From the Pharmacogenetics Section, Laboratory of
Reproductive and Developmental Toxicology and § Laboratory
of Structural Biology and NIEHS, National Institute of Health, Research
Triangle Park, North Carolina 27709 and ¶ Department of
Biochemistry, Kobe Pharmaceutical University, Higashinada-ku, Kobe
685-8858, Japan
Received for publication, October 15, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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EXTL2, an
Proteoglycans are composed of core proteins and covalently linked
glycosaminoglycan side chains. Proteoglycans are distributed at cell
surfaces and in the extracellular matrices of most animal tissues.
Heparan sulfate and heparin are uronic acid-containing sulfated
glycosaminoglycan chains that are comprised of repeat disaccharide
units consisting of alternating GlcNAc and GlcUA or iduronic acid.
Heparan sulfates and heparin have been implicated in blood coagulation,
cell proliferation and differentiation, tissue morphogenesis, and viral
and bacterial infection (1, 2). Accordingly, disruption of enzymes in
heparan sulfate biosynthesis can have severe biological consequences in
mammals (3). Human hereditary multiple exostoses disorder,
characterized by benign tumors of bony outgrowths, has been attributed
to mutations in EXT1 and EXT2 of the
EXT
(exostosin)
gene family that encode various enzymes in heparan biosynthesis (4-6).
EXT1 and EXT2 are bifunctional enzymes with
In addition to the EXT enzymes, the EXT family also includes so-called
EXT-like enzymes EXTL1, EXTL2, and EXTL3 (7, 8). Purified recombinant
EXTL2 displays activity for transfer of GlcNAc and GalNAc from the
respective UDP-sugars to an acceptor analog, [glucuronic
acid] Here we have determined the ternary complex structure of mouse EXTL2
(mEXTL2)1 with UDP and the
acceptor substrate analog
GlcUA Protein Expression and Purification--
The upstream primer
5'-CGCGGATCCCATCATCATCATCATCATCATCATCATCATACCAACTTACTCCCCAACATCAAAG
and the downstream primer 3'-CCCTAGTCTAGATCACATTTTACTTTTATCGTT were used to amplify the coding region (T38-M330) of EXTL2 from mouse (liver) cDNA by PCR. The PCR product was cloned into the pMAL-2c vector using the BamHI and XbaI
restriction sites. The corresponding construct was used to transform
Top10 cells followed by retransformation into BL21 (DE3) cells. To
express the soluble protein, 100 µl of overnight cell stock was added
to 1 liter of 2YT medium plus 200 µg/ml ampicillin in a 2-liter flask
and placed on a shaker at 250 rpm at 37 °C for 14 h. 20 ml of
culture was added to each of 20 2-liter flasks containing 1 liter of
2YT plus 200 µg/ml ampicillin. These flasks were placed at 37 °C
on a shaker set at 250 rpm. When the A600
reached 0.8, the temperature was set to 23 °C, and
isopropyl-1-thio-
For mammalian expression of mutated EXTL2 enzymes, each expression
plasmid (6 mg) was transfected into COS-1 cells in a 100-mm plate using
FuGENETM 6 (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Two days after transfection, 1 ml of the
culture medium was collected and incubated with 10 ml of IgG-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The beads
recovered by centrifugation were washed with and resuspended in 50 mM MES buffer, pH 6.5, containing 20 mM
MnCl2 and 171 mM the sodium salt of ATP.
Protein Crystallization, Data Collection, and
Refinement--
Crystals were obtained using the hanging drop method
by mixing 4 µl of the digested protein in 25 mM Hepes, pH
7.5, 100 mM NaCl with 4 µl of the reservoir solution
containing 0.1 M sodium cacodylate, pH 7.5, 10-12%
polyethylene glycol 3000 (PEG3000), and 200 mM
MgCl2. These crystals diffract at 2.4-Å resolution and
were suitable for structural studies. For data collection of the
apoprotein, crystals were transferred to a cryo-solution containing 0.1 M sodium cacodylate, pH 7.5, 17% PEG3000, 200 mM MgCl2, and 12.5% ethylene glycol and
mounted in a cryo-loop and flash-frozen on the goniometer in a stream
of nitrogen gas cooled to
Crystals of selenomethionine-labeled protein were obtained using the
same protocol as the native crystals but with an addition of 1 mM dithiothreitol present in the protein solution. Crystals of the CH3HgCl and HgCl2 derivative data sets
were obtained by soaking native crystals in the reservoir solution
containing saturated CH3HgCl or HgCl2 for ~15
min and then back-soaking into the cryo-solution and flash-frozen
(Table I). The isomorphous difference data between the selenomethionine
data set and the native data set were used to find 16 selenium sites
(eight sites per monomer with two monomers in the asymmetric unit) with
the real space heavy atom search protocol in the program CNS
(14). Phases produced from these data were then used to find six
mercury sites in both the CH3HgCl and
HgCl2 data sets. Using the isomorphous difference data from all three derivatives and anomalous difference data from the mercury derivatives initial phases were calculated to 2.9 Å. The phases were
extended to 2.4 Å using the solvent-flipping and phase-extension options in the density modify protocol of CNS (14). The original model
of the apo-enzyme of mEXTL2 was built into electron density calculated
from these phases using the program O (15). The model was subsequently
refined using iterative cycles of model building and refinement in CNS
utilizing the minimization, torsion angle refinement, and individual
B-factor refinement protocols at each step. When all of the protein
density had been accounted for with model building, water molecules
were added using the water_pick protocol in CNS. Criteria for water
selection were based on a 2.5- Site-directed Mutagenesis--
A mouse EXTL2 cDNA fragment
that lacks the first 53 N-terminal amino acids of the EXTL2 was
amplified by PCR with the full-length cDNA template and
oligonucleotides 5'-CGGGATCCGAGATCAAATCCCCGAGCAAG-3' and
5'-CGGGATCCACACAGCATGGAGAAGCCACTG-3' for 5'- and 3'-primers, respectively. The 5'-primer contained an in-frame BamHI
site, whereas the intrinsic BamHI site was located 41 bp
downstream of the stop codon. A two-stage PCR mutagenesis method was
used to construct EXTL2 mutants. Two separate PCR reactions were
performed to generate two overlapping gene fragments using the
full-length mouse EXTL2 cDNA as a template. In the first PCR, the
sense 5'-primer described above and one of the antisense internal
mutagenic primers listed below were used: N243A,
5'-GCGATGTCGTCACAGGCCTGCGTCTCATC-3'; D246A,
5'-CATAGCGATGGCGTCACAGTTCTGCGTCTC-3'; H289A,
5'-CTCTGCAGAAAGGCCTCCGCCCGGTGCC-3'; and R293A,
5'-CTTATTTATACAGTAGGATGCCTGCAGAAA-3'. In the second round of
PCR, the respective sense internal mutagenic primer (complementary to
the antisense internal mutagenic primer) and the antisense 3'-primer
described above were used. These two PCR products were gel-purified and
then used as a template for a third PCR reaction containing the sense
5'-primer and the antisense 3'-primer described above. This produced
the full-length version of the gene with the mutation incorporated. All
PCR reactions were carried out with KOD DNA polymerase (TOYOBO).
