From the Departments of Chemistry and
Molecular and Cell Biology and the ** Howard Hughes
Medical Institute, University of California, Berkeley, California 94720 and the
Center for Advanced Materials,
Materials Science Division, Lawrence Berkeley National Laboratory,
Berkeley, California 94720, and the ¶ Department of
Bioengineering, Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan
Received for publication, November 27, 2002, and in revised form, December 23, 2002
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ABSTRACT |
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Sialic acid is a major determinant of
carbohydrate-receptor interactions in many systems pertinent to human
health and disease. N-Acetylmannosamine (ManNAc) is the
first committed intermediate in the sialic acid biosynthetic pathway;
thus, the mechanisms that control intracellular ManNAc levels are
important regulators of sialic acid production. UDP-GlcNAc 2-epimerase
and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc
from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has
been shown to direct metabolic flux toward sialic acid in
vivo, the function of the latter enzyme is unclear. Here we study
the effects of GlcNAc 2-epimerase expression on sialic acid production
in cells. A key tool we developed for this study is a cell-permeable,
small molecule inhibitor of GlcNAc 2-epimerase designed based on
mechanistic principles. Our results indicate that, unlike UDP-GlcNAc
2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc
2-epimerase can serve a catabolic role, diverting metabolic flux away
from the sialic acid pathway.
Carbohydrate-receptor interactions participate in numerous
cell-cell recognition events in eukaryotes (1-4). Of the nine monosaccharides that constitute mammalian polysaccharides, the common
terminal residue sialic acid stands out as a major determinant of
cell-cell interactions. Sialic acid is a component of sialyl Lewis x, a
tetrasaccharide that binds the selectin family of adhesion molecules
and initiates inflammatory leukocyte adhesion (5). Sialic acid is also
the major binding determinant of Siglecs (sialic acid-binding immunoglobulin superfamily
lectins), a family of sialic acid-binding
lectins that are involved in many processes including B-cell signaling
and activation (6).
Because of the prominent role of sialic acid in cell surface
recognition, there is considerable interest in the regulatory mechanisms that control its biosynthesis and presentation on the cell
surface (Fig. 1) (7). The first committed intermediate in the
biosynthesis of sialic acid is N-acetylmannosamine
(ManNAc),1 which is
phosphorylated by ManNAc 6-kinase (8, 9) to initiate sialic acid
biosynthesis in the cytosol. The subsequent action of sialic acid
synthase (10), followed by an unknown phosphatase, yields sialic
acid, which is then transformed into CMP-sialic acid by CMP-sialic acid
synthetase in the nucleus (11, 12). After transport into the Golgi
compartment, the sialyltransferases utilize this substrate to sialylate
the terminal position of oligosaccharide chains. Transcriptional
regulation of sialyltransferases is one mechanism for controlling the
production of certain sialylated epitopes such as polysialic acid (13),
sialyl Lewis x (5), and The overall production of sialic acid in mammalian cells appears to be
regulated by the availability of ManNAc, which, in principle, can be
synthesized through two possible routes.
UDP-N-Acetylglucosamine (UDP-GlcNAc) 2-epimerase (Fig.
1) catalyzes the conversion of UDP-GlcNAc
to ManNAc and has been implicated as a major source of ManNAc in
mammalian cells (14-16). High levels of CMP-sialic acid inhibit
UDP-GlcNAc 2-epimerase, thereby reducing flux in the sialic acid
biosynthetic pathway (17). A second enzyme, GlcNAc 2-epimerase, can
also produce ManNAc by catalyzing the reversible epimerization of
GlcNAc to ManNAc (18-20). However, GlcNAc is thermodynamically favored
with an equilibrium ratio of 3.9:1 (21). Thus, it is possible that
GlcNAc 2-epimerase functions to divert ManNAc away from sialic acid
biosynthesis and into other glycosylation or glycolytic pathways.
Additionally, human GlcNAc 2-epimerase binds the protease renin (22),
but as of yet no functional significance for this interaction has been
demonstrated (23). In summary, the role of UDP-GlcNAc 2-epimerase in
the biosynthesis of sialic acid has been clearly established, but the
cellular role of GlcNAc 2-epimerase remains unclear.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-2,6-linked sialyllactosamine (6).
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Fig. 1.
Sialic acid biosynthesis. ManNAc is
synthesized from UDP-GlcNAc by UDP-GlcNAc 2-epimerase, but the role of
GlcNAc 2-epimerase is unclear. ManNAc or a ManNAc analog is
phosphorylated by ManNAc 6-kinase to yield ManNAc 6-phosphate
(ManNAc 6-P). ManNAc 6-phosphate is subsequently condensed
with phosphoenolpyruvate (PEP) to yield sialic acid
9-phosphate (sialic acid 9-P) in a reaction catalyzed by
sialic acid-9-phosphate synthase. Dephosphorylation of sialic acid
9-phosphate by an unknown phosphatase and transport to the nucleus
enables CMP-sialic acid synthetase to produce CMP-sialic acid.
