Metabolism of vertebrate amino sugars with N-glycolyl groups: Intracellular β-O-linked N-glycolylglucosamine (GlcNGc), UDP-GlcNGc,and the biochemical and structural rationale for the substrate tolerance of β-O-linked& β-N-acetylglucosaminidase

Article abstract of DOI:10.1074/jbc.M112.363721

The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.

Full text of DOI:10.1074/jbc.M112.363721

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 34, pp. 28882–28897, August 17, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Metabolism of Vertebrate Amino Sugars with N-Glycolyl
Groups
INTRACELLULAR ␤-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND
THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF
□
S
␤-O-LINKED ␤-N-ACETYLGLUCOSAMINIDASE*
Received for publication, March 22, 2012, and in revised form, May 25, 2012 Published, JBC Papers in Press, June 12, 2012, DOI 10.1074/jbc.M112.363721
Matthew S. Macauley‡1, Jefferson Chan‡, Wesley F. Zandberg‡, Yuan He§2, Garrett E. Whitworth‡, Keith A. Stubbs¶3,
Scott A. Yuzwaʈ, Andrew J. Bennet‡, Ajit Varki**, Gideon J. Davies§4, and David J. Vocadlo‡ʈ5
From the Departments of ‡Chemistry and ʈMolecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia
V5A 1S6, Canada, the §York Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD York, United
Kingdom, the ¶School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia 6009, Australia,
and the **Departments of Medicine and Cellular and Molecular Medicine, University of California San Diego,
La Jolla, California 92093-0687
Background: The active site of O-GlcNAcase (OGA) is larger than needed to process O-GlcNAc from proteins.
Results: GlcNGc is assimilated to form UDP-GlcNGc and to generate O-GlcNGc, which can be removed by OGA.
Conclusion: OGA has a conserved active site that enables processing of O-GlcNGc.
Significance: Substrate tolerance of OGA occurs to process metabolic variants of O-GlcNAc.
The O-GlcNAc modification involves the attachment of single incorporation and removal of O-GlcNGc in cells occur in a
␤-O-linked N-acetylglucosamine residues to serine and threo-
nine residues of nucleocytoplasmic proteins. Interestingly, pre-
vious biochemical and structural studies have shown that
O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from
proteins, has an active site pocket that tolerates various N-acyl
groups in addition to the N-acetyl group of GlcNAc. The
remarkable sequence and structural conservation of residues
comprising this pocket suggest functional importance. We
hypothesized this pocket enables processing of metabolic vari-
ants of O-GlcNAc that could be formed due to inaccuracy within
the metabolic machinery of the hexosamine biosynthetic path-
way. In the accompanying paper (Bergfeld, A. K., Pearce, O. M.,
Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287,
28865–28881), N-glycolylglucosamine (GlcNGc) was shown to be a
catabolite of NeuNGc. Here, we show that the hexosamine sal-
vage pathway can convert GlcNGc to UDP-GlcNGc, which is
then used to modify proteins with O-GlcNGc. The kinetics of
dynamic manner on a time frame similar to that of O-GlcNAc.
Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc
glycoside reveals OGA can process glycolyl-containing sub-
strates fairly efficiently. A bacterial homolog (BtGH84) of OGA,
from a human gut symbiont, also processes O-GlcNGc sub-
strates, and the structure of this enzyme bound to a GlcNGc-
derived species reveals the molecular basis for tolerance and
binding of GlcNGc. Together, these results demonstrate that
analogs of GlcNAc, such as GlcNGc, are metabolically viable
species and that the conserved active site pocket of OGA likely
evolved to enable processing of mis-incorporated analogs of
O-GlcNAc and thereby prevent their accumulation. Such plas-
ticity in carbohydrate processing enzymes may be a general
feature arising from inaccuracy in hexosamine metabolic
pathways.
In nature, the biosynthesis of centrally important biomol-
ecules, such as DNA and protein, is prone to mis-incorporation
of monomeric units arising from a low level of inaccuracy in the
biosynthetic machinery (1, 2). In some cases, such mis-incor-
poration errors have been proposed to offer evolutionary ben-
efits (3, 4), while in other cases it can be detrimental. As a result
* This work was supported in part by National Institutes of Health Grant
R01GM32373(toA. V.). ThisworkwasalsosupportedbytheCanadianInsti-
tutes for Health Research and the Natural Sciences and Engineering
Research Council of Canada (to D. J. V.).
This article contains supplemental Fig. S1, Scheme 1, and Table S1.
The atomic coordinates and structure factors (codes 4aiu and 4ais) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
□S
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/). of the apparently unavoidable laxness in the accuracy of even
1 Recipient of a senior scholarship from the Michael Smith Foundation for
the most efficient enzymes, editing mechanisms are critical to
Health Research and Natural Sciences and Engineering Research Council
ensure that the accumulation of such errors in proteins and
of Canada.
2 Recipient of Ph.D. support from the Wild Fund of the University of York.
3 Recipient of support from the Australian Research Council.
nucleic acids is minimized (5–7). Carbohydrates, unlike DNA
and protein, are not synthesized using a chemically defined
template but instead their biosynthesis is governed by the tis-
4 Royal Society/Wolfson Research Merit Award recipient and supported by
the Biotechnology and Biological Sciences Research Council.
5 Canada Research Chair in Chemical Glycobiology, a scholar of the Michael sue-specific expression of the enzymes responsible for their
Smith Foundation for Health Research, and an E. W. R. Steacie Memorial
Fellow. To whom correspondence should be addressed: 8888 University
Drive, Burnaby, British Columbia V5A 1S6, Canada. Tel.: 604-291-3530; Fax:
778-782-3765; E-mail: dvocadlo@sfu.ca.
biosynthesis (the glycosyltransferase superfamily) as well as the
cellular localization of these enzymes and their substrates (8).
Glycosyltransferases have been found in many cases to be sur-
28882 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 34•AUGUST 17, 2012

A Metabolic Rationale for the Substrate Tolerance of OGA
FIGURE 1. The Hexosamine biosynthetic pathway leads to the formation of UDP-GlcNAc, which is the donor substrate used by OGT to form the
O-GlcNAc modification, which is in turn processed within the conserved active site pocket found in human OGA and homologs. A, O-GlcNAc consists
of serine and threonine residues modified with a single ␤-O-linked N-acetylglucosamine (GlcNAc) residue. Installation of O-GlcNAc is catalyzed by OGT, which
uses UDP-GlcNAc as a substrate. Removal of O-GlcNAc from modified proteins is catalyzed by OGA. UDP-GlcNAc is made from the hexosamine biosynthetic
pathway (box). GlcNAc can be salvaged by phosphorylation to generate the HBSP intermediate GlcNAc-6-P. UDP-GlcNAc is also used for the biosynthesis of
CMP-Neu5Ac and UDP-GalNAc. B and C, a conserved active site pocket is present around the 2-acetamido substituent in all GH84 family members. B, structure
of BtGH84 with NAG-thiazoline bound in its active site (Protein Data Bank code 2CHN). Highlighted are residues that line the active site including the pocket
formed beneath the 2Ј-methyl group of NAG-thiazoline. C, sequence alignment of several GH84 family members. Residues in boldface are those that line the
active site pocket. Note that Asp-242, Phe-240, Thr-310, and Val-314 are not noted in B because they are either out of plane (into the page) or obscured.
prisingly tolerant to variation in the structure of their preferred gosaccharides. The O-GlcNAc modification is found on pro-
substrates. Indeed, some organisms exploit this substrate teins with a wide range of functions and appears to be essential
promiscuity by diversifying the repertoire of available sub- for life at the single cell level (14). GlcNAc is installed by an
strates (9). These “errors,” which arise from the loose substrate enzyme termed O-GlcNAc transferase (OGT),6 which uses
specificity of the enzyme, can give rise to further diversification UDP-GlcNAc as a substrate (Fig. 1A) (15, 16). UDP-GlcNAc is
of an already heterogeneous population of glycoforms (10). itself a product of the hexosamine biosynthetic pathway
Such variations may offer evolutionary advantages (11) or, (HBSP) (Fig. 1A). This pathway involves four enzymes that con-
alternatively, remain as an evolutionary relic (12). It is impor- vert fructose 6-phosphate, an intermediate in glycolysis, to
tant to point out, however, that cells must be able to degrade the UDP-GlcNAc. Intermediates in the pathway such as glucosa-
full complement of glycoconjugates to recycle individual com- mine (GlcN) and GlcNAc can also be incorporated into the
ponents and avoid the deleterious accumulation of these spe- pathway by a salvage pathway (the hexosamine salvage pathway
cies. This requirement is underscored by the existence of dis- (HSP)) involving enzymes that phosphorylate these species to
ease states, such as the set of lysosomal storage diseases, in form GlcN-6-P and GlcNAc-6-P, respectively. Another enzyme
which various glycoconjugates accumulate to toxic levels due to termed O-GlcNAcase (OGA) catalyzes hydrolysis of the glyco-
the inability of cells to catabolize certain glycoconjugates (13). sidic linkage between GlcNAc and the protein to return pro-
Heterogeneity is well established in forms of glycosylation
6 The abbreviations used are: OGT, O-GlcNAc transferase; HBSP, hexosamine bio-
synthetic pathway; GlcNGc, N-glycolylglucosamine; OGA, O-GlcNAcase; HSP,
hexosamine salvage pathway; NeuNGc, N-glycolylneuramic acid; ManNGc,
N-glycolylmannosamine; ddH2O, double distilled H2O; Hex, hexosaminidase;
rcf, relative centrifugal force; pNP-GlcNGc, 4-nitrophenyl 2-deoxy-2-hydroxy-
acetamido-␤-D-glucopyranoside; GalNGc, N-glycolylgalactosamine.
that exists on the extracellular side of the plasma membrane.
