of Drosophila GMII are reported here to resolutions between
2.03 and 1.10 Å, with Rfree between 20% and 15.5%. The
statistics for the refinement and PDB codes are listed in the
Supporting Information.
distorted S2 skew-boat or H5 half-boat could also be accom-
modated reasonably well. The high-energy 1,4B boat conforma-
tion with equatorial hydroxyls has been previously noted for
inhibitors such as kifunensine,49 ghavamiol,50 or manno-
(49) Shah, N.; Kuntz, D. A.; Rose, D. R. Biochemistry 2003, 42, 13812–
13816.
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Substrate Specificity of Golgi R-Mannosidase II
A R T I C L E S
associated -1 mannoside and 16.4 and 15.7 for the mannosides
in the +1 and +2′ sites, respectively). Furthermore, the sugar
4
rings of 2 adopted low-energy C1 conformations; however,
there was a slight twist in the C2, C3, C5, O5 plane of the
mannoside in the -1 site.
The trisaccharide 2 makes extensive water contacts (a total
of 10; Figure 3C). The +2′ site is formed primarily by Arg343,
Asp340, and Asp341, which make hydrogen bonds with the
O3 and O4 of the R(1,3)-S-linked mannoside of 2. Furthermore,
the O3 is at the apex of an interaction triangle consisting of the
Nε of Arg343, the carbonyl oxygen of Asp341, and a water
molecule (Wat2343 in PDB structure 3BVU). Water 2343 in
turn interacts with the terminal amino group of Lys288. The
O4 lies equidistant between the NH2 nitrogen of Arg343 and
the Oδ2 oxygen of Asp340 (2.8 Å). The O6 of the sugar bound
in the +2′ site is found in two almost equally populated
conformations, making contacts in either conformation with two
water molecules. One of the A-conformation interacting waters
makes contact in turn with the Ser926 backbone amine.
Apart from a hydrogen bond between the O4 and Asp341
Oδ2 (2.6 Å), the interactions with the central mannoside (+1
site or swivel position) are primarily with loosely bound (high
B-factors) water molecules, which are not directly attached to
the amino acid backbone. Only O2 of the swivel residue makes
a hydrogen bond to a well-defined water molecule (Wat2012),
which in turn hydrogen-bonds to the Oδ1 of Asp340.
Figure 2. Binding of mannose in the active site of (A) D341A dGMII
[PDB code 3BUP] or (B) D204A dGMII [PDB code 3BUQ]. Fo - Fc omit
electron density maps are shown contoured at 3σ (0.2 e/Å3). A magenta
ball represents the active-site zinc. (C) Superposition of the bound
mannosides. Superposition was based on the protein atoms, and the mannose
was extracted from the fit structures. Mannose in the active site of D341N
(yellow) is in a high-energy boat conformation, while in D204A (green) it
4
is in a C1 chair conformation.
1
noeuromycin51 bound to the wild-type enzyme, while the S5
conformation was observed in the trapped covalent intemedi-
ate.32 All these complexes lie on the same conformational
4
4
0
0
interconversion itinerary from C1 (i.e., C1 T H5 T S2 T
1
B2,5 T S5 T 1,4B).
The complex with disaccharide 1 (Supporting Information)
demonstrates that a thio-linked mannoside can be complexed
in the active site (-1) of the enzyme. In this case, the position
of the S-linked mannoside complexed to zinc in the -1 site
was clearly defined. In contrast, the poor quality of the density
for the mannoside in the +1 site and its higher B-factors
(average of 27.7 versus 10.9 for the mannoside in the -1 site)
indicates that it is loosely bound and probably somewhat mobile.
The cocrystal structures with oligosaccharides 3 and 4 further
support the notion that 1-thiomannosides are not well tolerated
by the catalytic site of dGMII. The structural details of these
complexes will be discussed in the next section because they
also provide evidence of the importance of the GlcNAc moiety
of compound 5 and the natural substrate as an anchoring residue.
Structure of Cocomplex of D204A with Pentasaccharide 5
and Comparison with the Mode of Binding of Thio-Linked
Oligosaccharides 3 and 4. In view of the difficulties encountered
in visualizing the thio-linked oligosaccharides in the active site of
D204A, the oxygen-containing derivative 5 was synthesized. The
high-resolution structure (1.10 Å) of crystals soaked with 5 showed
density for the complete pentasaccharide, with the density for the
swivel residue (+1 site) being somewhat less defined but assign-
able. The density is shown in Figure 3B, and a schematic illustration
of the protein and water contacts is shown in Figure 3D. The
interactions made by the mannoside in the -1 site are almost
identical to those observed in the other structures and involve
primarily amino acid contacts and tight complexation to the zinc.