Following transport into the Golgi compartment, CMP-sialic acid is
utilized by the sialyltransferases that append the sialic acid to
glycoconjugates ultimately destined for the cell surface or
secretion.
Northern analysis of the mRNAs encoding these epimerases has revealed some differences in their tissue distribution in mice. UDP-GlcNAc 2-epimerase is highly expressed in the liver (9, 24), whereas GlcNAc 2-epimerase is highly expressed in the kidney (23). The enzymes do appear to have some overlapping tissue distribution, however, suggesting either that one enzyme is redundant or the functions they perform are distinct.
To investigate the function of GlcNAc 2-epimerase in cells, we
identified a cell line lacking the enzyme, introduced the gene into
these cells, and studied the effects of GlcNAc 2-epimerase expression
on sialic acid biosynthetic flux. We found that GlcNAc 2-epimerase
expression suppressed sialic acid production in response to the
exogenous addition of ManNAc or ManNAc analogs. Furthermore, guided by
a proposed chemical mechanism (21), we prepared two novel
substrate-based inhibitors of GlcNAc 2-epimerase and demonstrated that
one of these inhibitors functions within cells to block the action of
the enzyme. Using the inhibitor as a tool, we confirmed that the
phenotype resulting from GlcNAc 2-epimerase expression was indeed due
to direct action of the enzyme. Based on these data, we propose that
GlcNAc 2-epimerase plays a catabolic role in sialic acid metabolism.
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EXPERIMENTAL PROCEDURES |
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Materials-- Pfu DNA polymerase was from Stratagene. All PCRs were performed on an MJ Research DNA Engine. The vector pDsred2-N1 was from Clontech. His-bind resin and pET28b(+) were from Novagen. All restriction enzymes and T4 DNA ligase were from New England Biolabs. DNA sequencing was performed by the University of California at Berkeley DNA Sequencing Facility or Davis Sequencing. RPMI 1640 medium, LipofectAMINE PLUS, Hygromycin, Zeocin, the Micro-FastTrack 2.0 mRNA isolation system, the Superscript first strand synthesis system for RT-PCR, and the FlpIn system were purchased from Invitrogen. FITC-avidin, penicillin, streptomycin, biotin hydrazide, and GlcNAc were purchased from Sigma. ManNAc was purchased from Pfanstiehl. Fetal calf serum was from Hyclone. Maackia amurensis-FITC and Limax flavus-FITC were purchased from EY Laboratories, and Tritrichomonas mobilensis-biotin was from Calbiochem. Cell densities were determined using a Coulter Z2 cell counter. Flow cytometry data were acquired using a Coulter EPICS XL-MCL flow cytometer or a BD Biosciences FACSCalibur flow cytometer equipped with a 488-nm argon laser. For flow cytometry experiments, 10,000 live cells were analyzed, and all data points were collected in triplicate.
All chemical reagents were obtained from commercial suppliers and used without further purification unless otherwise noted. All NMR spectra were measured with a Bruker AMX-300 MHz, AMX-400 MHz, or DRX-500 MHz spectrometer as noted. All coupling constants (J) are reported in Hz. Mass spectral data were obtained from the University of California at the Berkeley Mass Spectrometry Laboratory. Reversed-phase HPLC was performed using a Rainin Dynamax SD-200 HPLC system with 220-nm detection on a Microsorb C18 analytical column (4.6 × 250 mm) at a flow rate of 1.0 ml/min or a preparative column (25 × 250 mm) at a flow rate of 20 ml/min. Ac4ManLev (25), Ac4ManNAz (26), and compound 2 (see Fig. 6) (27) were synthesized as previously described.
Expression of GlcNAc 2-Epimerase in Jurkat Cells-- For mammalian expression, the GlcNAc 2-epimerase gene was cloned into the pcDNA5/FRT vector, a component of the FlpIn system, by digestion of pUKHRB6 (20) and pcDNA5/FRT with EcoRI and subsequent ligation with T4 DNA ligase. The correct product was confirmed by DNA sequencing, and the vector was denoted pSJLG2E/FRT. The gene encoding the red fluorescent protein (dsRed) was also cloned into pcDNA5/FRT. The vectors pcDNA5/FRT and pDsRed2-N1 were digested with HindIII and NotI, and the gene encoding dsRed was ligated into pcDNA5/FRT with T4 DNA ligase. The correct product was confirmed by DNA sequencing, and the vector was denoted pSJLRFP/FRT.