However, the modification of proteins within the nucleus and
cytoplasm with a single ␤-O-linked N-acetylglucosamine
(O-GlcNAc) has been, to date, described as a having an invari-
able structure and linkage and lacking elongation to form oli-
AUGUST 17, 2012•VOLUME 287•NUMBER 34
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A Metabolic Rationale for the Substrate Tolerance of OGA
teins to their unmodified state. The actions of OGA give the strate the molecular basis for the ability of OGA to process a
O-GlcNAc modification a dynamic nature since it has been glycolyl substrate through the crystal structure of a trapped
demonstrated that the lifetime of O-GlcNAc on a protein is less GlcNGc reaction intermediate. We propose that OGA has
than the polypeptide backbone of the protein to which it is evolved this ability to compensate for metabolic promiscuity in
attached (17).
the HSP and HBSP, as well as in OGT, and that without such a
Through a series of biochemical and structural studies (18– proofreading function, O-GlcNGc could accumulate on pro-
23), it has been firmly established that OGA harbors an unusu- teins in an irreversible manner.
ally large active site pocket located directly below the 2-acet-
EXPERIMENTAL PROCEDURES
amido group of the substrate (Fig. 1B). The residues that line
this pocket are conserved among eukaryotic homologs of
General
human OGA and even among homologs from pathogenic and
CTD110.6 was obtained from Covance, and the RL2 and
symbiotic bacteria (Fig. 1C). The near complete conservation of
HGAC85 antibodies were obtained from Abcam. Secondary anti-
these residues suggests functional importance. Yet, within
bodies were obtained from Santa Cruz Biotechnology. Human
mammals, no derivative of GlcNAc bearing a larger substituent
placental hexosaminidase B (HexB) was obtained from Sigma.
than the 2-acetamido group has been shown to occur naturally.
Interestingly, however, the HBSP as well as OGT have been
Cell Culture
shown to have some plasticity in substrate recognition because
ϩ/Ϫ
Ϫ/Ϫ
EMEG
and EMEG
cells were cultured in standard
they can readily use a substrate with an azido substituent
appended off the 2-acetamido group of GlcNAc (24).
DMEM (Invitrogen) containing 5% fetal bovine serum
(HyClone). Cells were passaged every 2–3 days. The growth
rate of EMEG cells was determined as follows: 5000 cells were
plated onto 12-well plates and counted 5 days later using a
hemocytometer. During these 5 days of growth, the media were
replaced after 2 and one-half days to ensure that nutrients
and/or supplements were not depleted. All counting was car-
ried out in a minimum of three replicates. For experiments
involving HPLC analysis of sugars digested from proteins by an
We were stimulated to look for a derivative of GlcNAc and
found a number of older studies pointing toward the possible
existence of one such species. One intriguing study has shown
that glucosamine 6-phosphate acetyltransferase (GNAT) can
accept glycolyl-CoA as a substrate to form N-glycolylglucos-
amine 6-phosphate (GlcNGc) (Fig. 1a). Given that glycolyl-
CoA is itself naturally produced in cells from ␻-hydroxylated
fatty acids (25) makes the production of a GlcNGc species plau-
sible. However, as shown in an accompanying paper (26), met-
abolic feeding of glycolic acid does not lead to the formation of
GlcNGc suggesting that this route is improbable. More com-
pelling is another possible route. Catabolism of N-glycolyl-
neuramic acid (Neu5Gc) proceeds to generate N-glycolylman-
nosamine (ManNGc) via an enzyme termed N-acylneura-
minate pyruvate lyase (27). In the preceding accompanying
paper (26), it is clearly shown that ManNGc can be converted to
GlcNGc by GlcNAc 2-epimerase and to GlcNGc 6-phosphate.
The demonstration that GlcNGc and GlcNGc 6-phosphate are
true catabolic intermediates within cells is significant. In vitro
studies using sheep tissue extracts have shown that GlcNGc can
be converted to GlcNGc-6-P and then to GlcNGc-1-P (28), and
purified GlcNAc-6-P phosphomutase from pig could convert
GlcNGc-6-P to GlcNGc-1-P with only a 3-fold decrease in cat-
alytic efficiency compared with GlcNAc-6-P (29). Given that
OGT can modify protein with 2-azidoacetamido-2-deoxy-D-
glucopyranose (24), which is similar in size to GlcNGc, it
seemed probable that if UDP-GlcNGc could be formed in cells
it could be transferred to proteins as O-GlcNGc.
N-acetylglucosaminidase from Bacteroides thetaiotaomicron
Ϫ/Ϫ
(BtGH84), EMEG
cells (5 ϫ 106) were plated onto 175-cm
flasks and treated with 5 mM GlcNAc, GlcNGc, or PBS as a
control. The cells were grown for 48 h, at which time the cells
reached confluence. The cells were washed with 5 ml of PBS,
3 ml of trypsin solution was added, and the cells were incubated
for 3 min at 37 °C to detach the cells from the plate. The buffer
and cells were then transferred to a 15-ml conical tube and
pelleted by centrifugation (250 rcf, 8 min), and the supernatant
was carefully removed. The cell pellet was stored at Ϫ80 °C
until further processing.
Immunoblotting
Procedures for Western blotting are the same as those
described previously (31). The dilutions of the three anti-
O-GlcNAc antibodies used were 1:4000, 1:500, and 1:1000,
respectively, for CTD110.6, RL2, and HGAC85. To block bind-
ing of the CTD110.6 antibody using either GlcNAc or GlcNGc,
the antibody was first made up to the appropriate dilution in
PBS-T containing 1% BSA. The appropriate amount of sugar
was then incubated with this antibody solution for 10 min at
room temperature, and this solution was used directly to probe
the membranes.
With these thoughts in mind, studies were initiated to test
two hypotheses. The first hypothesis is that GlcNGc can be
salvaged through the HSP and HBSP to generate UDP-GlcNGc
within cells. The second is that GlcNGc can enter into the
O-GlcNAc cycle to form O-GlcNGc-modified proteins through
the action of OGT and then be removed by the action of OGA.
Cloning
The cDNA encoding human GalE was obtained from Ori-
Here, we report multiple lines of evidence supporting both of gene and amplified by PCR using the following two primers:
these hypotheses. The results offer a clear rationale for the pres- 5Ј-AGCAGCCATATGATGGCAGAGAAGGTGCTGG-3Ј (NdeI
ence of the highly conserved active site pocket found in OGA cut site shown in boldface) and 5Ј-AGCAGCCTCGAGTCAG-
and other GH84 enzymes (for a review of the classification sys- GCTTGCGTGCCAAAGCC-3Ј (XhoI cut site shown in bold-
tem of glycoside hydrolases see Ref. 30). We further demon- face). The PCR product was digested with NdeI and XhoI and
28884 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 287•NUMBER 34•AUGUST 17, 2012

A Metabolic Rationale for the Substrate Tolerance of OGA
ligated into pET28a to produce a construct with an N-terminal (Fluka) and was filtered through a 0.22-␮m filter (Millipore)
His6 tag. The clone for SpHex, in pET15 encoding an N-termi- prior to use. Before samples were introduced, the capillary was
nal His6 tag, was obtained as a gift from Dr. Brian Mark (Uni- conditioned by washing sequentially with 1 N NaOH (2 min,
versity of Manitoba). The gene encoding human Agx1, in the 20 p.s.i.), ddH2O (3 min, 20 p.s.i.), and running buffer (5 min,
pTrcHisB expression vector encoding an N-terminal His6 tag, 40 p.s.i.). To ensure reproducible migration times, the running
was obtained as gift from Véronique Piller (Université buffer was replaced after every second run. After injecting a
d’Orléans). Cloning of human OGA and BtGH84 into pET28a short (ϳ15 nanoliters) H2O plug, samples were electrostatically
to encode fusion proteins with an N-terminal His6 tag has been introduced and pre-concentrated at the anode by applying a
described previously (18, 32).
potential of Ϫ10 kV for 10 s according to a field-amplified sam-
Protein Expression—All vectors were transformed into ple injection technique described previously (37). Electropho-
TunerTM(␭DE3) cells (Stratagene) and selected with either 50 resis was carried out at a constant voltage of 30 kV (which pro-
␮g/ml kanamycin or 100 ␮g/ml ampicillin with the exception duced a current of about 90 ␮A), and the capillary was
that SpHex was selected with only 25 ␮g/ml ampicillin. Cells thermostated at 22 °C. Electrophoretograms were derived by
containing the plasmids were grown until an A600 of ϳ1.0 was measuring the absorbance at 254 (Ϯ10) nm at a rate of 4 Hz.
reach, after which time protein expression was induced by addi- Peaks were integrated using 32 Karat 5.0 software (Beckman-
tion of 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside for 3–4 h Coulter), and all data were normalized to the GDP-Glc internal
at 25 °C. Bacteria were pelleted, lysed, and purified by nickel che- standard. All NDP-sugar standards, as well as GlcNAc-1-P,
late affinity chromatography using a previously reported proce- were obtained from Sigma, except for UDP-GlcNGc and UDP-
dure (32).
GalNGc, which were made as part of this work.