The three central mannosyl residues of 5 in the +1, +2, and +3
sites make only a few direct hydrogen bonds with the protein, and
most interactions are by water contact. Thus, the O4 of the
mannoside in the +1 site interacts with Asp341 Oδ2 and the O4
of the mannoside in the +2 site interacts with Tyr267 OH.
Furthermore, three amino acids (Asp340, Arg410, and Glu875)
form water-mediated hydrogen bonds with the mannosides in the
+1, +2, and +3 sites.
Interestingly, mannose complexed in the nucleophile mutant
(D204A) adopted the low-energy 4C1 conformation (Figure 2B),
previously unobserved for dGMII binding. The change in
conformation of zinc-bound mannose is a reflection of the
difference in the environment around the zinc in the D204A
mutant. Superposing the two structures and extracting the bound
mannose indicates that the position of the hydroxyls other than
O2 also differ slightly. (Figure 2C). While D204A can bind
some compounds in its active site (albeit in a nonnative
conformation), we have failed to find evidence of binding of a
number of inhibitors, including the nanomolar inhibitor
swainsonine.
Crystallography of D204A dGMII with Thioglycoside-
Containing Oligosaccharides. Successful visualization of the
oligosaccharides in the active-site pocket required extended
soaking (16-24 h) of the synthetic oligosaccharides with
phosphate or MOPS-washed D204A crystals. As will be
discussed below, the thio-linked mannosyl moiety of compounds
1-4 was not well tolerated by the active site of the mannosidase,
and when possible the binding mode was altered such that this
moiety was complexed in another subsite of the enzyme. The
latter feature is best illustrated in the complex of dGMII with
compound 2.
The D204A/2 crystal diffracted to high resolution (1.12 Å
with R and Rfree of 11.4% and 15.5%, respectively) and the
density was exceptionally clear for the complete trisaccharide
(Figure 3A). The R(1,6)-O-linked mannosyl residue of com-
pounds 2 was complexed in the active site (-1) and the R(1,3)-
S-linked mannosyl moiety was bound in a newly identified
pocket, which is designated as the +2′ site (to distinguish it
from the +2, +3, and +4 sites, which bind with the other branch
of the natural substrate). The B-factors for the trisaccharide were
comparable along its length (average of 9.9 for the zinc-
The GlcNAc residue of 5 is accommodated in an important
binding site (+4), and as expected for its role as an anchor
(51) Kuntz, D. A.; Liu, H.; Bols, M.; Rose, D. R. Biocatal. Biotransform.
2006, 24, 55–61.
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A R T I C L E S
Zhong et al.
Figure 3. Binding of 2 and 4 to D204A: stereoviews (divergent) of (A) 2 bound in Fo - Fc omit map contoured at 4σ (0.38 e/Å3) or (B) 4 contoured at
1.6σ (0.15 e/Å3) to visualize density in the +1 and +2 positions. The MPD displaced by GlcNAc binding is shown in panel A. The orientation in both panels
is the same to illustrate the differences in position of each compound. (C, D) Interactions less than 3.2 Å are shown for (C) 2 or (D) 3. Distances are given
in angstroms. Interacting waters are shown as orange spheres. Zinc is represented as a magenta sphere.
residue, it makes extensive interactions with the protein. There
is a direct hydrogen bond between His273 and O3 of the
GlcNAc moiety as well as three single water molecule-mediated
hydrogen bonds to the protein. Furthermore, an important
stacking interaction is present between the Tyr267 ring and
GlcNAc ring.
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Substrate Specificity of Golgi R-Mannosidase II
A R T I C L E S
Figure 5. Comparison of binding of 2 (green) and 3 (slate) to D204A.
Compound 2 has an R(1,6)-linked mannoside while 3 has an R(1,3)-linked
mannoside bound in the -1 site. Compound 3 is a tetramannoside but only
three mannosides are visible in the electron density; the terminal thio-linked
mannose cannot be assigned. The thio bonds are colored orange, and zinc
in the active site is represented by a magenta ball.