Jurkat cell lines stably expressing GlcNAc 2-epimerase were generated with the FlpIn system according to the manufacturer's instructions. Briefly, Jurkat cells were transfected with pFRT/lacZeo using LipofectAMINE PLUS. After selection using Zeocin, stable transfectants were isolated that contain Flp recombinase sites flanking the Zeocin resistance gene. Clonal populations were isolated using "feeder" Jurkat cells as previously described (28), and the gene encoding dsRed was introduced into selected clones to determine whether the site of incorporation yielded high, homogeneous gene expression. The vectors pSJLRFP/FRT and pOG44 (encoding the Flp recombinase) were introduced into various Zeocin-resistant clones, and cells were selected for Hygromycin resistance. The cells were analyzed by flow cytometry, and one Zeocin-resistant cell line was selected as the host for GlcNAc 2-epimerase (denoted wt*). The vectors pSJLG2E/FRT and pOG44 were introduced into wt* cells using LipofectAMINE PLUS. After selection in the presence of Hygromycin, the resulting population was denoted G2E*.
Jurkat and Jurkat-derived cell lines were maintained in a 5.0% CO2, water-saturated atmosphere at 37 °C and grown in RPMI 1640 medium supplemented with penicillin (100 units/ml) and streptomycin (0.1 mg/ml). wt* cells were grown in the presence of Zeocin (100 µg/ml), and G2E* cells were grown in the presence of Hygromycin (300 µg/ml). Typically, both cell lines were grown in T-25 flasks, and cell densities were maintained between 2.0 × 105 and 1.6 × 106 cells/ml.
RT-PCR on wt* and G2E* Cells--
Messenger RNA from 5.0 × 106 of Jurkat, wt*, and G2E* cells was isolated using the
Micro-FastTrack 2.0 mRNA Isolation kit according to the
manufacturer's instructions. cDNA was synthesized using the
SuperScript first strand synthesis kit and oligo(dT) primers. The
GlcNAc 2-epimerase gene was amplified using forward primer (5'-GCAGGTATGGATGTATTGTCG-3') and reverse primer
(5'-CTGTCACTGTAACCCATGAGG-3'), and the -actin gene was detected with
forward primer (5'-GTGGGCCGCTCTAGGCACAA-3') and reverse primer
(5'-CTCTTTGATGTCACGCACGATTTC-3'). PCR was performed using
Pfu DNA polymerase and the following cycling conditions: 96 °C for 3.0 min; 30 cycles of 96 °C for 45 s, 55 °C for
45 s, 72 °C for 90 s; and then 72 °C for 5.0 min.
Flow Cytometry-- Cells were seeded at a density of 2.0 × 105 cells/ml and incubated for 3 days with the compounds indicated. Cells were then washed and stained with either biotin hydrazide and FITC-avidin to detect ketones (29) or with phosphine-FLAG and anti-FLAG-FITC to detect azides (30). For lectin staining, wt* and G2E* cells were washed twice with wash buffer (PBS, pH 7.1, containing 1.0% fetal calf serum) and incubated with T. mobilensis-biotin for 1.0 h at room temperature. Following incubation, cells were washed three times with wash buffer and stained with FITC-avidin (1:250 dilution in wash buffer) for 30 min at 4.0 °C. M. amurensis-FITC (1:200 dilution in wash buffer) and L. flavus-FITC (1:50 dilution in wash buffer) staining was performed for 30 min at 4.0 °C. Cells were washed twice and then analyzed by flow cytometry.
Periodate-Resorcinol Determination of Sialic Acid Concentration-- wt* or G2E* cells were incubated with the indicated substrate or with no substrate. Cells were grown to a density between 6.0 × 105 and 9.0 × 105 cells/ml over 3 days, and the periodate-resorcinol assay was performed as described (28, 31).
Synthesis of Compound 1--
Hydroxylamine hydrochloride
(4.0 g, 0.057 mol) and sodium methoxide (3.0 g, 0.057 mol) were stirred
in methanol for 30 min. The resulting white salts were removed by
filtration. H2O (30 ml) and GlcNAc (5.0 g, 0.022 mol) were
added to the filtrate, and the reaction was stirred for 18 h at
40 °C. The reaction was concentrated and filtered through a plug of
silica, eluting with 5:1 CHCl3/MeOH to yield the desired
product (5.2 g, 100%). Compound 1 was further purified for
enzymatic assays by recrystallization from acetonitrile and methanol
and then characterized as a mixture of E and Z
isomers. 1H NMR (500 MHz, D2O): 1.89 (3H,
s), 1.89 (3H, s), 3.35 (1H, dd, J = 2.0, 8.5), 3.39 (1H, dd, J = 2.0, 8.5), 3.47 (2H, dd, J = 6.5, 12.0), 3.60 (2H, m), 3.68 (2H, J = 3.0, 12.0),
3.92 (1H, dd, J = 2.0, 7.5), 3.96 (1H, dd,
J = 2.0, 7.5), 4.54 (1H, dd, J = 6.0, 7.5), 5.02 (1H, app t, J = 7.3), 6.64 (1H, d,
J = 6.5), 7.32 (1H, d, J = 6.0).