Nucleotide Diphosphate Extraction
Large Scale Digestion of Lysates with BtGH84
Nucleotide diphosphate sugars were extracted from cells
Cell pellets were thawed on ice and resuspended in 1 ml of
Ϫ/Ϫ
according to established procedures (33, 34). EMEG
cells buffer consisting of 10 mM sodium phosphate and 10 mM
were plated onto 10-cm plates and grown to 50% confluence sodium chloride, pH 6.5. Solutions were made up very carefully
and then treated with either 5 mM GlcNAc or 5 mM GlcNGc or with the highest quality salts and water to avoid small amounts
PBS as a control. Each growth condition was carried out in of contaminant that would be concentrated over the following
triplicate. After 48 h, a time when the cells have reached con- steps. Low amounts of salts and buffer were used to minimize
fluence, the plates of cells were washed once with PBS before the salt content. The cells were lysed by passing the solution up
the addition of 1 ml of PBS containing 10 mM EDTA. Cells were and down through a 27-gauge needle three times. This suspen-
incubated at 37 °C for 4 min, scraped from the plates using a sion was sonicated (two times for 20 s at 15% duty) followed by
rubber policeman, transferred into 1.5-ml centrifuge tubes, and centrifugation (17,900 rcf, 20 min) to clarify the supernatant.
placed on ice prior to pelleting by centrifugation (100 rcf, 10 The supernatant was made up to 2.5 ml with the same low salt
min, 4 °C). Cell pellets were immediately lysed by adding 750 ␮l buffer and desalted using a PD-10 column equilibrated in same
of pre-chilled (Ϫ20 °C) 75% ethanol and sonicating these sam- low salt buffer as the eluent. One round of desalting did not
ples using a W-375 ultrasonic processor (Heat Systems Ultra- efficiently desalt the samples; therefore, the 3.5 ml of elution
sonics, Inc.) using two blasts of 30 s each. Insoluble material was obtained after the first column was further diluted to 5 ml and
removed from the lysates by centrifugation (21,000 rcf, 15 min, split into two 2.5-ml aliquots, which were desalted a second
4 °C), and the supernatants, which contained the sugar nucleo- time. These eluted proteins were combined (7 ml in total), and
tides, were snap-frozen in liquid nitrogen and then lyophilized. fucose and maltose were added as internal standards (1 ␮M final
To this crude extract was added GDP-Glc (200 pmol) as an concentration) to enable a percentage recovery to be calculated.
internal standard, dissolved in 0.5 ml of ddH2O, and extracted These solutions were treated at 37 °C for 1 h with 200 ␮l of 1
using Envi-Carb graphite solid-phase extraction cartridges (200 mg/ml BtGH84 (that had been dialyzed in the same low salt
mg) (Supelco) exactly as described previously (35). Sugar nucle- buffer) or 200 ␮l of buffer as a control. One set of lysates was not
otides were eluted three times using 1 ml of 25% CH3CN in 50 incubated at 37 °C but instead was kept on ice to control for any
mM triethylammonium acetate, pH 7.0, passed through a molecules that were not removed by desalting and/or released
0.22-␮m filter (Millipore), lyophilized, and stored in dry form at by endogenous glycosidases during the 1-h 37 °C incubation
Ϫ20 °C until analysis by capillary electrophoresis.
step. Following glycosidase digestion, the samples were con-
centrated down to 1 ml on a speed vac (Thermo) heated to
55 °C, and the protein was then precipitated from the reactions
Capillary Electrophoresis
Lyophilized sugar nucleotides were dissolved in 200 ␮l of by the addition of 7 ml of 95% cold ethanol, and the resulting
ddH2O, and an aliquot was diluted 1:4 prior to characterization mixtures were incubated at Ϫ20 °C for 30 min. The precipitated
by capillary electrophoresis by adapting a previously reported protein was pelleted by centrifugation (4000 rpm, 15 min). The
method (36). Capillary electrophoresis was performed using a supernatant was gently decanted and then lyophilized (over-
ProteomeLab PA800 (Beckman-Coulter) equipped with fused night). The precipitate was resuspended in 2 ml of methanol by
silica capillaries of 50 ␮m internal diameter ϫ 44 cm length (to extensive vortexing. Any precipitate that did not dissolve was
the detector). The running buffer, prepared from components removed by centrifugation (17,900 rcf, 10 min), and the super-
of the highest available purity, was 40 mM Na2B4O7 (Sigma), pH natant was evaporated using a speed vac heated to 55 °C. The
9.5, containing 1.0% (w/v) polyethylene glycol (Mr 20,000) residue was taken back up in 150 ␮l of ddH2O. These solutions
AUGUST 17, 2012•VOLUME 287•NUMBER 34
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A Metabolic Rationale for the Substrate Tolerance of OGA
were vortexed and then centrifuged (17,900 rcf, 5 min) to a total of 100 ␮l with various concentrations of substrate and
remove any insoluble debris, and the supernatant was collected 100 nM enzyme in 96-well plates. Reactions were initiated by
for HPLC analysis of GlcNAc or GlcNGc content.
the addition of enzyme and monitored continuously at either
400 nM for OGA-catalyzed reactions or 340 nM for HexB-cata-
lyzed reactions on a 96-well plate reader. Initial rates of reaction
HPLC Analysis of Sugars Liberated by BtGH84 Digestion
Carbohydrates were separated by HPLC anion exchange chro- were obtained between the 2nd and 5th min of the reaction.
matography using an ICS 3000 HPLC equipped with an ASI 100 Rates were converted into absolute amount of product released
automated sample injector (Dionex) and detected using an elec- by comparing the absorbance values to a standard curve of p-ni-
trochemical detector (ED50, Dionex) using a gold working elec- trophenol made up in the same buffer conditions.
trode and an Ag/AgCl reference electrode. To detect GlcNAc, a
Chemical Syntheses-4-Nitrophenyl 2-deoxy-2-
hydroxyacetamido-␤-D-glucopyranoside (pNP-GlcNGc) 7
(Scheme 1)
CarboPac PA20 column was used, and isocratic elution was car-
ried out using 20 mM NaOH. Preliminary experiments designed to
guide optimization of the assay showed that a GlcNGc standard
was retained by the PA20 column and eluted very late in the chro-
1,3,4,6-Tetra-O-acetyl-2-acetyloxyacetamido-2-deoxy-gluco-
matogram. The long retention time of GlcNGc reflects strong pyranose 2—Triethylamine (2.2 ml, 16 mmol) was added to a
interaction with the solid phase of the column (immobilized qua- solution of the hydrochloride salt of 2-amino-2-deoxy-1,3,4,6-
ternary ammonium ion), and this interaction is much stronger tetra-O-acetyl-␤-D-glucopyranose 1 (39) (2.0 g, 5.2 mmol) in
than GlcNAc because the highly basic mobile phase likely results CH2Cl2 (20 ml), at which time the starting material dissolved.
in partial deprotonation of the acidic group present on the glycolyl The reaction mixture was cooled to 0 °C, and acetoxyacetyl
moiety. Therefore, for better detection of GlcNGc, a CarboPak chloride (0.7 ml, 6.5 mmol) was added. The resultant mixture
PA100 column was used, and isocratic elution was carried out was stirred for ϳ2 h at room temperature. When the reaction
using 100 mM NaOH. In each case, 20 ␮l of sample was injected; mixture was judged complete by TLC analysis, the mixture was
this was followed by 20 min of isocratic elution, followed by a diluted with EtOAc. The resulting organic phase was washed
5-min wash with 500 mM NaOH to clean the column between the successively with water, 1 M NaOH, and saturated sodium chlo-
samples. After washing, the column was equilibrated for a mini- ride. The organic phase was dried over MgSO4, filtered, and
mum of 15 min in the same concentration of NaOH used to run concentrated to yield a white crystalline solid. The material was
the isocratic elution. A constant flow rate of 0.4 ml/min was used recrystallized using a mixture of ethyl acetate and hexanes to
with the PA20 column, whereas a flow rate of 0.5 ml/min was used yield the desired compound 2 as a white solid (2.0 g, 86%). For
withthePA100column, asperthemanufacturer’sinstructions. To 1H NMR (600 MHz, CDCl3), ␦ ϭ 6.25 (1H, d, JNH-H2 ϭ 9.2 Hz,
assess the percent recovery and absolute amount of GlcNAc and NH), 5.71 (1H, dd, JH1-H2 ϭ 8.8 Hz, H-1), 5.19–5.11 (2H, m,
GlcNGc present in the samples, standard curves of fucose, malt- H-3/4), 4.53 (1H, A part of ABq, JH-H ϭ 15.4 Hz, CH2), 4.40 (1H,
ose, GlcNAc, and GlcNGc were constructed for concentrations A part of ABq, CH2), 4.32 (1H, ddd, JH2-H3 ϭ 9.1 Hz, H-2), 4.25
ranging from 0.5 to 50 ␮M. GlcNAc (Bioshop), ManNAc (Jülich), (1H, dd, JH5-H6 ϭ 4.3 Hz, JH6-H6 ϭ 12.3 Hz, H-6), 4.12 (1H, dd,
maltose (Sigma), and fucose (Carbosynth) were obtained com-
JH5-H6 ϭ 2.2 Hz, H-6Ј), 3.81 (1H, ddd, JH4-H5 ϭ 9.2 Hz, H-5),
mercially and carefully prepared in ddH2O to construct the stan- 2.15 (3H, s, OAc), 2.10 (3H, s, OAc), 2.08 (3H, s, OAc), 2.02 (3H,
dard curves.
s, OAc), and 2.01 (3H, s, OAc). For 13C NMR (150 MHz,
CDCl3), ␦ ϭ 171.3, 170.6, 169.7, 169.6, 169.2, 167.5 (C ϭ O),
92.4 (C-1), 73.0, 72.1, 67.6 (C-3, C-4, C-5), 62.6, 61.6 (C-6, CH2),
Comparative Digests of Cell Lysates with SpHex and BtGH84
SpHex and BtGH84 were prepared as 1 mg/ml stocks in a 52.7 (C-2), 20.8, 20.7, 20.5 (OAc). For mass spectrometry, FAB
buffer consisting of 50 mM sodium phosphate and 100 mM calculated for C18H26NO12 was 448.1455, and found was
Ϫ/Ϫ
sodium chloride at pH 6.5. Cell lysates from EMEG
cells 448.1445. The melting point ϭ 168–169 °C. For C18H25NO12,
cultured for 48 h with either GlcNAc or GlcGc were treated that calculated was C 48.32, H 5.63 and found was C 48.48, H
with either SpHex or BtGH84 at a final concentration of 0.1 5.59.
mg/ml and incubated at 37 °C. After various times, an aliquot of
2-Deoxy-2-hydroxyacetamido-D-glucopyranose (GlcNGc)—
the reaction was removed and quenched by addition of SDS- Following a published synthetic strategy (40), a solution
PAGE loading buffer and heated at 95 °C for 5 min. Samples of 1,3,4,6-tetra-O-acetyl-2-acetyloxyacetamido-2-deoxy-␤-D-
were analyzed by Western blot using the CTD110.6 glucopyranose 2 (1.5 g, 3.35 mmol) in MeOH (50 ml) was
anti-O-GlcNAc antibody as described above. Densitometric treated with 0.5 M NaOMe (5 ml). The solution was stirred
ϩ
analysis was performed using ImageQuant software.
overnight at room temperature. Amberlite resin (H form) was
added to neutralize the reaction and was filtered. The filtrate
was concentrated under reduced pressure to afford the desired
Enzyme Kinetics
The rates of OGA- and HexB-catalyzed hydrolysis of product as a white solid (711 mg, 90%) and m.p. ϭ 179.0–
pNP-GlcNAc and pNP-GlcNGc (7) were measured using a 180 °C. Spectral properties of the desired product (GlcNGc)
continuous UV/visible assay as described previously (38). matched those described previously (40).