Each final PCR fragment of the mutants, as well as the wild-type EXTL2,
was subcloned into the BamHI site of pGIR201protA (17),
resulting in the fusion of the mutants, as well as the wild-type EXTL2, to the insulin signal sequence and the protein A sequence present in
the vector. An NheI fragment containing each of the above
fusion protein sequences was inserted into the XbaI site of
the expression vector pEF-BOS (19). The nucleotide sequence of
the amplified cDNA was determined with a 377 DNA sequencer
(PerkinElmer Life Sciences).
Enzyme Assay--
GlcNAc transferase activities using 0.1 µM UDP-[3H]GlcNAc (39.7 Ci/mmol;
PerkinElmer Life Sciences) and GalNAc transferase activities using 0.1 µM UDP-[3H]GalNAc (7 Ci/mmol; Sigma) were
tested as described below. The assay mixture (25 µl) for GlcNAc
transferase and GalNAc transferase contained 25 mM PIPES,
pH 6.5, 15 mM MnCl2, 0.1% (v/v) Triton X-100,
a radioactive donor substrate, 1 mM
GlcUA
To measure the activity of the mutants, the assay mixture contained 10 µl of the resuspended beads (enzyme source), 250 nmol of
GlcUA Enzyme Expression and Characterization--
EXTL2 is a type II
Golgi membrane protein that contains a hydrophobic membrane binding
domain at the N terminus, followed by a stem region that connects the
membrane binding domain to the C-terminal catalytic domain. Protease
digestion of the recombinant EXTL2 generated the catalytic domain with
an apparent molecular mass of ~30 kDa on an SDS-gel electrophoresis
(data not shown). This catalytic domain lacks, at most, 62 N-terminal
residues based on the first residue with significant electron density.
Using GlcUA Overall Fold--
The catalytic domain of mEXTL2 crystallized in
this study is a globular domain consisting of residues Ala-63 to
Lys-327. This domain can be divided into two subdomains, referred to as
the UDP binding subdomain, comprised of residues Ala-63 to Val-150, and
the acceptor binding subdomain, comprised of residues Thr-154 to
Lys-327 (Fig. 1, A and
B). These two subdomains are connected by the signature
sequence motif for UDP-sugar-dependent
glycosyltransferases, the DXD motif (Asp-151-Asp-153). This
motif interacts with the Mn2+ metal ion and UDP-sugar donor
and thus helps position them in the proper orientation for catalysis.
The UDP binding subdomain consists of a Rossmann-like fold with four
parallel Donor UDP-Sugar Binding--
Upon binding donor substrate certain
residues in the active site undergo conformational changes. In the
apo-structure atom OE1 of Glu-288 is within hydrogen bonding distance
of OH of Tyr-193 (2.7 Å), and the side chain of His-289 is directed
toward the surface of the protein. The position of Glu-288 in the
apo-structure is in direct conflict with the position of the donor
sugars and the acceptor substrate in the UDP-GlcNAc, UDP-GalNAc, and
UDP plus acceptor substrate structures. In the holo-structures these residues take on different conformations such that His-289 is directed
toward the position of the donor sugar, and Glu-288 is disordered on
the surface of the protein.
The positions of the UDP portions of the UDP-GalNAc and UDP-GlcNAc
molecules are very similar. The UDP-sugars bind at the C terminus of
Although the UDP portion of the donor substrates UDP-GalNAc and
UDP-GlcNAc form the same interactions with the protein, the donor
sugars bind in slightly different orientations. As with other
glycosyltransferase structures, the first Asp in the DXD motif, Asp-151, forms interactions with both a Mn2+
coordinated water, as well as direct interactions with the donor sugar.
For UDP-GalNAc binding, other residues such as Arg-135, Asp-245, and
Arg-293 are all in position to form hydrogen bonds with the donor sugar
(see Fig. 2A and Table II). In the UDP-GlcNAc structure, the
GlcNAc binds in an orientation such that the 4-hydroxyl and 6-hydroxyl
groups occupy similar locations as the 3-hydroxyl and 4-hydroxyl groups
of the GalNAc moiety of UDP-GalNAc, respectively (Fig. 2B
and Table II). In this conformation, Arg-135, Asp-245, and
Arg-293 can form different interactions with the donor sugar than seen
for UDP-GalNAc. In addition, Asp-246 is within hydrogen bonding
distance of the 3-hydroxyl group of the donor sugar. Despite the
different interactions between the GlcNAc and GalNAc moieties, the C1
carbons of the UDP-GalNAc and UDP-GlcNAc molecules only differ in
position by 0.8 Å in the superposition, suggesting that both molecules
could be binding in catalytically relevant conformations.