13C NMR (125 MHz, D2O):
20.9, 47.5, 52.0, 63.0, 69.5, 70.2, 71.1, 149.5, 150.0, 174.2. FAB-HRMS calcd. for (M + H+) 237.2305, found 237.1087.
Synthesis of Compound 3--
Acetic anhydride (5.0 ml,
0.055 mol) was added to a solution of 2 (1.0 g, 0.0045 mol)
in pyridine (10 ml), and the reaction was stirred overnight at room
temperature. The solution was concentrated, dissolved in
CHCl3, and washed with 1.0 M HCl, NaHCO3, and saturated NaCl. The organic phase was dried
over MgSO4, filtered, and concentrated to give the crude
product (0.64 g, 33%). This material was purified for metabolic
experiments by reversed-phase HPLC, eluting with a gradient of
CH3CN (10-60%) and H2O. The HPLC-purified
compound was dissolved in ethanol (5.0 mM stock solution)
and filtered (0.25-µm sterile filter) prior to incubation with cells.
1H NMR (300 MHz, MeOD): 1.98 (3H, s), 2.00 (3H, s),
2.02 (3H, s), 2.04 (3H, s), 2.05 (3H, s), 2.09 (3H, s), 3.91 (1H, dd,
J = 6.6, 11.3), 4.12 (2H, m), 4.29 (1H, dd,
J = 3.9, 12.2), 4.51 (1H, m), 5.08 (1H, m), 5.38 (1H,
m). 13C NMR (125 MHz, MeOD):
19.1, 19.2, 19.2, 19.3, 21.1, 61.1, 62.4, 69.0, 69.0, 69.7, 169.9, 170.1, 170.2, 170.7, 170.8, 172.1. FAB-HRMS calcd. for (M + H+) 434.4071, found
434.1662.
Protein Expression and Purification-- For bacterial expression, the human GlcNAc 2-epimerase gene was cloned into pET28b(+). The GlcNAc 2-epimerase gene was amplified by PCR using Pfu DNA polymerase, MgSO4 (5.0 mM), and the following cycling conditions: 96 °C for 5.0 min; 30 cycles of 96 °C for 45 s, 60 °C for 45 s, and 72 °C for 90 s; and then 72 °C for 5.0 min. Primer sequences were as follows: 5' primer, 5'-GGTGGTGGTCATATGATGGAGAAAGAGCGAGAGACTCTGCAGG-3'; 3' primer, 5'-CCGCCGGAATTCTTAGGAGCGGACTCAGCCTTTATTCCGCGC-3'. The PCR product and pET28b(+) were digested with NdeI and EcoRI and ligated with T4 DNA ligase. A 24-nucleotide C-terminal deletion in the gene was discovered when the resulting vector, pSJL41392, was sequenced. At that point, pSJL41392 was digested with KpnI and EcoRI to excise the incorrect 850-bp C-terminal fragment of the GlcNAc 2-epimerase gene. The vector pUKHRB6 was digested with KpnI and EcoRI to obtain the correct C-terminal fragment. Upon ligation with T4 DNA ligase, the correct C terminus of the GlcNAc 2-epimerase gene was installed. DNA sequencing of the resulting vector, pSJL737, confirmed this result.
The vector pSJL737 was transformed into the E. coli strain
BL21(DE3) and expression of GlcNAc 2-epimerase was induced with 1.0 mM isopropyl-1-thio--D-galactopyranoside for
5.0 h at 37 °C. The cells were lysed by sonication at 4.0 °C
(5 × 20 s at 5.0 min intervals, constant duty cycle, level 3 on a Branson Sonifier 450) and centrifuged, and the supernatant was
purified with His-bind resin according to the manufacturer's
instructions. The protein was eluted with 1.0 M imidazole,
and fractions containing protein were dialyzed against a storage buffer
(8 liters, 20 mM sodium phosphate (pH 7.0), 1.0 mM EDTA, 5.0% sucrose, and 1.0%
-mercaptoethanol (20))
for 24 h at 4.0 °C. For NMR experiments, the protein solution was lyophilized once from H2O and twice from
D2O and concentrated 2.0-fold. For colorimetric assays,
GlcNAc 2-epimerase was lyophilized once and concentrated 2.0-fold. The
protein was stored at
80 °C and found to be stable for at least
6.0 months.
Colorimetric Assay for GlcNAc 2-Epimerase-- The colorimetric assay to detect the epimerization of ManNAc by GlcNAc 2-epimerase was performed as described (32), except that the enzymatic reactions were monitored continuously on a Molecular Devices SpectraMax 190 spectrophotometer to acquire initial rates. Trend lines for inhibition by 1 and 2 were determined from nonlinear regression analysis using the program GraFit 4.0, and the data were fit to a competitive inhibition model using the nonlinear regression analysis program, SAS. Trend lines did not converge to fit a noncompetitive pattern for either compound. There was a small background rate that could be attributed to the side reaction of ManNAc with the coupling enzymes, as previously reported (32). In all assays, this background signal was subtracted to establish the actual rate of the reaction. In addition, at the highest inhibitor concentrations and lowest substrate concentrations, there was a significant background signal that was attributed to reaction of the inhibitor directly with the coupling enzymes. This value was subtracted but probably contributes some error to the rates measured using these data points.