Briefly, 10 mM stocks of the two substrates were made up in
4-Nitrophenyl 2-deoxy-2-hydroxyacetamido-␤-D-glucopy-
either PBS or low pH citrate buffer (50 mM sodium citrate, 100 ranoside 7—A solution of 1,3,4,6-tetra-O-acetyl-2-acetyloxyac-
mM sodium chloride, pH 4.5) for monitoring the OGA- and etamido-2-deoxy-D-glucopyranose 2 (250 mg, 0.56 mmol) and
HexB-catalyzed reactions, respectively. Reactions consisted of 4-nitrophenol (117 mg, 0.84 mmol) in anhydrous CH2Cl2 (10
28886 JOURNAL OF BIOLOGICAL CHEMISTRY
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A Metabolic Rationale for the Substrate Tolerance of OGA
SCHEME 1. (i) AcOCH2C(O)Cl, Et3N, CH2Cl2; (ii) NaOMe, MeOH; (iii) BnNH2, THF; (iv) (PhO)2P(O)Cl, DMAP, CH2Cl2; (v) a; PtO2, THF; b, MeOH:H2O:Et3N (5:2:1);
(vi) 4-nitrophenol, BF3ꢀOEt2, CH2Cl2.
ml) was cooled to 0 °C in an ice bath. The solution was treated Ϫ10 °C. Diphenyl chlorophosphate (0.9 ml, 4.3 mmol) was
with boron trifluoride etherate (177 ␮l, 1.4 mmol) and stirred at
room temperature for 3 h. The reaction mixture was then
washed successively with saturated NaHCO3 (20 ml), water (20
ml), and brine (20 ml). The organic fraction was dried (Na2SO4)
and concentrated under reduced pressure to give a syrup. This
material was purified via flash chromatography (EtOAc/
hexanes, 1:1, v/v) to afford the intermediate 3 as an off-white
solid. Deacetylation of 3 under Zemplén conditions afforded
the desired compound 7 as a light-yellow solid (92 mg, 46% over
two steps). For 1H NMR (600 MHz, D2O), ␦ ϭ 8.19–8.18 (m,
2H, Ar-H), 7.15–7.11 (m, 2H, Ar-H), 5.33 (dd, J ϭ 8.4, 1.2, 1H,
H-1), 4.10–3.98 (m, 3H), 3.91–3.89 (m, 1H), 3.78–3.70 (m, 2H),
3.68–3.61 (m, 1H), 3.54 (t, J ϭ 13.9, 1H), and 1.21 (bs, 1H, NH).
For 13C NMR (151 MHz, D2O), ␦ ϭ 175.4 (C ϭ O), 161.2, 142.2,
125.6, 116.0 (Ar), 97.9 (C-1), 75.8 (C-5), 72.7 (C-3), 69.1 (C-4),
60.4 (CH2O2), 59.9 (C-6), and 54.5 (C-2). The melting point ϭ
188.5–189 °C. For C14H18N2O8, that calculated was C 49.12, H
5.30 and found was C 49.29, H 5.19.
added dropwise and the solution stirred (4 h), gradually warm-
ing to 10 °C. The solution was then diluted with CH2Cl2 (30 ml)
and washed sequentially with cold water, 0.5 M HCl, and satu-
rated sodium bicarbonate solution, then dried (MgSO4), and
concentrated. Flash chromatography (EtOAc/hexanes, 1:3) of
the residue gave the ␣-phosphate 5 as a colorless oil (730 mg,
83%). For 1H NMR (500 MHz, CDCl3), ␦ ϭ 7.38–7.32 (4H, m,
Ar), 7.24–7.18 (6H, m, Ar), 6.40 (1H, d, JNH-H2 ϭ 8.4 Hz, NH),
6.04 (1H, dd, JH1-H2 ϭ 3.2 Hz, JH1-P ϭ 6.0 Hz, H-1), 5.29 (1H, dd,
JH3-H4 ϭ 9.7 Hz, JH4-H5 ϭ 9.7 Hz, H-4), 5.22 (1H, dd, JH2-H3 ϭ
9.7 Hz, H-3), 4.50 (1H, A part of ABq, JH-H ϭ 15.5 Hz, CH2), 4.39
(1H, m, H-2), 4.18–4.14 (2H, m, CH2, H-6), 4.06 (1H, ddd,
J
H5-H6 ϭ 2.0 Hz, JH5-H6 ϭ 6.0 Hz, H-5), 3.89 (1H, dd, JH6-H6Ј
ϭ
12.6 Hz, H-6Ј), 2.06 (3H, s, OAc), 2.04 (3H, s, OAc), 2.03 (3H, s,
OAc), and 1.99 (3H, s, OAc). For 13C NMR (125 MHz, CDCl3),
␦ ϭ 171.7, 170.5, 169.5, 168.9, 167.6 (C ϭ O), 150.1, 129.9,
125.8, 119.8 (Ar), 96.6 (JC1-P ϭ 6.7 Hz, C-1), 70.1, 69.5, 66.7
(C-3, C-4, C-5), 62.3, 61.0 (C-6, CH2), 52.3 (JC2-P ϭ 8.0 Hz, C-2),
20.5, 20.4, and 20.3 (OAc). For mass spectrometry, FAB calcu-
lated for C28H33NO14P was 638.1624 and found was 638.1639.
For C28H32NO14P, that calculated was C 52.75, H 5.06 and
found was C 52.68, H 5.13.
Bis(triethylammonium) 2-Hydroxyacetamido-2-deoxy-␣-D-
glucopyranosyl Phosphate 6
3,4,6-Tri-O-acetyl-2-acetyloxyacetamido-2-deoxy-D-gluco-
pyranose 4—To acetate 2 (1.0 g, 2.2 mmol) dissolved in THF
(10 ml) was added benzylamine (0.4 ml, 3.5 mmol), and the
mixture was kept (2 h). The mixture was then diluted with
CH2Cl2 (30 ml) and washed sequentially with cold water, 0.5 M
HCl, and saturated sodium bicarbonate solution, then dried
(MgSO4), and concentrated affording a colorless oil (800 mg).
This material, presumably the hemiacetal 4, was used in the
next step without any further purification.
Bis(triethylammonium) 2-Hydroxyacetamido-2-deoxy-␣-D-
glucopyranosyl Phosphate 6—PtO2 (40 mg) was added to a solu-
tion of the ␣-phosphate 5 (110 mg) in THF (5 ml), and the
mixture was stirred under an atmosphere of hydrogen gas (RT,
48 h). When the reaction was judged complete by TLC analysis,
the mixture was filtered through Celite and concentrated in
vacuo. The crude material was then resuspended in MeOH/
H2O/triethylamine (5:2:1, 5 ml) and stirred for 16 h at 4 °C and
then concentrated in vacuo to yield the phosphate 6 as a color-
less oil with no further purification required (85 mg, 98%). For
Diphenyl (3,4,6-Tri-O-acetyl-2-acetyloxyacetamido-2-de-
oxy-␣-D-glucopyranosyl) Phosphate 5—A solution of the hemi-
acetal 4 (0.56 g, 1.4 mmol) and 4-dimethylaminopyridine
(DMAP, 1.00 g, 8.2 mmol) in CH2Cl2 (30 ml) was cooled to 1H NMR (500 MHz, D2O), ␦ ϭ 5.39 (1H, dd, JH1-H2 ϭ 3.3 Hz,
AUGUST 17, 2012•VOLUME 287•NUMBER 34
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A Metabolic Rationale for the Substrate Tolerance of OGA
J
H1-P ϭ 7.6 Hz, H-1), 4.15 (1H, A part of ABq, JH-H ϭ 16.5 Hz, BtGH84-glycolic acid structure, above. Details of data and
CH2), 4.11 (1H, A part of ABq, CH2), 3.99 (1H, ddd, JH2-P ϭ 2.1 final structure quality are shown in supplemental Table 1.
Hz, JH2-H3 ϭ 10.5 Hz, H-2), 3.94 (1H, ddd, JH5-H6 ϭ 2.2 Hz, Coordinates have been deposited with the Protein Data Bank
J
H5-H6 ϭ 5.1 Hz, JH4-H5 ϭ 10.1 Hz, H-5), 3.89–3.84 (2H, m, H-3, under the codes 4aiu and 4ais.