The residues that form specific interactions with the donor sugar
moieties are highly conserved (Fig. 3).
Interestingly, many of the residues are found clustered around the
totally conserved cysteines, Cys-244 and Cys-296. These residues form a
disulfide bond linking UDP Plus Acceptor Substrate Binding--
Binding of the acceptor
substrate analog, GlcUA
A number of residues other than Trp-284 appear to be involved in
binding the acceptor analog. Residues His-289, Tyr-193, and Arg-181 are
in position to form interactions with the carboxylate group of the
GlcUA moiety whereas Arg-181 is the only residue that is in position to
form a typical hydrogen bound with the Gal moiety (Table II). In
addition to these interactions, the side chains from totally conserved
Lys-213 and partially conserved Phe-290 and Trp-284 help orient the
acceptor through Van der Waals contacts. The most interesting
interactions with the acceptor are those between the acceptor and the
Although there is clear electron density for the GlcUA Similarities to Other Structures--
Despite little to no
sequence homology, the crystal structure of mEXTL2 reveals strong
similarity to two other retaining glycosyltransferases for which
structures have been determined, galactosyltransferases LgtC from
Neisseria meningitidis and bovine Catalytic Mechanism--
EXTL2 catalyzes a retaining transfer
reaction in which the
For the SNi-like reaction mechanism, the approach of the
acceptor hydroxyl would be from the same side of the C1 carbon as the
leaving group UDP, and the reaction would most likely proceed through
an oxocarbenium ion-like transition state (see Figs.
6 and 7). The SNi-like
reaction is mainly dissociative in nature; therefore increasing the
leaving group character of the UDP would help initiate the reaction. Three new interactions exist with the
It is also possible these residues may work together to help stabilize
the transition state. In this scenario, Arg-293 could stabilize the
partial positive charge increase on the oxocarbenium transition state
by either interactions with the O5' or C1 of the donor. Under normal
circumstances arginine is not a good electron donor. However, both NH1
and NH2 are within hydrogen bonding distances to OD1 of Asp-246, and
atom OD1 of Asn-243 is 3.2 Å from NE of Arg-293. These
interactions might be capable of neutralizing the positive charge
normally associated with arginine. This probably would not be enough to
make it a good nucleophile (as discussed in the double-displacement
mechanism) but may allow Arg-293 to delocalize the increase in positive
charge on the C1 atom in the transition state. This concept is also
supported by the site-directed mutagenesis studies. In the D246A mutant
a positive charge would exist on Arg-293 thus actually inhibiting the
reaction. This would explain why the D246A mutant has undetectable
activity, and the R293A mutant that only removes the arginine side
chain maintains minimal GlcNAc transferase activity (0.2 pmol/mg
protein/h). The N243A mutant, which displays only 1.4 pmol/mg of
protein/h of GlcNAc transferase activity could disrupt the hydrogen
bond network, greatly reducing the effectiveness of Arg-293. In
addition to these conserved interactions with Arg-293, in mEXTL2
Tyr-193 forms a hydrogen bond with the carboxylate group from the
acceptor and is 3.1 Å from NH1 of Arg-293. Thus for EXTL2 this
hydrogen bonding network, which might include the acceptor carboxylate
group, could contribute to stabilization of the transition state.
Conclusions--
The x-ray crystal structures of mEXTL2 represent
the first structures from the EXT gene family. These
structures reveal residues important for substrate donor and acceptor
bindings for the 1,4-N-acetylhexosaminyltransferase, catalyzes the
transfer reaction of N-acetylglucosamine and
N-acetylgalactosamine from the respective UDP-sugars
to the non-reducing end of [glucuronic acid]
1-3[galactose]
1-O-naphthalenemethanol, an
acceptor substrate analog of the natural common linker of various
glycosylaminoglycans. We have solved the x-ray crystal structure of the
catalytic domain of mouse EXTL2 in the apo-form and with donor
substrates UDP-N-acetylglucosamine and
UDP-N-acetylgalactosamine. In addition, a structure of the ternary complex with UDP and the acceptor substrate analog [glucuronic acid]
1-3[galactose]
1-O-naphthalenemethanol has
been determined. These structures reveal three highly conserved
residues, Asn-243, Asp-246, and Arg-293, located at the active
site. Mutation of these residues greatly decreases the activity. In the
ternary complex, an interaction exists between the
-phosphate of the UDP leaving group and the acceptor hydroxyl of the substrate that may
play a functional role in catalysis. These structures represent the
first structures from the exostosin gene family and provide important insight into the mechanisms of
1,4-N-acetylhexosaminyl transfer in heparan biosynthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1,4-glucuronyltransferase and
1,4-N-acetylglucosaminyltransferase activities. Known as heparan polymerases, EXT1 and EXT2 elongate the heparan sulfate chain by alternative additions of GlcUA and GlcNAc to the non-reducing ends. Although a better understanding of how these EXT enzymes function
is critical for elucidating their roles in heparan biosynthesis, no
protein structure from the EXT family of enzymes is available, and the
reaction mechanisms still remains elusive.
1-3[galactose]
1-O-naphthalenemethanol
(GlcUA
1-3Gal
1-O-naphthalenemethanol) (7).
Because of the
1,4-N-acetylglucosaminyltransferase
activity, EXTL2 is suggested to act as an enzyme that can
transfer GlcNAc to the common linker tetrasaccharide of a core protein
(8). On the other hand, the
1,4-N-acetylgalactosaminyltransferase activity is
speculated as an activity that could provide a way to terminate
glycosaminoglycan biosynthesis all together (9), although it is not
known whether EXTL2 is this termination enzyme. EXTL2, the smallest of
the known EXT homologues, consists of 330 amino acid residues and shows
extensive amino acid sequence homology to the C-terminal region of EXT1
and EXT2 that presumably constitutes the
1,4-N-acetylglucosaminyltransferase domain. Thus, EXTL2
provides an excellent model for investigating the structure-based
mechanism of the
1,4-N-acetylglucosaminyltransferase
reaction catalyzed by the EXT gene family.