1H NMR Assay for GlcNAc 2-Epimerase
Activity--
1H NMR assays of GlcNAc 2-epimerase activity
on ManNAc analogs were performed on a Bruker DRX-500 MHz spectrometer
equilibrated to 37 °C. D2O (225 µl), 4× enzyme buffer
(125 µl, 400 mM Tris buffer (pH 7.4), 40 mM
MgCl2, 16 mM ATP, lyophilized twice from D2O), and alanine (50 µl, 11.95 mg/ml in D2O,
internal standard) were added to the desired monosaccharide (0.073 mmol) to constitute the reaction mixture. GlcNAc 2-epimerase (100 µl;
enzyme concentration varied between preparations, but all rates were
normalized to the rate of conversion of ManNAc for each preparation)
was added immediately prior to acquisition of the first spectrum.
Spectra were acquired over various lengths of time depending on the
monosaccharide (1.0-10 h). The enzyme-catalyzed rate of epimerization
for each monosaccharide was measured by integration of the peaks
corresponding to H-1 of both anomers of ManNAc (or the ManNAc analog)
at various time points. Using Microsoft Excel, the rate of
disappearance of ManNAc (or the ManNAc analog) was calculated relative
to the internal standard (alanine) that remained unchanged throughout the course of the reaction.
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RESULTS |
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Introduction of GlcNAc 2-Epimerase into Human Cells-- Analysis of mRNA from the human T cell lymphoma line Jurkat by RT-PCR showed no detectable message for GlcNAc 2-epimerase, suggesting that ManNAc is produced exclusively by UDP-GlcNAc 2-epimerase in these cells. Accordingly, the mRNA encoding UDP-GlcNAc 2-epimerase was observed by RT-PCR (data not shown).
To achieve stable expression of GlcNAc 2-epimerase in Jurkat cells, we
used the two-step FlpIn system (Invitrogen). This approach to stable
transfection is particularly useful with genes that do not produce a
readily selectable phenotype, as is the case with GlcNAc 2-epimerase.
First, clonal populations of potential host Jurkat cells were generated
that contain a Zeocin resistance gene, flanked by Flp recombination
target (FRT) sites (DNA sequences recognized by Flp recombinase),
stably integrated into the genome. In order to determine which host
cell line would lead to the desired levels of gene expression, we
introduced a reporter protein (dsRed) into selected clonal populations
and screened the resulting cells by flow cytometry. One host cell line
was selected based on relatively high, homogeneous expression of dsRed;
this host cell line was denoted wt*. We expected that the expression of
GlcNAc 2-epimerase introduced by the same method into wt* cells would
yield a similar expression pattern. Like the parent Jurkat cells, wt*
cells had no detectable mRNA encoding GlcNAc 2-epimerase (Fig.
2, lane 3).
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Second, a plasmid (pSJLG2E/FRT) containing the GlcNAc 2-epimerase and Hygromycin resistance genes, flanked by FRT sites, was introduced into wt* cells along with a plasmid (pOG44) containing the gene for Flp recombinase. Flp recombinase-mediated DNA recombination and selection using Hygromycin yielded wt* cells with the GlcNAc 2-epimerase gene stably integrated into the genome (the resulting population was denoted G2E*). The expression of the mRNA encoding GlcNAc 2-epimerase in G2E* cells was confirmed by RT-PCR analysis (Fig. 2, lane 4).
Effect of GlcNAc 2-Epimerase on Metabolic Flux within the Sialic
Acid Pathway--
We next investigated the effect of GlcNAc
2-epimerase expression on cell surface sialic acid content. G2E* and
wt* cells were stained with the conjugated lectins M. amurensis II-FITC, which recognizes -2,3-linked sialic acid,
L. flavus-FITC, and T. mobilensis-biotin (followed by FITC-avidin), which recognize sialic acid in a
linkage-independent fashion and analyzed by flow cytometry. In all
cases, G2E* cells showed very little change in fluorescence compared
with wt* cells (data not shown).
Cell surface sialylation does not necessarily reflect the level of free
sialic acid within cells. For example, a buildup of sialic acid will
not be detected if downstream enzymes, such as the sialyltransferases,
are functioning at capacity. Previously, our laboratory has
demonstrated that ManNAc analogs bearing chemically detectable probes
can be used as tools to monitor the metabolic flux within the sialic
acid pathway. For example, analogs of ManNAc that contain a ketone
(ManLev; Fig. 3A) (33) or an
azide (ManNAz) (26) within the N-acyl group are converted by
cells to the corresponding sialic acids, SiaLev and SiaNAz,
respectively. When expressed on the cell surface, these modified sialic
acids can be reacted with chemical probes and quantified by flow
cytometry (Fig. 3B). Typically, ketones are detected with
biotin hydrazide followed by FITC-avidin (34), and azides are detected
with phosphines conjugated to the FLAG peptide
(NH2-DYKDDDDK-COOH) followed by an anti-FLAG antibody (35).