Figure 4. The presence of GlcNAc causes a radical rearrangement of
oligosaccharides bound to Golgi mannosidase II. Shown is the binding of
3 (blue, from PDB structure 3BVV) and GlcNAc-modified 4 (yellow, from
PDB structure 3BVW). For orientation purposes, one of the mannoside
residues is colored green. This mannoside binds in the -1 site of D204A/3
complex and in the +3 site of the D204A/4 complex. The active-site zinc
is colored magenta. Although 3 is a tetrasaccharide, only three sugars could
be assigned to the electron density.
(Supporting Information), the modeling was consistent with an
occupancy of 70%. In a second crystallization attempt, under
slightly different conditions, the occupancy was less than 10%,
and thus the affinity of 3 for D204A appears quite low. The
R(1,3)-O-linked and R(1,6)-O-linked mannosyl residues of 3
are complexed in the active site (-1) and the newly identified
+2′ site, respectively. A superimposition of 2 with 3 (Figure
5) demonstrates that the mannosides in the -1 and +2′ subsites
are in almost identical positions and make similar types of
interactions. However, the central swivel mannosides (+1),
which are R(1,3)-linked mannoside in 3 and R(1,6)-linked
mannoside in 2, are less clearly resolved and located in different
positions. In particular, the interactions made between the protein
and the mannoside of 3 in the +1 site differ from those of the
complexes with 1 and 2: O4 interacts with the presumptive
acid-base catalyst Asp341 Oδ2 (2.3 Å), while a close contact
(2.9 Å) is formed between the exocyclic oxygen of the R(1,3)-
glycosidic linkage and Oδ2 of Asp341. The O2 interaction of
the residue in the +1 site to a tightly bound water seen in the
complex with 2 is no longer present. Instead, it interacts with
the Arg876 backbone carbonyl, which in turn hydrogen-bonds
with the O6 of the mannoside in the -1 site.
The GlcNAc binding site has never been observed to be
unoccupied,24,32,49,53 containing either 2-methylpentane-2,4-diol
(MPD, Figure 3A) or glycerol depending on the cryocondition
used. The binding of free N-acetylglucosamine to this site has
also been observed in crystals of dGMII soaked with this
compound (D. Kuntz, unpublished observation). Previously, we
proposed that the non-reducing-terminal GlcNAc of the natural
substrate GlcNAcMan5GlcNAc2-Asn would bind in this location,
and the data presented here support this proposal.52
The crystal structures of compounds 3 and 4 with D204A
provide further support of the importance of the GlcNAc moiety
as an anchoring residue. Thus, the 1-thiomannosyl and GlcNAc
residues of 4 are accommodated in the -1 and +4 sites,
respectively, and superimpose with similar residues of 5
(Supporting Information). The middle mannosides, however,
diverge and the density for the mannoside in the +1 site was
ill-defined despite complete redundant data and the highly
refined nature of the model. This lack of density most likely
results from the unfavorable nature of complexation of a
thiomannoside and the very few interactions made by this
residue with the protein. The latter is a reflection of the role of
the mannoside in the +1 site as a swivel residue.
Enzymology. To establish the importance of the GlcNAc
binding subsite for hydrolysis, time course studies were first
performed with pyridylamine- (PA-) tagged derivatives of the
natural substrates GlcNAcMan5GlcNAc2-PA and Man5GlcNAc2-
PA. In addition, comparative assays were performed with human
lysosomal mannosidase (hLM), a GH38 enzyme with sequence
and structural similarity to dGMII.
The tetrasaccharide 3 has a similar structure as 4 except that
it does not contain a GlcNAc moiety (Figure 1). Interestingly,
the lack of the latter residue resulted in a completely different
mode of complexation (Figure 4). Thus, the electron density of
the soaked crystal structure does not show the thio-linked
mannoside and only a trimannoside moiety could be visualized.
Although the density of the trimannoside is well-defined
To determine relative activities of both enzymes, experiments
were carried out with 4-methylumbelliferyl R-mannoside (4MU-
R-Man) as substrate. This information was employed in time
course studies, which demonstrated rapid cleavage of GlcNAc-
Man5GlcNAc2-PA by dGMII through a GlcNAcMan4GlcNAc2-
PA intermediate to produce GlcNAcMan3GlcNAc2-PA, similar
to the established substrate specificity for the equivalent
mammalianenzyme.Insharpcontrast,dGMIIcleavedMan5GlcNAc2-
PA >80-fold more slowly (Figure 6).