1-3Gal
1-O-naphthalenemethanol, the binary
complex structures with UDP-GlcNAc and UDP-GalNAc, and the structure of
the apo-enzyme. These structures reveal conserved residues within the
EXT gene family that play a role in substrate binding and/or
catalysis. A site-directed mutagenesis study was performed to provide
functional evidence to support their roles in catalysis. EXTL2
catalyzes a transfer reaction in which the
-linkage of the C1-O1
bond in the donor UDP-sugar is retained in the reaction product. Two
different mechanisms for the retaining transfer reaction have been
proposed, a SN2-like double-displacement mechanism (10) and
a SNi-like reaction mechanism (11, 12). The
SN2-like double-displacement mechanism would likely involve a direct nucleophilic attack on the UDP-sugar at the C1 atom of the
sugar from the opposite side of the UDP moiety. This would invert the
stereochemistry at the C1, releasing the UDP moiety, to form a covalent
intermediate. The acceptor hydroxyl could then attack the C1 carbon in
the same manner releasing the protein side chain inverting the
stereochemistry at the C1 atom back to its original state to form the
final product. The SNi-like mechanism would involve a
dissociation of the UDP and attack on the transition state by the
acceptor from the same side as the leaving UDP to maintain the
stereochemistry at the C1 carbon. Our present mEXTL2 structures are
consistent with the SNi-like reaction mechanism for the
retaining glycosyltransferases. The ternary complex also suggests that
the UDP leaving group may facilitate sugar transfer by deprotonating
the acceptor substrate. These structures represent the first from the
EXT gene family and therefore provide important new
information for enzymes involved in heparan biosynthesis and provide
additional insight into glycosyltransferases in general.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-galactopyranoside was added to a
final concentration of 0.2 mM. Flasks were allowed to shake overnight. Cells were pelleted at 4000 rpm for 10 min and then resuspended in the sonication buffer containing 50 mM
Hepes, pH 7.5, 350 mM NaCl, 0.5 mM
dithiothreitol, 10 µg/ml
1-chloro-3-tosylamido-7-amino-L-2-heptanone (TLCK),
and 10 µg/ml leupeptin. Cells were disrupted by sonication on
ice and then spun down at 38,000 rpm for 30 min. The ~300 ml of
soluble faction was allowed to bind to amylose resin (New England Biolabs) in batch at 4 °C for 1 h with gentle agitation. Resin was spun down at 4 °C for 10 min at 500 × g. The
supernatant was poured off, and the resin was washed with 30 mM Hepes, pH 7.5, 100 mM NaCl at a volume ratio
of 10:1 (buffer:resin). Resin was pelleted and washed a total of five
times. Protein was then eluted from the resin with a buffer containing
25 mM Hepes, pH 7.5, 100 mM NaCl, 20 mM maltose. Protein was concentrated and dialyzed overnight
in 25 mM Hepes, pH 7.5, 100 mM NaCl. Because
treatment with factor Xa did not cleave the fusion protein, subtilisin
was used to digest the fusion protein at a 1 to 10,000 subtilisin to
EXTL2 mass to mass ratio. This cleavage was performed in 25 mM Hepes, pH 7.5, 100 mM NaCl, 1 mM
CaCl2 at room temperature for 1 h followed by
overnight cleavage at 4 °C. The cleavage resulted in producing EXTL2
fragments with molecular masses of ~30 kDa as determined by
SDS-PAGE. The protein was then loaded on to a Ni2+-agarose
column and eluted with a gradient elution of imidazole from 0.0 to 0.3 M. The protein was eluted from the Ni2+-agarose
with ~45 mM imidazole, dialyzed into 20 mM
Hepes, pH 7.5, 100 mM NaCl, and concentrated to 10 mg/ml.
Protein concentration was determined using Bradford protein assay
reagent (Bio-Rad). Selenomethionine-labeled protein was obtained by
transforming the plasmid containing the fusion protein into B834 (DE3)
cells. For expression, 1 ml of overnight culture was added to 1 liter of expression medium in a 2-liter flask containing 10 g of M9 minimal salts, 0.5 g of NaCl, 1.0% glucose, 2 mM
MgSO4, 0.1 mM CaCl2, 1 µg/ml
thiamine, 0.5 g of all essential amino acids (with the exception
of methionine), 7.5 mg of FeSO4, and 50 mg of
selenomethionine. 50 ml of 2YT was added to the flask for the starter
culture. This was allowed to shake at 37 °C at 250 rpm overnight.
200 ml of the starter culture was spun down at 4000 rpm for 10 min. The cells were washed with the expression medium (containing no 2YT) and
spun down again. Then the cells were resuspended in 25 ml of expression
medium, and 5 ml was added to each of 18 2-liter flasks, containing 1 liter of expression medium. These flasks were placed on a shaker at
37 °C and 250 rpm. When the A600 reached 0.6 the temperature was changed to 23 °C, and
isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 0.2 mM. Flasks were allowed to shake
overnight. The expressed selenium-labeled protein was purified
using the same protocol as the wild-type enzyme with the exception of
an addition of 0.5-1 mM dithiothreitol present at all steps.
177 °C by a Molecular Structure
Corp. X-stream device. Oscillation data for this and all
subsequent crystals were collected using phi scans on a rotating anode
RUH3R generator equipped with a RAXIS IV area detector and MSC confocal
blue mirrors. Data were processed using the programs Denzo and
Scalepack (13) (Table I).