Since the ManNAc analogs compete with endogenous ManNAc, changes in the
intracellular concentration of native sialic acid intermediates will
affect unnatural sialic acid expression on the cell surface. Thus,
unnatural sialic acid expression can serve as a reporter of cellular
metabolic flux. We have previously exploited this phenomenon to study
regulatory mechanisms of sialoside expression (28).
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In order to observe the effect of GlcNAc 2-epimerase expression on
metabolic flux within the sialic acid pathway, we incubated wt* and
G2E* cells with peracetylated forms of ManLev (Ac4ManLev; Fig. 3A) and ManNAz (Ac4ManNAz) and then analyzed them by
flow cytometry. The peracetylated sugars permeate cell membranes more readily than the unacetylated sugars and can therefore be used at lower
concentrations (25, 36). The acetyl groups are removed by nonspecific
esterases in the cytosol, liberating the free sugars inside cells. As
shown in Fig. 4, G2E* cells produced less
unnatural sialic acid on the cell surface than wt* cells when both were treated with either Ac4ManLev (Fig. 4A) or
Ac4ManNAz (Fig. 4B). In the case of
Ac4ManLev, the difference in cell surface fluorescence was
5-fold, whereas in the case of Ac4ManNAz this difference
was 17-fold. Thus, the presence of the epimerase reduces unnatural sialoside expression, and the effect is more dramatic with the substrate bearing the smaller N-acyl group.
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We have previously found that the size of the N-acyl group on ManNAc derivatives can affect the efficiency of unnatural sialic acid biosynthesis in cells (37) and substrate activity with isolated enzymes in vitro.2 Accordingly, we postulated that GlcNAc 2-epimerase catalyzes the epimerization of ManNAz to the corresponding gluco analog more efficiently than ManLev. To address this experimentally, we developed an in vitro 1H NMR assay to measure the relative rates of the GlcNAc 2-epimerase-catalyzed reaction with the two unnatural substrates. As shown in Table I, the enzyme epimerizes ManNAz 345 times more rapidly than ManLev, and both substrates react more slowly than ManNAc.
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The Effect of GlcNAc 2-Epimerase on Intracellular Free Sialic Acid
Concentrations--
To investigate the effect of GlcNAc 2-epimerase on
the flux of natural sialic acid biosynthesis, we analyzed free sialic
acid content in wt* and G2E* cells. Levels of glycoconjugate-bound and
total sialic acid can be measured using the
periodate-resorcinol assay, and the level of free sialic acid can be
determined by subtracting the former from the latter (31). The level of
free sialic acid in untreated Jurkat cells is close to the detection limit of the assay and is difficult to measure reproducibly.
Nonetheless, G2E* cells appeared to have lower concentrations of free
sialic acid than wt* cells (Fig. 5).
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More reliable measurements were obtained by the addition of ManNAc, which increases the amount of free sialic acid in the cell well above the limit of detection. When both cell lines were treated with 10 mM ManNAc, G2E* cells clearly produced less sialic acid than wt* cells (Fig. 5). Thus, in the presence of excess ManNAc, GlcNAc 2-epimerase acts to lower free sialic acid levels, presumably by diverting ManNAc to GlcNAc. The Jurkat cell lines were also both incubated with 10 mM GlcNAc and analyzed for the effect on sialic acid content. The addition of GlcNAc did not measurably increase the level of sialic acid in G2E* cells above that in wt* cells (Fig. 5), suggesting that intracellular ManNAc concentrations did not differ significantly between the two cell lines. To rule out the possibility that the different effects of ManNAc and GlcNAc on sialic acid production were due to differential uptake by the cells, we performed the same experiment with the peracetylated derivatives. These compounds should equally penetrate cell membranes by passive diffusion (36). The peracetylated compounds had the same relative effects on sialic acid content in wt* and G2E* cells as observed with free ManNAc and GlcNAc (data not shown).
Substrate-based Inhibitors of GlcNAc 2-Epimerase--
The previous
experiments establish that GlcNAc 2-epimerase expression correlates
with a reduction in sialic acid biosynthesis. In order to confirm that
this reduction was the direct result of enzyme activity, we designed an
inhibitor of GlcNAc 2-epimerase for use in cellular studies. A
mechanism describing the interconversion of GlcNAc and ManNAc by GlcNAc
2-epimerase was recently proposed by Samuel and Tanner (Fig.