H-6), 3.78(1H, dd, JH6-H6Јϭ12.4Hz, H-6Ј), 3.51(1H, dd, JH3-H4 ϭ9.2
Hz, H-4), 3.20 (12H, q, JCH2-CH3 ϭ 7.4 Hz, CH2CH3), and 1.28
(18H, t, CH2CH3). For 13C NMR (125 MHz, D2O), ␦ ϭ 176.3
RESULTS AND DISCUSSION
To begin our search for a viable metabolically-derived
(C ϭ O), 93.1 (JC1-P ϭ 5.5 Hz, C-1), 73.3, 71.9, 70.8 (C-3, C-4, GlcNAc analog, we felt that a cell line in which the biosynthesis
C-5), 62.0, 61.6 (C-6, CH2), 54.6 (JC2-P ϭ 7.7 Hz, C-2), 47.6 of UDP-GlcNAc is compromised would be useful. Because
(CH2CH3), and 9.2 (CH2CH3). For mass spectrometry, FAB cal- UDP-GlcNAc is generally abundant in cells, lower levels of
culated for C8H15NNa2O10P was 362.0229 and found was UDP-GlcNAc would facilitate the observation of an analog of
362.0224.
GlcNAc and enhance the effects of feeding exogenous GlcNAc
analogs that would compete with GlcNAc derived through
the HBSP. It is known, however, that the biosynthesis of
UDP-GlcNAc is necessary for life; deletion of the gene encod-
2-Deoxy-2-hydroxyacetamido-D-mannopyranose (ManNGc)
(supplemental Scheme 1)
Following a published synthetic strategy (41), a solution of ing glucosamine-6-phosphate acetyltransferase (EMEG32)
mannosamine hydrochloride 8 (1.46 g, 6.77 mmol) and results in embryonic lethality in mice (47). Despite the fatal
NaHCO3 (3.5 g, 42 mmol) in H2O (60 ml) was cooled in an consequence of deleting the EMEG32 gene at the organismal
ice-bath and treated with acetoxyacetyl chloride (3.9 ml, 36 level, mouse embryonic fibroblasts from EMEG32-deficient
mmol). After stirring for 1 h, the solution was neutralized with embryos have been generated and readily propagated (47).
ϩ/Ϫ
aqueous HCl. The reaction mixture was concentrated under Although heterozygous (EMEG32
) cells have normal
reduced pressure, and the crude residue was purified via flash growth rates and show levels of UDP-GlcNAc comparable with
Ϫ/Ϫ
chromatography (EtOAc/iPrOH/H2O, 27:8:4, v/v) to afford the those of WT cells, the growth rate of EMEG32
cells is dra-
intermediate 9 as a white solid. This material was dissolved in matically impaired, and their cellular UDP-GlcNAc levels are
ϩ/Ϫ
autoclaved H2O (20 ml), treated with amano lipase A from ϳ1/50th the concentration of those found in EMEG32
and
Ϫ/Ϫ
Aspergillus niger (500 mg, activity Ն12,000 units/g) and incu- WT cells. It is likely that EMEG32
cells derive this
bated at 37 °C for 48 h. The solution was filtered through an UDP-GlcNAc from scavenging free GlcNAc and degrading
Amicon filter and lyophilized to afford the title product as a GlcNAc-containing glycans present in the serum used during
white solid (952 mg, 59%). Spectral properties of the desired cell culture. Consistent with this view, we find that the growth
Ϫ/Ϫ
product matched those described previously (41).
rate of EMEG
cells is affected by serum concentration to a
ϩ/Ϫ
much larger extent than are EMEG
cells. Boehmelt et al.
cells could both be rescued by
Crystallography
(47) demonstrated that the slow growth rate and cellular levels
Ϫ/Ϫ
To obtain the complex structure of BtGH84-glycolic acid, of UDP-GlcNAc of EMEG32
apo-BtGH84 crystals, grown as described previously (18), were addition of GlcNAc to the media. Thus, we felt that
Ϫ/Ϫ
soaked in a drop containing 0.1 M NH4OAc, 15% (w/v) EMEG32
mouse embryonic fibroblasts would be an excel-
PEG3350, 20% glycerol, and 0.4 M glycolic acid at pH 6.0 over- lent tool for investigating the effects of exogenously added
night. The crystals were subsequently flash-frozen in liquid GlcNAc, or analogs thereof, on cells as well as evaluating the
nitrogen prior to data collection. X-ray data of BtGH84-glycolic tolerance of the HBSP to such compounds.
Ϫ/Ϫ
acid were collected from a single crystal to 2.0 Å on beamline
Rescue of the Growth Rate of EMEG
Cells by GlcNGc and
ID14-1 at the European Synchrotron Radiation Facility. All data ManNGc—Because OGA shows a highly conserved active site
were integrated using MOSFLM (42), followed by data scaling pocket that most likely accommodates structural variants of
and merging with SCALA from the CCP4 suite of program O-GlcNAc, we thought it would be interesting to see if cells
CCP4 suite (43). The structure was solved using molecular treated with metabolically viable analogs of GlcNAc could
replacement method using the apo structure (Protein Data incorporate these in place of O-GlcNAc. Earlier suggestions
Bank code 2CHO) as an initial searching model. Manual cor- regarding the existence of GlcNGc (25), and an accompanying
rections to the model were made with COOT (44), and refine- paper (26), which provides compelling evidence that both
ment cycles were performed with REFMAC (45).
GlcNGc and GlcNGc-6-phosphate are catabolic intermediates
The D242N-glycolyl oxazoline complex was obtained by derived from Neu5Gc, provide a clear rationale to evaluate
soaking of D242N crystals (46) in a drop containing crystalliza- whether GlcNGc could be incorporated as O-GlcNGc. Using
Ϫ/Ϫ
tion mother liquor supplemented with a minute amount of EMEG32
cells, we observe that the growth rate of these cells
pNP-GlcGc 7 for 5 min. Soaked crystals were then quickly approached that of heterozygous cells when 10 mM GlcNAc
transferred to a second drop comprising the same solution was added to the culture media (Fig. 2A). Heterozygous cells
with glycerol concentration raised to 20% (w/v) before the showed no enhancement in growth rate when GlcNAc was
crystals were flash-frozen in liquid nitrogen. Data for added to the culture media (Fig. 2B); results that are consistent
D242N-glycolyl oxazoline were collected at the European with previous studies using this cell line (47). In parallel, we
Synchrotron Radiation Facility (Grenoble) to 2.25 Å resolu- treated cells with varying concentrations of GlcNGc. In
Ϫ/Ϫ
tion on beamline ID29. Data processing and structure solu- EMEG32
cells, GlcNGc rescued the growth rate in a dose-
tion were performed essentially as described for the dependent manner up to a concentration of 10 mM, such that
28888 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287•NUMBER 34•AUGUST 17, 2012

A Metabolic Rationale for the Substrate Tolerance of OGA
passively diffuse into the cell. With these complications in
Ϫ/Ϫ
mind, the growth rate data for EMEG32
cells treated with
ManNGc does suggest that it can be epimerized to GlcNGc,
which together with the growth rate rescue observed using
ManNAc suggests that GlcNAc 2-epimerase is operative in
these cells. The data further suggest that the HBSP is likely
capable of converting GlcNGc to UDP-GlcNGc.
Analysis of UDP-GlcNAc Levels—To test if exogenously
added GlcNGc can be converted to UDP-GlcNGc within
Ϫ/Ϫ
EMEG32
cells, we used a capillary electrophoresis assay to
Ϫ/Ϫ
analyze levels of NDP-sugars from cells. When EMEG32
cells were incubated with 5 mM GlcNAc overnight, the levels of
UDP-GlcNAc dramatically increased as expected (Fig. 3A,
GlcNAc). On incubation with 5 mM GlcNGc, two new peaks
appeared in the electrophoretogram (Fig. 3A, GlcNGc, peaks 2
and 5). One peak corresponds to a species eluting slightly before
UDP-GlcNAc (Fig. 3A, peak 2). We confirmed that this was a
distinct species by mixing samples derived from cells treated
with a mixture of GlcNAc and GlcNGc (Fig. 3A, mixture). A
second new species gave rise to a peak (Fig. 3A, peak 5) eluting
between UDP-Glc and UDP-GalNAc. We hypothesized that
the two new peaks were UDP-GlcNGc and, because UDP-
GalNAc is made directly from UDP-GlcNAc, UDP-GalNGc. In
the absence of standards, however, the identity of these two
new peaks could not be unambiguously assigned. Accordingly,
synthetic standards of UDP-GlcNGc 12 and UDP-GalNGc 14
FIGURE 2. GlcNGc and ManNGc rescue the growth rate of EMEG32؊/؊
mouse embryonic fibroblasts. Approximately 5000 EMEG32Ϫ/Ϫ (A and C)
EMEG32ϩ/Ϫ (B and D) cells were plated onto 12-well plates and at the end of 5
days of growth; the cells were counted on a hemocytometer to obtain the
doubling time. Cells were cultured with the indicated concentration of
GlcNAc or GlcNGc (A and B) and ManNAc or ManNGc (C and D) through the
experiment. Each experimental condition was repeated in at least triplicate,
and the errors represent the standard deviation between these replicates.
the growth rate essentially matched that observed when using were prepared enzymatically from chemically synthesized
GlcNAc (Fig. 2A). Above 10 mM GlcNGc, cells began to grow GlcNGc-1-P 6 (Scheme 2). We used two recombinant enzymes
more slowly, and this appears to be specific to GlcNGc treat- for this biosynthesis as follows: Agx1, a UDP-HexNAc pyro-
ment in EMEG32-deficient cells, because we do not observe the phosphorylase that catalyzes the transfer of UMP from UTP to
ϩ/Ϫ
same effect when using GlcNGc in EMEG32
cells (Fig. 2B). GlcNAc-1-P, and GalE, a UDP-HexNAc 4-epimerase that cat-
These results suggested to us that it might be that GlcNGc is alyzes the interconversion of UDP-GlcNAc and UDP-GalNAc.