(52) van den Elsen, J. M. H.; Kuntz, D. A.; Rose, D. R. EMBO J. 2001,
20, 3008–3017.
(53) Heikinheimo, P.; Helland, R.; Leiros, H. K. S.; Leiros, I.; Karlsen,
S.; Evjen, G.; Ravelli, R.; Schoehn, G.; Ruigrok, R.; Tollersrud, O. K.;
McSweeney, S.; Hough, E. J. Mol. Biol. 2003, 327, 631–644.
9
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A R T I C L E S
Zhong et al.
Figure 7. Spatial clash between Asp204 and sugar bound in the low-energy
4C1 conformation. Coordinates for the D204A/5 complex (PDB code 3BVX)
were superimposed with those of the unliganded native enzyme (PDB code
3BVT). If the pentasaccharide bound to the native enzyme in an identical
manner to which it binds to D204A, the distance between the Asp204 Oδ2
and O2 of 5 would be only 1.2 Å. The pentasaccharide is colored in cyan,
while amino acid side chains of the native protein are shown in green, except
for Asp204, which is highlighted by coloring it magenta.
Figure 6. In Vitro digestion time course of Man5GlcNAc2-PA and
GlcNAcMan5GlcNAc2-PA by dGMII and hLM. Purified recombinant
dGMII (A, C) and hLM (B, D) were used in digestion time course studies
with GlcNAcMan5GlcNAc2-PA (A, B) or Man5GlcNAc2-PA (C, D) as
substrates. Cleavage of the substrates (GlcNAcMan5GlcNAc2-PA or
Man5GlcNAc2-PA, [) to smaller glycan structures (GlcNAcMan4Glc-
NAc2-PA or Man4GlcNAc3-PA, 9; GlcNAcMan3GlcNAc2-PA or
Man3GlcNAc3-PA, 2; GlcNAcMan2GlcNAc2-PA or Man2GlcNAc3-PA,
b) were quantitated by HPLC. dGMII cleaved GlcNAcMan5GlcNAc2-PA
∼80-fold faster than Man5GlcNAc2-PA at equivalent enzyme concen-
trations. Minimal digestion of Man5GlcNAc2-PA or GlcNAcMan5GlcNAc2-
PA by hLM was detected when equivalent enzyme activity units (based on
4MU-R-Man activity) of hLM and dGMII were employed (not shown).
Increasing the enzyme concentration of hLM in the in vitro assays by 100-
fold (B, D) resulted in detectable cleavage of Man5GlcNAc2-PA, but
cleavage of GlcNAcMan5GlcNAc2-PA remained ∼16-fold slower.
bound zinc, which interacts with a mannoside with the strongest
coordination through the C-2 hydroxyl. There is an unusual
architecture of the protein backbone around the -1 pocket, as
Trp95, which can be considered a flap residue, and the 204 residue,
whether catalytic aspartate or mutant alanine, always appears as
outliers in Ramachandran plot analyses. Access to the -1 site is
somewhat restricted as the pocket is covered by Trp95. However,
Trp95 demonstrates a degree of mobility in the various structures
and can reposition itself to maintain stacking interactions with
bound carbohydrates or inhibitors. The incoming substrate’s
approach path to the zinc is further restricted by the surrounding
amino acid side chains. Previous structures with a number of
inhibitors,24,32,50,51 as well as the structure of mannose bound to
D341N presented here, indicate that binding typically occurs in a
high-energy distorted B2,5 conformation.
Insufficient quantities of the natural substrates prevented
detailed Km analyses, but the time course data clearly indicate
that the non-reducing-terminal GlcNAc residue confers ad-
ditional binding affinity and substrate preference for the
GlcNAcMan5GlcNAc2-Asn glycan processing intermediate.
hLM cleaved Man5GlcNAc2-PA at a similar rate (based on
equivalent 4MU-R-Man hydrolytic activity) as dGMII, while
cleavage of GlcNAcMan5GlcNAc2-PA was undetectable at
equivalent enzyme concentrations (data not shown). When the
concentration of hLM was increased 100-fold, cleavage of
Man5GlcNAc2-PA to smaller structures was clearly evident, but
cleavage of GlcNAcMan5GlcNAc2-PA by hLM was ∼16-fold
slower (Figure 6). These results indicate that the non-reducing-
terminal GlcNAc in the GlcNAcMan5GlcNAc2-PA substrate
enhances the rate of glycan cleavage by dGMII, whereas the
same residue is inhibitory for glycan cleavage by hLM.