6A) (21) but has not been
addressed experimentally. The proposed reaction involves ring-opening
to enable abstraction of H-2 and formation of an enediol-like
intermediate, resembling reactions catalyzed by
D-ribulose-5-phosphate-3-epimerase (21, 39),
triose-phosphate isomerase (40), and phosphoglucose isomerase (41, 42).
Although analogs of the enediol intermediate have been shown to inhibit
these related enzymes (43-45), we opted for more synthetically
tractable mimetics based on the ring-opened form of GlcNAc. We
synthesized compounds 1 and 2 (Fig.
6B) and adapted a published enzyme-coupled colorimetric assay for GlcNAc 2-epimerase to measure their inhibitory activity. The
assay detects the production of GlcNAc by enzymatic oxidation of the
C-6 hydroxyl group and concomitant production of hydrogen peroxide,
which is subsequently consumed in a chromogenic peroxidase reaction
(32). For ease of analysis, we modified the assay to monitor the
reaction continuously.
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The value of Km obtained for ManNAc (4.3 mM) was close to the literature value (13.2 mM)
obtained using the end point assay (46). We then measured the
inhibition constants (KI) of compounds 1 and 2 to be 90 and 109 µM, respectively. By
varying the concentrations of substrate and inhibitor, we obtained kinetic data consistent with a competitive inhibition mechanism for
both compounds 1 and 2, indicating that both analogs compete with ManNAc for the active site (Fig.
7). To make an inhibitor useful in cell
culture (36), compound 2 was acetylated to form compound
3 (Fig. 6B).
|
GlcNAc 2-Epimerase Inhibitor Is Active in Cells--
Compound
3 was used to confirm that GlcNAc 2-epimerase activity was
responsible for the reduced conversion of Ac4ManLev to
SiaLev in G2E* cells compared with wt* cells. As shown in Fig. 8, compound 3 reverses the
effect of GlcNAc 2-epimerase in a dose-dependent fashion,
nearly restoring unnatural sialic acid biosynthesis to wt* levels at
200 µM. Importantly, the production of SiaLev in wt*
cells was not significantly affected by the addition of compound
3.
|
Finally, we incubated wt* and G2E* cells with ManNAc in the presence
and absence of compound 3 and analyzed free sialic acid
levels using the periodate-resorcinol assay (Fig.
9). Although the sialic acid level in
G2E* cells was still lower than that in wt* cells, the differential was
significantly reduced by the GlcNAc 2-epimerase inhibitor (100 µM). Thus, the inhibitor reverses the low sialic acid
phenotype characteristic of G2E* cells, confirming that the enzyme is
directly responsible for that phenotype.
|
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DISCUSSION |
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To investigate the effect of GlcNAc 2-epimerase on intracellular metabolic flux within the sialic acid pathway, we initially analyzed cell surface sialic acid expression. Sialic acid-binding lectins revealed little about the changes in sialic acid metabolism resulting from GlcNAc 2-epimerase expression in Jurkat cells. However, we were able to observe an effect on sialic acid biosynthesis by using unnatural metabolic substrates and by measurements of intracellular sialic acid levels.
G2E* cells, which express GlcNAc 2-epimerase, were unable to convert
two unnatural ManNAc analogs (Ac4ManLev and
Ac4ManNAz) to the corresponding sialosides (SiaLev and
SiaNAz) at levels comparable with wt* cells. This observation
is consistent with epimerization of the ManNAc analogs to the
corresponding GlcNAc analogs and, consequently, diversion from the
sialic acid pathway. GlcNAc derivatives that are produced in this
fashion could be utilized in other metabolic pathways that do not
contribute to cell surface fluorescence, or they may be dead end
biosynthetic intermediates. This model for the effect of GlcNAc
2-epimerase on sialic acid biosynthesis is summarized in Fig.
10A.
|
Alternatively, it may be possible that G2E* cells produce higher levels of ManNAc than wt* cells due to epimerization of GlcNAc, a model summarized in Fig. 10B. Indeed, ManLev was previously used by our laboratory as a tool to select a Jurkat cell line containing mutations in the UDP-GlcNAc 2-epimerase gene that make the protein resistant to inhibition by CMP-sialic acid (28). Lacking that regulatory mechanism, these cells overproduced ManNAc, which suppressed unnatural sialic acid expression by direct competition with ManLev. The models shown in Fig. 10 are not mutually exclusive; GlcNAc 2-epimerase may perform either of these functions depending on the relative availabilities of GlcNAc and ManNAc in the cell.
GlcNAc 2-epimerase is clearly capable of converting ManNAz and ManLev to their gluco counterparts in vitro. The superior efficiency of ManNAz epimerization in vitro is consistent with the dramatic effect of epimerase expression on SiaNAz biosynthesis in cells. Furthermore, intracellular sialic acid levels are reduced in cells expressing GlcNAc 2-epimerase, an effect that cannot be reversed by the addition of exogenous GlcNAc. Collectively, these data support the first model (Fig. 10A), in which GlcNAc 2-epimerase converts ManNAc and its analogs to the respective gluco isomers. In the case of the second model (Fig. 10B), epimerase expression would be expected to boost intracellular sialic acid levels in the presence of excess GlcNAc. Furthermore, the conversion of ManNAz to SiaNAz would be suppressed to a lesser extent than the conversion of ManLev to SiaLev, since ManNAz is a more capable competitor with ManNAc in the sialic acid pathway (30). The data presented show the opposite effects, arguing against the second model.