being converted to UDP-GlcNGc, which can compensate for As shown in Fig. 3B, Agx1 yielded UDP-GlcNAc 11 when incu-
decreased UDP-GlcNAc and thereby rescue growth. Following bated with GlcNAc-1-P and UTP (Scheme 2). When GalE was
the idea that GlcNGc might be derived from Neu5Gc, we added to the resulting reaction mixture, a second peak could be
Ϫ/Ϫ
treated EMEG32
cells with various concentrations of observed in the electrophoretogram that corresponded to
ManNAc and found that high concentrations of ManNAc also UDP-GalNAc 13, which indicates that these enzymes are
rescued their growth rate (Fig. 2C). The high concentration of active. When GlcNGc-1-P was used as a substrate for Agx1, a
ManNAc required to observe this effect is in keeping with ear- peak was observed that eluted from the capillary slightly before
lier reports showing that ManNAc is not as readily taken up UDP-GlcNAc, and importantly, this peak has the same electro-
into the cell as is GlcNAc (48) and that uptake of ManNAc is phoretic mobility as the first new peak (peak 2) from the cells
nonsaturable up to a concentration of 20 mM (49). When using treated with GlcNGc. When GalE was added to this reaction
ManNGc, we found a minor increase in growth rate at 20 mM; mixture (Scheme 2), another species eluted from the capillary at
however, the difference did not reach statistical significance the same time as the second new peak (peak 5) observed in the
(p ϭ 0.09) (Fig. 2C). Furthermore, we found that 20 mM sample obtained from cells treated with GlcNGc. Carrying out
ManNGc had a slightly deleterious effect on the growth rate of a reaction with GlcNAc-1-P and GlcNGc-1-P at equimolar
ϩ/Ϫ
EMEG32
cells (Fig. 2D). This latter observation suggested concentrations in the presence of Agx1 and GalE resulted in the
to us that the effect of ManNGc on the growth rate of production of the expected four product peaks (Fig. 3B, peaks 2,
Ϫ/Ϫ
EMEG32
cells might be complicated by two countervailing 3, 5, and 6). It should be noted that the two enzymes we use
effects. The first is that epimerization of ManNGc to GlcNGc here, Agx1 and GalE, are recombinant human enzymes. These
might result in increased UDP-GlcNGc levels, and this could observations therefore support the ability of these enzymes to
rescue the growth rate by substituting for UDP-GlcNAc. The process substrates having the glycolyl moiety in human cells
second is that high levels of ManNGc or some metabolic inter- and other eukaryotes, such as mice, in which these enzymes are
mediate derived from ManNGc might have a deleterious effect highly conserved at the amino acid level. Although a quantita-
on growth rate. An additional complication is that because tive kinetic analysis was not performed, it should be noted that
ManNAc appears to only gain access to the cell through passive in Fig. 3B both UDP-GlcNAc 11 and UDP-GlcNGc 12 were
diffusion (49), the additional hydroxyl group of the glycolyl unit produced quantitatively. Also, the absolute amount of UDP-
of ManNGc might compromise the ability of this molecule to GlcNGc produced in cells treated with 5 mM GlcNGc is similar
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A Metabolic Rationale for the Substrate Tolerance of OGA
plementary evidence for the metabolic promiscuity of the
HBSP and GalE by showing exogenous GalNGc is converted to
both UDP-GalNGc and UDP-GlcNGc in cultured mammalian
cells.
Immunoblot Evidence for Modification of Proteins with
O-GlcNGc—Having established that UDP-GlcNGc 12 can be
biosynthesized within cells, it follows that it may be used as a
substrate by OGT. OGT has been previously shown to use uri-
dine diphosphate N-azidoacetylglucosamine UDP-GlcNAz as a
substrate (24), which suggests that it might be able to use UDP-
GlcNGc 12 with reasonable efficiency because the azide and
hydroxyl substituents of the compounds are similar in size. To
Ϫ/Ϫ
test this possibility, we treated EMEG32
cells for 24 h with 5
mM GlcNAc or GlcNGc, after which O-GlcNAc levels were
analyzed by Western blot using the CTD110.6 anti-O-GlcNAc
antibody (Fig. 4A). As measured this way, we find that
O-GlcNAc levels increased substantially in cells treated with 5
mM GlcNAc compared with control cells, which is in line with
the large increase in UDP-GlcNAc levels observed when these
cells were treated with 5 mM GlcNAc (Fig. 4A). In contrast, the
immunoreactivity of lysates derived from cells treated with 5
mM GlcNGc was greatly diminished compared with control
cells despite equivalent loading of all samples. Initially, this
result was surprising to us and prompted the investigation of
two other anti-O-GlcNAc antibodies (RL2 and HGAC85). We
therefore tested these antibodies and observed a similar phe-
Ϫ/Ϫ
nomenon (Fig. 4, B and C). Interestingly, EMEG32
cells
treated with 20 mM of either ManNAc or ManNGc showed a
similar trend except that the differences in levels of immunore-
activity were not as dramatic (Fig. 4D). Two possibilities could
account for the observed decreases in immunoreactivity. The
first possibility is that these antibodies have decreased binding
affinity to possible O-GlcNGc-modified proteins. The second
possibility is that some GlcNGc metabolite could act as an
inhibitor of OGT. To test the first possibility, the CTD110.6
anti-O-GlcNAc antibody was analyzed for its ability to bind
GlcNAc and GlcNGc by preincubating it with different concen-
trations of GlcNAc or GlcNGc prior to using it in a Western
blot. We found that at least 100-fold more GlcNGc is required
to block antibody binding (Fig. 4E). Thus, the CTD110.6 anti-
body has a preference for the 2-acetamido group of GlcNAc
FIGURE 3. UDP-GlcNGc and UDP-GalNGc can be biosynthesized from
exogenously supplied GlcNGc in EMEG32؊/؊ mouse embryonic fibro- over the 2-N-glycolyl group of GlcNGc, and the decrease in
blasts. A, EMEG32Ϫ/Ϫ cells were treated for 24 h with either 5 mM GlcNAc or
Ϫ/Ϫ
immunoreactivity observed in lysates from EMEG32
cells
GlcNGc. Cells were then harvested, and nucleotide mono-, di-, and triphos-
phate-containing compounds were isolated from cell extracts and analyzed
by capillary electrophoresis. Commercially available standards (upper trace)
established the electrophoretic mobilities of the following: 1) GDP-Glc (8.6
min;usedasaninternalstandard);3)UDP-GlcNAc11(8.9min);4)UDP-Glc(9.3
min); 6) UDP-GalNAc 13 (9.85 min); and 7) UDP-Gal (10.2 min). Peaks labeled 2
(8.8 min) and 5 (9.75 min) are unique to cells treated with GlcNGc and mixing
samples from GlcNAc- and GlcNGc-treated cells (mixture) strongly suggest-
ing that peak 2 is not the peak arising from UDP-GlcNAc. B, to establish the
identity of peaks 2 and 5, GlcNAc-1-P and GlcNGc-1-P were converted to
UDP-GlcNAc and UDP-GlcNGc, respectively, by the enzyme Agx1. Addition-
ally, UDP-GlcNAc and UDP-GlcNGc 12 were converted to UDP-GalNAc 13 and
UDP-GalNGc 14, respectively, by the epimerase GalE.
treated with GlcNGc or ManNGc could represent the accumu-
lation of ␤-O-linked GlcNGc on nucleocytoplasmic proteins.
Evidence for Modification of Proteins with O-GlcNGc by
HPLC Analysis—To obtain more direct evidence for the pres-
ence of O-GlcNGc-modified proteins, we monitored GlcNAc
or GlcNGc enzymatically liberated from proteins by anion-ex-
change HPLC. GlcNAc or GlcNGc was released from proteins
using a bacterial homolog of OGA from B. thetaiodomicron
(BtGH84). This enzyme was used because of the following: (i) it
can efficiently remove O-GlcNAc from modified proteins (18);
to the amount of UDP-GlcNAc formed when cells were treated (ii) it can be expressed recombinantly at high levels, which
with 5 mM GlcNAc. These data indicate that the enzymatic allows the use of large quantities to drive reactions toward com-
machinery of the HBSP in mammals is capable of efficiently pletion (18), and (iii) it hydrolyzes GlcNGc glycosides only
Ϫ/Ϫ
converting GlcNGc to UDP-GlcNGc 12 as well as slightly less efficiently than GlcNAc (see below). EMEG32
UDP-GalNGc 14. An accompanying paper (50) provides com- cells (5 ϫ 107) were treated with either 5 mM GlcNAc or
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A Metabolic Rationale for the Substrate Tolerance of OGA
SCHEME 2. (i) UTP, Agx1; (ii) GalE.
FIGURE 4. Effect of GlcNGc and ManNGc on O-GlcNAc levels in EMEG32؊/؊ mouse embryonic fibroblasts. Cells were treated for 24 h with either 5 mM of
GlcNAcorGlcNGc(A–C)and20mM ManNAcorManNGc(D). O-GlcNAclevelswereassessedbyWesternblotanalysesusingthreedifferent O-GlcNAcantibodies.
In all cases, cells treated with GlcNGc or ManNGc show lower immunoreactivity using these antibodies (A and D, CTD110.6; B, RL2; C, HGAC85). The upper panels
represents Western blots with the specified anti-O-GlcNAc antibody, and the lower panels are Western blots obtained using an anti-␤-actin to demonstrate equal
loading of samples. E, CTD110.6 anti-O-GlcNAc antibody binds GlcNGc with lower affinity than GlcNAc. The antibody was incubated with the specified concentration
of either GlcNAc or GlcNGc for 10 min prior to as well as during probing of the blots containing cell lysates from EMEG32Ϫ/Ϫ cells treated with GlcNAc.