Crystallographic data statistics
1-3Gal
1-O-naphthalenemethanol
(GlcUA
1-3Gal
1-n), UDP-GlcNAc, and UDP-GalNAc. Also shown are the
data statistics for the derivative data sets used to phase the apo
native data (seleno-methionine, CH3HgCl, and HgCl2).
cutoff in peak height of a
Fo
Fc density map and
a hydrogen bond distance criteria between 2.3 and 3.5 Å to an oxygen
or nitrogen in the protein model. Additional water molecules were added
manually in the program O based on Fo
Fc density maps and the presence of hydrogen
bonding partners. Additional cycles of refinement and model building
were carried out to obtain the final model. The crystal used for the
UDP plus GlcUA
1-3Gal
1-O-naphthalenemethanol data set
was obtained by soaking an apo-crystal for 20 min in a solution
containing three parts of 11% PEG3000, 0.1 M sodium cacodylate, pH 7.5, 0.2 M MgCl2 to one part
12.5% monomethyl ether PEG2000, 9% PEG3000, 0.1 M sodium
cacodylate, 0.2 M MgCl2, 50 mM MES,
pH 6.5, 5.0 mM UDP, 2.5 mM MnCl2,
and 10 mM
GlcUA
1-3Gal
1-O-naphthalenemethanol. The crystal was
then transferred in two steps into the final cryo-solution containing
12.5% monomethyl ether PEG2000, 9% PEG3000, 13% ethylene glycol, 0.1 M sodium cacodylate, pH 7.5, 0.2 M
MgCl2, 50 mM MES, pH 6.5, 5 mM UDP,
2.5 mM MnCl2, and 10 mM
GlcUA
1-3Gal
1-O-naphthalenemethanol. The crystal was
flash-frozen, and data were collected. For the UDP-GlcNAc data set, a
crystal was soaked in three parts mother liquor to one part
cryo-solution containing 10 mM MnCl2, 10 mM UDP-GlcNAc for 20 min. The crystal was transferred in
two additional steps into the cryo-solution containing 10 mM MnCl2, 10 mM UDP-GlcNAc and
flash-frozen, and data were collected. For the UDP-GalNAc data set, the
same procedure was used as for the UDP-GlcNAc data set with the
substitution of 20 mM UDP-GalNAc for 10 mM
UDP-GlcNAc. For refinement of the data sets containing bound substrates
and analogs, the apo-model was refined against the data in CNS using the same methods as for the apo-protein. Substrates and analogs were
manually built into Fo
Fc density maps followed by additional rounds of
refinement in CNS. Water molecules for the substrate structures were
then added using the same criteria as for the apo-structure followed by
additional rounds of model building and refinement. All data were
always refined at the maximum resolution reported except for the
UDP-GlcNAc structure, which was completely refined to 2.3 Å and then
the data were extended to 2.1 Å for additional rounds of model
building and refinement. The quality of the models was analyzed with
the program Procheck (16). The Ramachandran statistics of the model are
as follows: apo (87% most favored region, 12.3% additional, 0.7%
generous, and no disallowed), UDP plus acceptor (88.6% most favored,
11.2% additional, 0.2% generous, and no disallowed), UDP-GlcNAc
(89.9% most favored, 11.2% additional, and no generous or
disallowed), and UDP-GalNAc (87.4% most favored, 12.6% additional,
and no generous or disallowed).
1-3Gal
1-O-naphthalenemethanol (custom-synthesized by the Peptide Institute, Inc., Osaka, Japan), 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml each
leupeptin and pepstatin A, and 0.5 µg of recombinant EXTL2 protein.
The reaction mixtures were incubated at 37 °C for 0.5 h.
Radiolabeled products were then separated from
UDP-[3H]GlcNAc or UDP-[3H]GalNAc by passage
of the reaction mixture through a Sep-Pak® Vac RC C18 cartridge
(Waters), as described previously (18). The recovered labeled products
were quantified by liquid scintillation counting. To determine
Km of an acceptor substrate for GalNAc and GlcNAc
transferase activities, increasing concentrations (0, 0.03, 0.1, 0.3, 1, and 3 mM) of
GlcUA
1-3Gal
1-O-naphthalenemethanol were added to the
reaction mixtures. Data were fitted by a s/v
s
plot to calculate Km value.
1-3Gal
1-O-naphthalenemethanol (250 nmol), 250 mM UDP-[3H]GlcNAc (8.21 × 105 dpm), 100 mM MES buffer, pH 6.5, 10 mM MnCl2, and 171 mM the sodium
salt of ATP in a total volume of 30 µl. Radiolabeled products were
then separated from UDP-[3H]GlcNAc by passage of the
reaction mixture through HPLC on a Nova-Pak® C18 column
(3.9 × 150 mm; Waters), respectively. The recovered labeled
products were quantified by liquid scintillation counting. In addition,
the specific activities of all mutants, along with wild-type EXTL2,
were calculated based on the transferase activities and the amounts of
the purified fusion enzymes that were quantified using the BCA protein
assay reagent (Pierce).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1-3Gal
1-O-naphthalenmethanol as an
acceptor substrate and the method established previously (18), we
characterized the
1,4-N-acetylhexosaminyltransferase
activity of the catalytic domain. The domain increased the activity as
a function of the acceptor concentration and reached a plateau at ~3
mM concentration. As reported previously (7), this domain
catalyzed both UDP-GalNAc and UDP-GlcNA transferase activities. The
specific activity (7.55 ± 0.71 nmol/mg EXTL2/h) of the UDP-GalNAc
transfer reaction was more than 10-fold higher than that (0.62 ± 0.03 nmol/mg EXTL2/h) of the UDP-GlcNAc transfer reaction. On the other
hand, the Km values for the acceptor substrate were
similar between the two reactions: 320 and 560 µM when
UDP-GalNAc and UDP-GlcNAc were used as the donor substrates,
respectively. These results indicated that the bacterially expressed
30-kDa recombinant domain retains the functional characteristics of EXTL2.