The X-ray crystallographic studies indicate that the active
site of dGMII poorly recognized thio-linked mannosides. To
support this observation, the rate of hydrolysis of different
concentrations of 4MU-R-Man alone and in the presence of
different concentrations of compounds 3 or 4 was measured
fluorometrically, and Ki values and IC50 values were estimated
to be larger than 5 mM, confirming that the thio derivatives are
poorly recognized by dGMII.
In the case of the D204A nucleophile mutant studied here,
changing the aspartate to the smaller-sized alanine allows more
free space in the cleavage pocket and a more unrestricted
approach to the zinc. As a result, the sugars bound in the -1
pocket of D204A retained a low-energy 4C1 conformation. An
overlay of the bound structure of compound 5 onto the native
enzyme indicates that a spatial clash between Asp204 Oδ2 and
O2 would occur if the -1 mannoside was bound in a 4C1
conformation (Figure 7). In native structures of dGMII, Asp204
does not show positional flexibility because Oδ1 is tightly
associated with the zinc (2.1-2.3 Å in various native structures)
and Oδ2 makes close contacts with the Nꢀ1 and NH2 of Arg228.
Thus, the data presented here indicate that sugar distortion is
being imposed by the spatial restraints of the cleavage pocket.
This distortion is further assisted by the formation of strong
hydrogen bonds to Asp204 when mannose is bound in the high-
energy conformation. Conformational distortion driven by steric
constraints has been demonstrated previously. For example, an
X-ray crystal structure of an inverting endoglucanase with a
nonhydrolyzable substrate showed the glucosyl unit in the
catalytic binding site adopting a 2S0 conformation.35 It appears
that steric hindrance by Tyr73 forces the glucoside into the
distorted conformation, as mutation of Tyr73 to a less bulky
serine gives an enzyme to which the glucoside binds in a low-
Discussion
The crystal structures of various synthetic oligosaccharides
complexed to Golgi R-mannosidase II containing a mutation of
the active-site nucleophile Asp204 define two previously unidenti-
fied carbohydrate-binding subsites and provide a model for natural
substrate recognition. The binding cleft of Golgi mannosidase II
consists of a long solvent-accessible groove with a buried catalytic
pocket, the -1 site. A defining feature of the -1 site is a tightly
4
energy C1 conformation. Thus, steric hindrance imposed by
the side chain of a catalytic residue or other amino acid in the
binding pocket of a glycosidase, in conjunction with favorable
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Substrate Specificity of Golgi R-Mannosidase II
A R T I C L E S
the -1 site of the D204A/3 complex is now bound in the +3 site
of the D204A/4 complex, the +1 mannose is in the +2 site, the
+2′ sugar is now in the +1 site, and the previously undefined thio-
linked sugar is in the -1 site) (Figure 4).
Studies on mammalian GMII have demonstrated the requirement
for the prior action of N-acetylglucosaminyltransferase I (GnT I)
to add a non-reducing-terminal GlcNAc residue to the core
R(1,3)mannosyl residue prior to cleavage of the non-reducing-
terminal R(1,3)- and R(1,6)-mannosyl residues by GMII.34 These
results are extended here for the homologous Drosophila GMII,
which demonstrates a ∼80-fold preference for cleavage of
GlcNAcMan5GlcNAc2 over structures lacking the terminal GlcNAc
residue. The structural basis for this specificity, first suggested
through the observed interaction of cryoprotectant molecules, is
confirmed in the present studies with the description of the anchor
site. Recently, the structure of dGMII D204A with the natural
substrate has confirmed that the observations reported here on
synthetic compounds are relevant to the true substrate as well (N.
Shah, D. A. Kuntz, and D. R. Rose, unpublished results).
Time course studies presented here, examining the cleavage
of glycan substrates by the catabolic GH38 hydrolase hLM (an
enzyme with sequence and structural similarity to GMII) indicate
that hLM prefers oligomannoside substrates lacking the terminal
GlcNAc residue, a finding that is consistent with the absence
of the extended GlcNAc binding subsite in bovine LM.53
The observation that the wild-type dGMII does not accom-
modate thio-substituted glycosides remains an important but
unresolved question. Evidently, there are some chemical char-
acteristics of the catalytic site, the thioglycosidic linkage, or
both that preclude binding. Possibly the distortion of the
substrate that occurs on binding, mediated by the nucleophile
Asp204, is not achievable in the substrate analogues, because
of either increased rigidity or a greater repulsion of the
S-substituent in the binding region.