GlcNGc for 48 h, and desalted cell lysates were incubated with The interpretation was initially complicated by the fact that in
active BtGH84 (WT BtGH84) or inactive mutant BtGH84 all the assays, no peak had the same retention time as the
(BtGH84-D242A). Despite two rounds of desalting, we find GlcNGc standard. There was, however, a peak in all the samples
sample lysates from cells treated with GlcNAc contained a treated with GlcNGc that eluted ϳ0.4 min earlier than the
small amount of GlcNAc even without adding BtGH84 (Fig. 5A, GlcNGc standard does, and significantly, this peak was larger
Enzyme (t ϭ 0)). When the lysates were incubated with the when analyzing lysates from cells incubated with GlcNGc and
inactive mutant BtGH84-D242A, the size of the peak corre- then digested with WT BtGH84 (Fig. 5B). To investigate
sponding to GlcNAc remained unchanged (Fig. 5A, ϩ Mut whether this peak corresponded to GlcNGc, and whether it
Enzyme). However, digestion of lysates with WT BtGH84 simply eluted earlier because of other factors, such as the com-
greatly increased the size of the peak corresponding to GlcNAc position of the sample, we carried out an additional experiment
(Fig. 5A, ϩ WT Enzyme). The results obtained for the lysates (Fig. 5C). As mentioned above, analysis of lysates from
derived from cells treated with GlcNGc appear initially to be GlcNAc-treated cells, which were never exposed to exoge-
more challenging to interpret (Fig. 5B); therefore, we carried nously added GlcNGc, revealed no peaks having an elution time
out several additional controls (Fig. 5C) to clarify the results. corresponding to either the GlcNGc standard or the peak
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A Metabolic Rationale for the Substrate Tolerance of OGA
GlcNGc and its retention time differs from that of the standard
due to the composition of the samples; the standard is in water,
and the experimental sample contains other small molecules
that can affect electrophoretic mobility.
Dynamic Addition and Removal of GlcNGc—The results of
Ϫ/Ϫ
the HPLC assay shown above indicate that EMEG32
cells
treated with GlcNGc have terminal ␤-O-linked GlcNGc within
their glycoconjugates. It is possible that some of this GlcNGc is
contained within soluble glycoconjugates other than O-
GlcNGc present on nucleocytoplasmic proteins. Yet, as many
other forms of glycoconjugates are found on proteins stably
associated with membranes and on secreted proteins, many of
these would have been removed by washing the cells and by the
centrifugation step following cell lysis. One hallmark of the
O-GlcNAc modification of nucleocytoplasmic proteins is its
dynamic nature (14). To assess the time-dependent addition
Ϫ/Ϫ
and removal of GlcNGc from proteins in EMEG32
cells, we
cultured cells for 48 h in the presence of either 5 mM GlcNAc or
GlcNGc. After this time, the cells were washed, and the media
were changed to media containing the other compound. Cells
were then harvested at various times after switching the media
to analyze O-GlcNAc levels by Western blot. When cells were
initially grown in GlcNGc and then switched to GlcNAc, we
found a time-dependent increase in O-GlcNAc immunoreac-
tivity up to a maximum at 48 h (Fig. 6A). This increase in immu-
noreactivity represents the removal of GlcNGc from proteins
and subsequent addition of GlcNAc. Based on these data, we
estimate the half-life for this process to be 24 h. Interestingly, to
our knowledge only two published studies have attempted to
quantify the dynamics of O-GlcNAc; one study estimated a
half-life for O-GlcNAc of 48 h on a cytokeratin (51), and
another study estimated a half-life of 12 h on an ␣-crystallin
(17). Although many factors likely contribute to the residency
time of O-GlcNAc (or O-GlcNGc) on proteins, the global rates
that are estimated from these data are in line with the rates
determined previously on individual proteins. When we first
cultured cells in media containing GlcNAc and then switched
to media containing GlcNGc, O-GlcNAc immunoreactivity
showed a time-dependent decrease over 48 h (Fig. 6B). In this
experiment, the decrease in signal represents the combined
processes of removal of GlcNAc from proteins and subsequent
addition of GlcNGc. These results support the view that the
GlcNGc can be added to and removed from proteins in a
dynamic manner consistent with that observed for O-GlcNAc.
O-GlcNGc in Cells Having an Intact HBSP—The evidence we
present thus far for O-GlcNGc modification of nucleocytosolic
FIGURE 5. Direct support for the attachment of GlcNGc to proteins via a
␤-O-linkage. EMEG32Ϫ/Ϫ cells were treated with either 5 mM GlcNAc (A) or 5
mM GlcNGc (B) for 24 h. Cell lysates were incubated with 1 mg/ml BtGH84 for
60 min, and protein was removed by ethanol precipitation. The samples were
then analyzed by anion-exchange HPLC. For cells treated with GlcNAc, sam-
pleswereanalyzedonaPA20(strongbinding)withamobilephaseconsisting
of 20 mM NaOH and an internal standard of 20 ␮M fucose. For cells treated
with GlcNGc, samples were analyzed on a PA100 (weaker binding) with a
mobile phase consisting of 100 mM NaOH and an internal standard of 20 ␮M
maltose. The internal standards were added into the samples prior to BtGH84
digestion to assess the percent recovery through handling steps. The reten-
tion times of GlcNAc (7.4 min) and GlcNGc (11.1 min) standards were used to
determinethepeakcorrespondingtothesespeciesfromthereactions. C, var-
iation in the retention time of GlcNGc observed when assaying standards
compared with samples derived from cell lysates was resolved by adding a
standard to lysates. Samples derived from cells treated with GlcNAc, but with-
out added BtGH84, were assayed both with and without added 1 ␮M GlcNGc.
Ϫ/Ϫ
proteins in cells treated with GlcNGc comes from EMEG32
cells, which lack a functional HBSP and cannot therefore inde-
pendently biosynthesize GlcNAc. To investigate if exogenously
added GlcNGc is able to compete with the regular flux through
ϩ/Ϫ
the HBSP in cells containing a functional HBSP, EMEG32
cells were used because they have normal UDP-GlcNAc levels
observed in the digested GlcNGc-treated cell lysates. When a (47); consistent with this, their growth rate is not dependent on
small amount (2 ␮M) of GlcNGc was added to this cell lysate, a exogenously added GlcNAc (Fig. 2B). We speculated that if
new peak appeared that eluted 0.4 min earlier than did the GlcNGc can compete with GlcNAc endogenously produced via
GlcNGc standard, and significantly, this new peak aligned with the HBSP, then exogenously added GlcNGc should result in
the peak hypothesized to be GlcNGc in the lysates obtained decreased O-GlcNAc immunoreactivity. To test this proposal,
ϩ/Ϫ
from GlcNGc-treated cells. Accordingly, this peak arises from we treated EMEG32
cells for 48 h with either 5 mM GlcNAc
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A Metabolic Rationale for the Substrate Tolerance of OGA
FIGURE 6. Time course for addition of and removal of GlcNGc from EMEG32؊/؊ mouse embryonic fibroblasts. Cells were grown for 48 h in the presence
of 5 mM GlcNGc (A) or GlcNAc (B), and then the media were replaced with media containing 5 mM of the other compound. At various times, the cells were
harvested, and O-GlcNAc levels were assessed by Western blot. The upper panels represent Western blots obtained using the CTD110.6 anti-O-GlcNAc
antibody, and the lower panels are Western blots obtained using an anti-␤-actin, which demonstrates equivalent loading of samples.
glycolyl-containing substrates as compared with the lysosomal
HexB, thestructure of whichrevealsthereisnopocket beneath the
2-acetamido group (53, 54). This highly conserved pocket (Fig. 1, B
and C) found in N-acetylglucosaminidases from GH84 is highly
conserved and is likely present to ensure that any O-GlcNGc, or
other adventitiously incorporated GlcNAc analogs, formed
within cells can be removed and not accumulate on proteins.
Molecular Basis for the Ability of OGA to Process of GlcNGc
Substrate—In an effort to provide structural insight into
GlcNGc binding to OGA, we solved the structure of BtGH84 in
complex with glycolic acid. BtGH84 is a good model of human
OGA because active site residues are entirely conserved
between these two enzymes. The rationale for using glycolic
acid was that the initial “apo” BtGH84 structure had shown a
molecule of acetate binding in the N-acetyl binding “pocket”
and glycerol (used as a cryo-protectant) binding in place of the
O4-C4-O5-C5-C6-O6 atoms of N-acetylglucosamine; the two
fragments mimicked well the position of the natural substrate
(18). When glycolate was soaked into the apo-crystal, we
observed it bound in a position previously occupied by an ace-
tate molecule, suggesting this is representative of the binding of
the N-glycolyl moiety of GlcNGc (together also with the bind-
ing of glycerol, Fig. 9, A and B (supplemental Table 1)). Inter-
estingly, the hydroxyl group of glycolic acid displayed different
binding modes in the two molecules found within the unit cell
of the crystal. In molecule A, it forms OH-␲ interactions with
Tyr-282 (3.4 Å from ring center) and makes one H-bond with
the thiol functionality of Cys-278. In molecule B, this hydroxyl
group is within hydrogen bond distance to carboxyl group of
Asp-242 and also makes OH-␲ interactions with Trp-337 (2.6
Å from ring center). The different binding modes observed for
this hydroxyl group implies high mobility of the molecule
within the crystal, which is also reflected by its relatively higher
B-factor values. To obtain more direct structural evidence for a
catalytically relevant GlcNGc species, we aimed to obtain a
BtGH84 bound GlcNGc structure. To achieve this aim, we built
upon our previous observation of a catalytically competent oxa-
zoline intermediate (46), and thus, by using the D242N mutant
of BtGH84 in combination with the substrate pNP-GlcNGc 7,
FIGURE7. EffectofGlcNGcandManNGcon O-GlcNAclevelsinEMEG32ϩ/Ϫ
mouse embryonic fibroblasts. Cells were treated for 48 h with either 5 mM
GlcNAc or GlcNGc (A) and either 20 mM ManNAc or ManNGc (B). The upper
panels represent Western blots obtained using the CTD110.6 anti-O-GlcNAc
antibody, and the lower panels are Western blots obtained using an anti-␤-
actin to demonstrate equivalent loading of samples.
or GlcNGc, and CTD110.6-positive immunoreactivity present
in lysates was assessed by Western blot (Fig. 7A). Cells treated
with GlcNAc showed only a slight increase in O-GlcNAc
immunoreactivity, whereas cells treated with GlcNGc showed a
decrease that is consistent with the studies presented above and
can be accounted for by poor recognition of GlcNGc by the
ϩ/Ϫ
antibody. Treatment of EMEG32
cells with either 20 mM
ManNAc or ManNGc produced a similar outcome (Fig. 7B).