-strands, ordered
3-
2-
1-
4, each separated in
sequence by an
-helix. The central core of the acceptor binding
subdomain is comprised of two
-sheets with the planes of the sheets
oriented relative to each other by ~90°. The first
-sheet is a
mixed
-sheet composed of strands
10-
6-
11-
13. These
strands form a continuous
-sheet from the UDP binding subdomain
resulting in a mixed eight-stranded
-sheet. The second
-sheet in
the acceptor binding subdomain is a twisted anti-parallel sheet
comprised of strands
8-
7-
9-
13. Residues at the interface of
the two sheets from the acceptor binding subdomain form a hydrophobic
core. These sheets are flanked by
-helix 4 on one side and an
-helix cluster consisting of
5,
6,
7, and
8 on the other
side. A disulfide bond formed by the conserved residues Cys-244 and
Cys-296 tethers
-helices 7 and 8. In addition to these structural
elements, a small two-stranded anti-parallel
-sheet exists between
-strands 5 and 12. In the apo-form of the enzyme the majority of
residues between
-strand 12 and
-helix 8 are disordered.
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Fig. 1.
A, ribbon diagram of
the catalytic domain (Ala-63-Lys-327) of mEXTL2 with bound UDP
(orange) and
GlcUA 1-3Gal
1-O-naphthalenemethanol acceptor substrate
analog (blue). The UDP-donor binding subdomain is colored
lavender, and the acceptor binding subdomain is colored
green. The catalytic Mn2+ ion (green)
is pictured with water molecules (red), Asp-153 (of the
DXD motif), and phosphate oxygens from UDP that define the
inner coordination sphere. B, the same figure as Fig.
2A but rotated ~90° about a horizontal axis. These
figures were created using Molscript and Raster3D (24, 25).
-strands 1 and 4 of the UDP subdomain (Fig. 1, A and
B). The orientation of the uridine ring is dictated by a number of interactions (Fig. 2,
A and B). The position of the uridine ring is
determined by hydrogen bonding interactions with Gln-72, Asn-101, and
Asn-130, as well as through a ring stacking interaction with Tyr-74
(Table II). The ribose ring forms
hydrogen bonds with Gln-72 and Asp-152, the middle residue of the
conserved DXD motif. The third Asp of the DXD
motif, Asp-153, forms an indirect interaction with the
- and
-phosphates through its interaction with the catalytic
Mn2+ ion. This Mn2+ ion is also coordinated to
oxygen atoms from both the
- and
-phosphates. The remaining
ligands to the octahedral coordinated Mn2+ are three water
molecules. In the donor-bound structures, Arg-76 forms the only direct
protein phosphate interactions.
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Fig. 2.
Stereo diagram of the donor
binding site of mEXTL2 with bound UDP-GalNAc
(orange). A, residues that
form direct interactions with the donor substrate are pictured
(khaki). An Fo Fc annealed omit map of the UDP-GalNAc molecule
was calculated at 2.1 Å resolution and is pictured contoured at 3
.
B, stereo diagram of the donor binding site of
mEXTL2 with bound UDP-GlcNAc (orange). Residues that form
direct interactions with the donor substrate are pictured
(khaki). An annealed Fo
Fc omit map of the UDP-GlcNAc molecule was
calculated at 2.3 Å and is pictured contoured at 2.5
. These figure
were created using Molscript and Raster3D (24, 25).
Hydrogen bonding partners of donor and acceptor substrates
-helices 7 and 8. In mouse and human EXT1,
EXT2, EXTL1, EXTL2, EXTL3, and EXTL2, as well as Tout-Velu from
Drosophila, Arg-135, Asp-151, and Asp-246 are invariant.
Residues Asp-153 and Arg-293 only differ in the mouse and human EXTL1
sequence. This is also true for His-289, which is oriented such
that atom NE2 is only 4.0 and 4.4 Å from the C1 carbons of the GlcNAc
and GalNAc moieties, respectively. In addition to these highly
conserved amino acids, the amino acid equivalent to Asp-245 in mEXTL2
is either an Asp or Glu in all the above sequences except the EXTL1s. This suggests that the donor binding pocket for UDP-sugar in other members of the EXT family should be very similar to that of mEXTL2.
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Fig. 3.
Alignment of the amino acid sequence of
mEXTL2 with the sequences of the C-terminal domains of the human
heparan polymerases hEXT1 (GenBankTM accession
number Q16394) and hEXT2 (GenBankTM accession
number AAB07008) and the Drosophila EXT homolog
Tout-Velu (GenBankTM accession number
NP034292). Secondary structure elements from the mEXTL2
structures are displayed above the corresponding residues
with -strands represented by green rectangles and
-helices represented by purple cylinders. Residues
involved in binding UDP based on the crystal structure are shown in
orange, and those that interact with the donor sugar of the
UDP-donor are in red. Residues that line the acceptor
substrate binding site are displayed in light blue. The two
conserved cysteines involved in a disulfide bond are colored
green whereas Asp-246, a proposed catalytic residue, is
colored purple. The loop that becomes ordered upon acceptor
substrate binding is highlighted in yellow. This alignment
was created using ClustalW (26).
1-3Gal
1-O-naphthalenemethanol,
results in the ordering of residues in a loop from Glu-275 to Glu-288.
Residues Glu-275 to Thr-277 and Ser-281 to Glu-288 become ordered upon
binding of the acceptor substrate analog. Residues from Gly-282 to
His-285, along with Trp-284, essentially bury the active site and thus
may serve to exclude excess water from the active site during
catalysis. Binding of the acceptor results in two new interactions
between the protein and the UDP molecule. Backbone amide nitrogens,
from Trp-284 and Met-283 that become ordered upon acceptor substrate
binding, form interactions with the
- and
-phosphates of UDP,
respectively (see Fig. 4 and Table II).