To confirm that GlcNAc 2-epimerase was the causative agent of the observed changes in sialic acid biosynthesis in G2E* cells, we designed a family of inhibitors (compounds 1-3) based on a proposed chemical mechanism of the enzyme (21) (Fig. 6). The KI values of compounds 1 and 2 (90 and 109 µM, respectively) were ~40-fold lower than the measured Km value for ManNAc (4.3 mM) and 200-fold lower than the reported Km value for GlcNAc (21.3 mM) (20). Since the inhibitors were designed to mimic the ring-opened form of GlcNAc, this lends support to the proposed mechanism in Fig. 6A. The hydrophilicity of compounds 1 and 2 would probably frustrate their activity in cell culture experiments. However, compound 3, a peracetylated analog of 2, demonstrated inhibitory activity in cells at micromolar concentrations.
Introduction of compound 3 into G2E* cells in the presence of Ac4ManLev resulted in a reversal of the phenotype attributed to GlcNAc 2-epimerase, indicating that the action of the enzyme was indeed responsible for the low cell surface expression of unnatural sialic acids (Fig. 8). Additionally, unnatural sialic acid expression in wt* cells was essentially unaltered in the presence of compound 3, indicating that the inhibitor does not cross-react with other proteins in the cell that affect sialic acid biosynthesis. The low intracellular sialic acid level maintained by G2E* cells was increased by compound 3, suggesting again that the inhibitor can reverse the phenotype associated with GlcNAc 2-epimerase expression.
It is possible that the primary function of GlcNAc 2-epimerase is
to divert metabolic flux from sialic acid biosynthesis into other
pathways reliant on GlcNAc or UDP-GlcNAc. Numerous glycoproteins and
proteoglycans include GlcNAc as a component, and several cytosolic and
nuclear proteins are modified with -O-GlcNAc in a
possible regulatory fashion (47, 48). Increases in cellular GlcNAc may
affect the flux in these pathways. Alternatively, phosphorylation of
GlcNAc followed by deacetylation of GlcNAc 6-phosphate to glucosamine 6-phosphate generates a substrate for glycolysis (38). It is possible
that GlcNAc 2-epimerase serves to increase glycolytic flux at the
expense of protein glycosylation.
We conclude that GlcNAc 2-epimerase catalyzes the conversion of ManNAc
(and ManNAc analogs) to GlcNAc (and GlcNAc analogs) in human cells, but
the reverse process is unobservable, even in the presence of added
GlcNAc. Furthermore, analogs of the ring-opened form of GlcNAc are
effective inhibitors of GlcNAc 2-epimerase in vitro, lending
support to a proposed chemical mechanism. One of these inhibitors
reversed the sialic acid suppression induced by GlcNAc
2-epimerase, confirming that the phenotype observed was directly
attributable to activity of the enzyme and demonstrating the utility of
the inhibitor in human cells. Based on the results presented here, we
propose that GlcNAc 2-epimerase is not an alternate route to ManNAc
production in cells. Rather, the enzyme diverts metabolic flux away
from sialic acid biosynthesis, performing a function distinct from that
of UDP-GlcNAc 2-epimerase.
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ACKNOWLEDGEMENT |
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We thank Hector Nolla for assistance with flow cytometry.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM58867 and by the Director, Office of Science, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering and the Office of Energy Biosciences of the United States Department of Energy under Contract DE-AC03-76SF00098 (to C. R. B.).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.
§ Present address: Whitaker Biomedical Engineering Institute, The Johns Hopkins University, Baltimore, MD 21218.
§§ To whom correspondence should be addressed. Tel.: 510-643-1682; Fax: 510-643-2628; E-mail: bertozzi@cchem.berkeley.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M212127200
2 C. L. Jacobs, S. Hinderlich, S. Goon, K. Viswanathan, J. W. Weedin, A. Blume, M. J. Betenbaugh, W. Reutter, and C. R. Bertozzi, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: ManNAc, N-acetylmannosamine; FITC, fluorescein isothiocyanate; Ac4ManLev, peracetylated N-levulinoylmannosamine; ManLev, N-levulinoylmannosamine; Ac4ManNAz, peracetylated N-azidoacetylmannosamine; ManNAz, N-azidoacetylmannosamine; FRT, Flp recombinase target; SiaLev, N-levulinoylneuraminic acid; SiaNAz, N-azidoacetylneuraminic acid; HPLC, high pressure liquid chromatography.
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