Collectively, these data indicate that GlcNGc can feed into the
HBSP via the HSP and be incorporated as O-GlcNGc in the
presence of an intact HBSP.
Activity of Human OGA toward pNP-GlcNGc 7—Biochemi-
cal studies of human OGA and the structures of OGA
homologs (18–23) have revealed the active site contains a
pocket that might enable processing O-GlcNGc-modified pro-
teins and other GlcNGc glycosides. To address this question,
we prepared (Scheme 1) pNP-GlcNGc, 7 and tested it as a sub-
strate for both human OGA and HexB. Consistent with the
cell-based studies, we find that OGA is able to catalyze cleavage
of this substrate (Fig. 8A) with a catalytic efficiency (Vmax/[E]ꢀKm)
toward pNP-GlcNGc that is 48-fold lower compared with
pNP-GlcNAc (Fig. 8Aand Table 1). However, the drop in catalytic
efficiency of human HexB toward pNP-GlcNGc as compared with
pNP-GlcNAc was much greater (550-fold) (Fig. 8B and Table 1).
Therefore, it appears that the large active site pocket of OGA we were able to trap a glycolyl-oxazoline reaction intermediate
revealed previously (18–20, 52) makes it more tolerant toward bound within the active site. Unambiguous electron density
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A Metabolic Rationale for the Substrate Tolerance of OGA
FIGURE 8. Activity of human OGA and HexB toward pNP-GlcNGc (7). The catalytic efficiency of human OGA (A) and HexB (B) was assessed by obtaining full
Michaelis-Menten plots against pNP-GlcNAc (squares) and pNP-GlcNGc (circles). Insets show that rates with the glycolyl derivative were slower but did show
Michaelis-Menten behavior.
TABLE 1
Enzymatic parameters describing the hydrolysis of pNP-GlcNAc and pNP-GlcNGc 7 by human OGA and HexB
Enzyme
Substrate
V
max/[E]0
Km
V
max/[E]0Km
(Vmax/[E]0 Km)Ac/(Vmax/[E]0Km)Gc
Ϫ1
Ϫ1
␮mol minϪ1 mg
mM
0.55
2.2
␮mol minϪ1 mgϪ1mM
OGA
HexB
pNP-GlcNAc
pNP-GlcNGc
pNP-GlcNAc
pNP-GlcNGc
1.8
3.3
48
0.15
25
0.068
36
0.71
4.9
550
0.32
0.065
revealed the glycolyl oxazoline derivative (Fig. 9C) bound in a conservation among mammals, it appears likely that GlcNGc
4C1 chair conformation that is essentially identical to that of could be found in a wide range of tissues and mammals.
previously described D242N-oxazoline (C␣ root mean square
The consequence of these observations and the biological
deviation ϭ 0.23 Å), with almost invariant active site interac- significance of O-GlcNGc and how it might interact with
tions (Fig. 9D). In brief, the C6 hydroxyl (carbohydrate num- O-GlcNAc are difficult to predict. Clearly, GlcNGc can substi-
bering) donates one H-bond to Asp-344. The C4 hydroxyl is tute for GlcNAc as measured by its ability to rescue the growth
Ϫ/Ϫ
H-bonded to Asp-344 and Asn-372. The C3 hydroxyl rate of EMEG32
cells (Fig. 2A). One interesting possible
receives one H-bond from the main chain carbonyl of Gly- scenario is that cells under certain metabolic pressures might
135. The oxazoline ring nitrogen is H-bonded to Asp-242, produce various short chain fatty acids that could be incorpo-
although the oxazoline oxygen receives one H-bond from rated to form analogs of GlcNAc, and this may either confer
Asn-339. One notable feature of the new structure lies in the some adaptive benefit or be maladaptive. It is interesting in this
glycolyl hydroxyl group, which is closely sandwiched by Trp- regard that short chain N-acyl variants have been found on the
337 and Tyr-282, to form an H-bond with the thiol of Cys- lysine residues of histones (55). These alternative modifications
278 at the bottom of the active site. These two structures are likely derived from cellular pools of propionyl-CoA and
therefore support the hypothesis that BtGH84 is able to bind butyryl-CoA as acetyltransferases can use these substrates with
and process metabolically derived O-GlcNGc and also offer a reasonable efficiency (for review see Refs. 56, 57). Whether such
structural rationale for this binding.
variants have biologically significant roles, or are the conse-
Conclusions—These collective observations support the view quence of the imperfection of enzymatic processes in their abil-
that O-GlcNGc modification of nucleocytoplasmic proteins ity to distinguish small structural variants of substrates,
can occur from metabolites such as GlcNGc and GlcNGc remains a topic of interest. In this regard, it should be noted that
6-phosphate that are generated by catabolism of Neu5Gc as although we find good support for O-GlcNGc being able to
shown in an accompanying paper (26). Less likely, based on data occur, there is no evidence to support that it is an abundant or
from another accompanying paper (50) is that GlcNGc could functionally distinct species. In this regard, however, the
arise by de novo synthesis of GlcNGc via the HBSP. We also find accompanying paper (26) provides clear evidence for GlcNGc
that GlcNGc introduced to cells having a functional HBSP can and GlcNGc 6-phosphate as true catabolic intermediates
compete with the normal flux through the HBSP and become derived from Neu5Gc, which is abundant in nonhuman verte-
covalently attached to nucleocytosolic proteins as O-GlcNGc. brates. Indeed, even if GlcNGc levels are significantly lower
Interestingly, O-GlcNGc can turn over within a similar time than GlcNAc levels and the efficiency of processing of GlcNGc
frame as O-GlcNAc, indicating that the enzymes involved have is lower than that of GlcNAc, principles of enzyme kinetics
a fair level of tolerance for this monosaccharide and for O- dictate that inevitably O-GlcNGc will form at some level. Given
GlcNGc. Enzyme kinetic and structural studies reveal the quan- that we show here that UDP-GlcNGc and UDP-GalNGc are
titative and structural basis for the tolerance of OGA toward O- formed within cells from GlcNGc, it seems very possible that
GlcNGc, supporting the cell-based observations. Given the ubiq- other glycan structures could incorporate N-glycolyl-hexo-
uitous nature of the HBSP, OGA, and OGT in tissues and their samines. Consistent with this view, in an accompanying paper
28894 JOURNAL OF BIOLOGICAL CHEMISTRY
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A Metabolic Rationale for the Substrate Tolerance of OGA
FIGURE 9. Three-dimensional structures of glycolic acid and glycolyl oxazoline bound within the N-acyl binding pocket of BtGH84 support the
catalytic competence of OGA toward O-GlcNGc. A and B, binding modes of glycerol and glycolic acid (cyan) to two BtGH84 molecules in the crystal structure
to mimic interactions with the substrate. Electron densities are maximum likelihood weighted 2Fobs Ϫ Fcalc countered at 1␴. Hydrogen bonds, within 3.2 Å, to
enzyme active site residues are shown as dashed lines. C, the glycolyl-oxazoline intermediate (magneta) bound to the D242N variant of BtGH84. D, overlay of
D242N-glycolyl oxazoline with D242N-oxazoline (green), highlighting invariant interactions in both structures. These figures were drawn with PyMOL.
(50), we show that GalNGc can be incorporated into various cell possibility is a topic of interest given the observations regarding
surface glycans. Notably, even enzymatic activities that are both
ancient and critical for life, including DNA and protein biosyn-
thesis, show less than complete fidelity. In response to such
enzymatic promiscuity, adaptive mechanisms have evolved to
correct and cope with such errors (5–7). Here, we propose that
the active site pocket of OGA and the substrate promiscuity of
OGA are highly conserved exactly because of the lack of complete
fidelity of the HBSP and OGT. OGA therefore acts to “proofread”
the incorporation of variants of GlcNAc. More generally, these
observations set precedent for the possibility that other carbohy-
drate-processing enzymes may show similar plasticity. This
metabolic promiscuity of glycosylation pathways and glycosyl-
transferases made in this and the accompanying papers (26,
50).
Acknowledgments—We thank Prof. Tak Mak of the Ontario Cancer
Institute for providing EMEG32 cells and Prof. Mario Pinto for
providing access to capillary electrophoresis instrumentation. We
thank Dr. Véronique Piller of Université d’Orléans for the
pTrcHisB expression vector encoding human Agx1 bearing an
N-terminal His6 tag.
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A Metabolic Rationale for the Substrate Tolerance of OGA
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AUGUST 17, 2012•VOLUME 287•NUMBER 34
JOURNAL OF BIOLOGICAL CHEMISTRY 28897

Glycobiology and Extracellular Matrices:
Metabolism of Vertebrate Amino Sugars
with N-Glycolyl Groups:
INTRACELLULAR β-O-LINKED
N-GLYCOLYLGLUCOSAMINE
(GlcNGc), UDP-GlcNGc, AND THE
BIOCHEMICAL AND STRUCTURAL
RATIONALE FOR THE SUBSTRATE
TOLERANCE OF β-O-LINKED β
-N-ACETYLGLUCOSAMINIDASE
Matthew S. Macauley, Jefferson Chan,
Wesley F. Zandberg, Yuan He, Garrett E.
Whitworth, Keith A. Stubbs, Scott A. Yuzwa,
Andrew J. Bennet, Ajit Varki, Gideon J.
Davies and David J. Vocadlo
J. Biol. Chem. 2012, 287:28882-28897.
doi: 10.1074/jbc.M112.363721 originally published online June 12, 2012
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