Although there are no typical hydrogen bonds between residues in this
loop and the acceptor, Trp-284 is oriented so that the plane of the
side chain is parallel to the imaginary plane of the two sugars in the
acceptor. The positions of the C1 carbon of the acceptor GlcUA and the
C3 of the Gal moiety are such that the hydrogens from these carbons
point directly into the center of the five- and six-member aromatic
rings (3.9 Å). This orientation allows for hydrogen bonding between
the aliphatic carbons of the sugar and the pi orbitals of the side
chain Trp-284 forming what are known as pi-CH hydrogen bonds (20).
View larger version (30K):
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Fig. 4.
Stereo diagram of the acceptor
binding site of mEXTL2 with
GlcUA 1-3Gal
1 of the
GlcUA
1-3Gal
1-O-naphthalenemethanol
substrate analog (blue) and UDP
(orange) pictured. A Fo
Fc omit map of the analog was calculated at
2.7-Å resolution and is pictured contoured at 3
(light
blue). Possible hydrogen bonds are displayed as dashed black
lines. Interactions to the catalytic Mn2+
(green) are displayed as solid black lines. This
figure was created using Molscript and Raster3D (24,
25).
-phosphate of the UDP product. In this structure, the 3- and
4-hydroxyls of GlcUA are in position to form hydrogen bonds with the
-phosphate. This suggests that the UDP portion of the donor may
participate in acceptor binding and possibly aid in catalysis.
1-3Gal
portion of the GlcUA
1-3Gal
1-O-naphthalenemethanol
analog, there is no interpretable density for the naphthalenemethanol moiety. This suggests that this moiety is disordered and forms no
specific interactions with the protein. The 1-hydroxyl group of the
galactose sugar that is connected to the naphthalenemethanol moiety is
directed away from the protein toward the solvent. Thus, based on
position of the analog, it is likely that the acceptor binding is
determined by the interactions between mEXTL2 and the disaccharide
GlcUA
1-3Gal portion. The recognition by mEXTL2 of the disaccharide
portion of the acceptor analog is reminiscent of that observed in
acceptor binding to GlcAT-1, the
1,3-glucuronyltransferase that completes the synthesis of the common glycosaminoglycan
linker region. In the crystal structure of GlcAT-1, electron density was found only for the Gal1
-3Gal portion of the acceptor substrate Gal1
-3Gal1
-4Xyl (21). This suggests that for glycosyltransferases similar to EXTL2 and GlcAT-1, only the disaccharide portion of the
non-reducing end of a given acceptor substrate may need to be
accommodated for catalytic recognition.
-1,3
galactosyltransferase,
3GT (10-12). Unfortunately, one report of
the
3GT structure does not include an acceptor substrate, whereas
coordinates are not available at the present time from the other. The
structure of LgtC provides an ideal comparison, because both a complete
donor substrate analog UDP-2-deoxy-2-fluoro-galactose and an acceptor analog 4'-deoxylactose are present in the crystal structure. The root
mean square deviation on 171 C
s between residue Phe-67 to Leu-273 of
mEXTL2 and 1 to Tyr-245 of LgtC is 3.1 Å as determined by the program
Dali (22). The superposition of the mEXTL2 structure, with UDP-GalNAc
bound to the LgtC structures, reveals that the orientation of the donor
sugar molecules with respect to the UDP are quite similar (Fig.
5). In both cases the sugars are not in an extended conformation but rather tucked under the diphosphates of
the UDP. Many of the interactions with the galactose moiety are
conserved, as well. The first Asp in the DXD motif forms a hydrogen bond with the 3-hydroxyl, as well as Arg-86 from LgtC and
Arg-135 from EXTL2. In addition, the 4-hydroxyl group is within hydrogen bonding distance to Asp-188 in LgtC and Asp-245 in EXTL2. Although the UDP-sugar donors bind in a similar orientation, the acceptor substrate in LgtC is oriented so that the planes of the sugars
are rotated 90° and thus perpendicular with respect to those of
mEXTL2 structure. The substrate in LgtC is translated ~4.5 Å out of
the plane of the acceptor in the EXTL2 structure, as well. Despite this
large difference in overall position, the position of the two acceptor
hydroxyls (when O4 is modeled onto the acceptor for LgtC) with respect
to the C1 carbon of the respective donor sugars, is very similar. These
positions in the retaining enzymes differ from that observed in the
structure of GlcAT-1, an inverting glycosyltransferases where the
leaving group and acceptor are found on directly opposite sides of the
C1 of the donor sugar (23). In the EXTL2 ternary complex, the acceptor 4-hydroxyl is within hydrogen bonding distance of the UDP molecule. This interaction has also been reported for
3GT (12). In all three
structures, LgtC,
3GT, and mEXTL2 the acceptors appear to be located
on the same side of the donor sugar as the leaving group suggesting
these enzymes catalyze reactions that may proceed by a similar
mechanism.
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Fig. 5.
Stereo diagram of the
superposition of the active sites of mEXTL2 with UDP-GalNAc
(orange) modeled onto the UDP in the
UDP/ GlcUA 1-3Gal
1-O-naphthalenemethanol
(blue)/mEXTL2 structure (khaki) to
the crystal structure of the retaining glycosyltransferase LgtC
(green) with bound donor and acceptor analogs
UDP-2-deoxy-2-fluoro-galactose and 4'-deoxylactose, respectively.
The position of the 4'-hydroxyl of the acceptor lactose has been
modeled for this figure. Residues with similar interactions with the
donor sugar are displayed. This figure was created using
Molscript and Raster3D (24, 25).