Figure 8. The swivel sugar is observed in a large number of positions.
The two mannosides closest to the active-site zinc are shown for the R(1,6)-
linked compounds 2 (green) and 5 (yellow) and for the R(1,3)-linked
compounds 1 (cyan) and 3 (slate). In all cases, the position of the zinc-
bound -1 mannoside is almost invariant, whereas the mannoside in the
+1 site, which is described as the swivel residue, is highly variable. The
oxygen at the cleavage site, indicated by an asterisk, is in almost the same
position in all complexes.
interactions elsewhere, can drive a conformational change
required for glycosidic bond cleavage.
The present study focused on the identification of additional
sugar binding subsites within the binding cleft of dGMII in
support of the unique substrate specificity for this enzyme. The
mode of binding of the synthetic oligosaccharides confirmed
the presence of two previously suspected sugar-binding subsites
and provide a model for substrate cleavage and product release.
On the basis of the presence of bound 2-methylpentane-2,4-
diol or glycerol in the crystal structure of dGMII, we previously
proposed a GlcNAc binding site at one end of the binding
groove, which would anchor the natural substrate GlcNAcMan5-
GlcNAc2-Asn into the groove.52 The intact substrate can be
thought of as a Y, where each arm of the Y is cleaved off in
successive reactions. The arms of the Y are made up of
respectively the R(1,6)-linked (initially bound in the -1 site)
and R(1,3)-linked (initially bound in the +2′ site) non-reducing-
end mannosides. The intervening mannoside residue at the base
of the Y (+1 site) swivels to reposition the arms between the
two cleavage steps. To fulfill its function as a swivel residue,
the mannoside in the +1 site must be loosely associated with
the enzyme. The oligosaccharide chain would remain tethered
in the binding site during the rotational event due to the presence
of the GlcNAc anchor, but the product would be released, as
binding sites are lost due to the cleavage events.
The complexes of D204A with compounds 2 and 5 best define
the +2′ and +4 carbohydrate binding sites, respectively, while the
other complexes provide support for the findings as well as
providing a clear indication of the positional mobility of the swivel
mannoside (Figure 8). Binding at the +2′ site is primarily facilitated
by direct contacts to Arg343, Asp340, and Asp341, while Lys288
and Ser296 are also involved through intermediary waters. The
GlcNAc (+4) binding site involves a direct hydrogen-bond
interaction with His273, and a tightly bound water-mediated contact
with the Val61-Gln64 main chain. The tight binding of the
GlcNAc is primarily due to a ring-stacking interaction between
the GlcNAc ring and Tyr267. The importance of the GlcNAc
binding site for anchoring the full-length GMII substrate is clearly
shown in the complexes with compounds 3 and 4, which adopt
completely different binding positions (e.g., the sugar formerly in
The data presented here extend the focus for selective GMII
inhibitor design through the identification of a GlcNAc binding
site that is open, accessible, and amenable to virtual and
experimental inhibitor screening as a potential route to identify-
ing selective antimetastatic agents without the secondary
complications from lysosomal storage disorders.
Acknowledgment. Funding was provided by National Institutes
of Health Grants 5P41RR05351-18 (G.-J.B. and K.W.M.) and
GM47533 (K.W.M.) and by the Canadian Institutes of Health
Research (D.A.K. and D.R.R.). We thank Ruth-Ann Stewart and
Tara Signorelli for help with mutagenesis and preparation of
D204A, and Tamika Hamlet and Kayla Shea for crystal screening.
This work is based upon research conducted at the Cornell High
Energy Synchrotron Source (CHESS), which is supported by the
National Science Foundation under Award DMR-0225180, using
the Macromolecular Diffraction at CHESS (MacCHESS) facility,
which is supported by Award RR-01646 from the National Institutes
of Health, through its National Center for Research Resources.
Supporting Information Available: Experimental procedures
and analytical data for all new compounds; crystallization
conditions, data collection and refinement statistics, and refine-
ment protocols for Drosophila Golgi mannosidase II complexes;
and detailed structural information for dGMII/water, D204A/
water, D204A/Tris, D204A/1, D204A/2, and D204A/4 com-
plexes. This information is available free of charge via the
Internet at http://pubs.acs.org.
JA711248Y
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J. AM. CHEM. SOC. VOL. 130, NO. 28, 2008 8983