-linkage of the C1-O1 bond of the donor
UDP-sugar is maintained in the acceptor product. The retaining reaction
has been suggested to proceed via either a double-displacement or
SNi-like reaction mechanism (10-12). The
double-displacement mechanism involves a SN2 in-line attack
on the C1 carbon of the donor to create a
-configuration intermediate either covalently linked to the protein or
non-covalently-bound, followed by a subsequent SN2 attack
on the C1 carbon by the acceptor, converting back to an
-linkage.
The crystal structures of mEXTL2 do not appear to be consistent with a
double-displacement mechanism. In the donor-bound structures, only atom
NH2 of Arg-293 is within 4 Å of the C1 carbon, in line with the
leaving group, so that, based on position, it could act as a
nucleophile and attack the C1 carbon to form a covalent intermediate.
Despite interactions with Asp-246 that could neutralize the positive
charge, Arg-293 is still unlikely to be a good nucleophile. In addition
we could find no examples in the literature where an arginine served as a nucleophile. Another highly conserved residue His-289 is also on the
correct side of the donor sugar for nucleophilic attack. However, NE2
of His-289 is 4.0 and 4.4 Å, respectively, from the C1 carbon in the
UDP-GlcNAc and UDP-GalNAc structures. In addition, the site-directed
mutant H289A maintained full GlcNAc transferase activity (74.4 pmol/mg
protein/h) as compared with the wild-type (73.1 pmol/mg protein/h).
This suggests His-289 is not required for catalysis and therefore
unlikely to function as a catalytic nucleophile. A third possibility
for a double-displacement reaction scheme would involve a non-covalent
intermediate whereby a water molecule serves as the nucleophile in the
first SN2 reaction. Asp-246 could serve as a base in such a
reaction. However, Arg-293 would likely require a conformational change
to accommodate the water for attack on the C1 carbon of the donor, and
the salt bridges between Asp-246 and Arg-293 would have to be
broken for Asp-246 to be a good base. In the apo, donor
substrate-bound, and acceptor substrate-bound structures, there is no
evidence of a water molecule at this position or a conformational
change of Arg-293. Therefore the structural and mutagenesis data
combined suggest that mEXTL2 is unlikely to proceed via the
double-displacement mechanism.
-phosphate in the UDP plus acceptor structures that do not exist in
the UDP-GlcNAc or UPD-GalNAc structures. They are the hydrogen bond
with the amide nitrogen of Trp-284 and between the 3- and 4-hydroxyls
of the acceptor (Fig. 4). These interactions may help stabilize the
increase in negative charge on the leaving oxygen in the transition
state. Superpositions of the UDP-sugar donor structures with the UDP
plus acceptor place the
-phosphate oxygens in slightly different
positions. In these superpositions the 4-hydroxyl of the acceptor is
not within hydrogen bonding distance of the oxygen in the phosphate-C1
bond. Thus it is unclear whether this interaction exists in the
pre-catalytic state with UDP-sugar plus acceptor-bound. If,
however, the interaction does exist, it would not only stabilize the
increase in charge on the leaving group but would also prime the
4-hydroxyl of the acceptor for a same-side attack on the C1. Otherwise
this interaction could form after dissociation of the UDP leaving group
in which case the
-phosphate could still act as a base to
deprotonate the acceptor hydroxyl through the phosphate 4-hydroxyl
interaction seen in the UDP plus acceptor structure. The similar
catalytic implications whereby the UDP can act as a base to deprotonate
the acceptor substrate were suggested previously (12) to interpret the
active site structure of
3GT. If acceptor binding stabilizes the
leaving group and primes the acceptor for catalysis, it is possible
that the main role of the hydrogen bonding network between conserved residues Asn-243, Asp-246, and Arg-293 is to orient the substrates in
the proper position for catalysis.
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Fig. 6.
Stereo diagram of the
superposition of the UDP-GalNAc donor substrate
(orange) onto the active site of the
UDP/GlcUA 1-3Gal
1-O-naphthalenemethanol
(blue)/mEXTL2 structure. Residues for which
site-directed mutagenesis were attempted are shown in khaki.
This figure was created using Molscript and Raster3D (24, 25).
View larger version (16K):
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Fig. 7.
Possible catalytic mechanism for mEXTL2.
The question mark denotes the possible interaction of
Arg-293 with the C1 carbon that could help stabilize the transition
state.
1,4-N-acetylhexosaminyltransferase activity performed by this enzyme. Based on the data presented here,
the most likely mechanism for the
1,4-N-acetylhexosaminyltransfer reaction appears to be
the SNi-like mechanism in which interactions between the
conserved residues Asn-243, Asp-246, and Arg-293 could help correctly
position the substrates for catalysis, as well as stabilize the
transition state. However, further analysis and more structures of
other retaining glycosyltransferases will likely be required for
conclusive determination of the mechanism. Nevertheless, our present
study sheds light on the mechanism by which members of the
EXT gene family carry out the transfer of GlcNAc from
UDP-GlcNAc to an acceptor polysaccharide chain in heparan sulfate
biosynthesis and provides additional data for determining how this and
other retaining glycosyltransferases function.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Thomas A. Darden and Traci M. T. Hall for critical reading of the manuscript and for many thoughtful discussions on the analysis of the data.
![]() |
FOOTNOTES |
---|
* The work at Kobe Pharmaceutical University was supported in part by a grant from the Science Research Promotion Fund of the Japan Private School Promotion Foundation, a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan, and by the Human Frontier Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1OMX, 1OMZ, 1ON6, 1ON8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
To whom correspondence may be addressed. Tel.:
81-0-78-441-7570; Fax: 81-0-78-441-7569; E-mail:
k-sugar@kobepharma-u.ac.jp.
** To whom correspondence may be addressed. Tel.: 919-541-2404; Fax: 919-541-0696; E-mail: negishi@niehs.nih.gov.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M210532200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
m, mouse;
MES, 4-morpholineethanesulfonic acid;
PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid);
PEG, polyethylene glycol;
3GT,
-1,3 galactosyltransferase.
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