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INTRODUCTION |
The deaminoneuraminic acid residue
KDN1
(2-keto-3-deoxy-D-glycero-D-galacto-nononic
acid) is a distinct member of the sialic acids that shares many
features in common with N-acylneuraminic acids (Neu5Acyl),
including variations in the 
-ketosidic linkages to the penultimate
sugar residues and their occurrence in vertebrate glycoproteins
(1-20), glycosphingolipids (5, 21-23), and bacterial capsular
polysaccharides (24). The enzymes involved in KDN metabolism, including
CMP-KDN synthetase (25), KDN-transferase (26-28), and KDN
residue-cleaving sialidases (29-31), have been identified and partially characterized, and their similarity and dissimilarity to the
metabolism of Neu5Acyl residues have been described. In marked
contrast, nothing is known concerning how the KDN monosaccharide is synthesized.
Two pathways are possible for synthesis of the KDN monomer. First, it
could be formed directly from Neu5Ac by deacylation and
deamination. Alternatively, it could arise by de novo
synthesis via enzymes different from those required for synthesis of
Neu5Ac. However, no evidence for the direct conversion of Neu5Ac to KDN has been obtained in any tissue homogenates examined to date. Thus,
de novo synthesis by a separate pathway appears to be
plausible by analogy with synthesis of other ulosonic acids. For
example, 3-deoxy-D-arabino-heptulosonic acid
7-phosphate, a biosynthetic precursor of aromatic amino acids (32),
3-deoxy-D-manno-octulosonic acid, a constituent
of bacterial lipopolysaccharides (33, 34) and Neu5Ac (35) are
synthesized through a series of similar reactions involving a
condensation of phosphoenolpyruvate (PEP) and the relevant sugar
phosphates, either erythrose 4-phosphate for
3-deoxy-D-arabino-heptulosonic acid 7-phosphate,
arabinose 5-phosphate for
3-deoxy-D-manno-octulosonic acid 8-phosphate, or
N-acetylmannosamine 6-phosphate (ManNAc-6-P) for Neu5Ac
9-phosphate. Accordingly, the de novo biosynthesis of KDN is
hypothesized to involve the condensation of PEP and mannose 6-phosphate
(Man-6-P), giving rise to KDN 9-phosphate (KDN-9-P). Two additional
reactions, the 6-O-phosphorylation of mannose (Man) and the
dephosphorylation of KDN-9-P would presumably be required for KDN
synthesis if Man and PEP were the intracellular substrates. It is
conceivable, however, that these two reactions could be catalyzed by
intracellular hexokinases and phosphatases, two ubiquitously occurring
enzymes known to have broad substrate specificities (36, 37). Thus, the
purpose of this study was to elucidate the biosynthesis of the KDN
monomer in rainbow trout ovaries and testis, tissues rich in expressing
KDN-containing glycoconjugates (6-8, 21-23). Our initial studies have
led to the discovery of a KDN-9-P synthetase, an activity that
condenses Man-6-P and PEP in cytosolic fractions from both tissues.
This activity was purified more than 50-fold from testis. Substrate
competition experiments were used to show that the KDN-9-P synthetase
activity was distinct from Neu5Ac-9-P synthetase, an activity
responsible for condensing ManNAc-6-P and PEP.
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EXPERIMENTAL PROCEDURES |
Materials
[1-14C]Man (2.07 GBq/mmol) was purchased from
American Radiolabeled Chemicals, Inc.
1,2-Diamino-4,5-methylenedioxybenzene (DMB), CMP-Neu5Ac, ATP,
hexokinase (bakers' yeast), and N-acetylneuraminic acid
aldolase from Escherichia coli were purchased from Wako Pure Chemicals (Japan). Sodium phosphoenolpyruvate, alkaline phosphatase from calf intestine, and Man-6-P were purchased from Sigma. Mannosamine hydrochloride was purchased from Nacalai Tesque (Japan). CMP-KDN and
KDN were prepared as described previously (25, 28). Rainbow trout
ovaries, testes, and liver were collected a month before ovulation or spermiation.
Quantitation of Free, Lipid-bound, and Protein-bound Sialic Acids
(Sia) in Rainbow Trout Ovary and Testis
All procedures were carried out at 4 °C unless otherwise
stated. Ovaries and testes (2 g each) were homogenized separately in 2 ml of 20 mM Tris-HCl buffer, pH 8.0, containing 0.1 M NaCl using a Polytron homogenizer (Kinematica,
Switzerland). One ml of each homogenate was mixed with 4 ml of water
and centrifuged at 100,000 × g for 60 min. The
supernatants were collected and designated the "cytosolic
fraction." The precipitates were extracted serially with
chloroform:methanol, 2:1 (v/v) and 1:2 (v/v), as described previously
(21). The extract and the resultant precipitate were designated as the
"lipid fraction" and the "protein fraction," respectively. The
lipid and protein fractions were each resuspended in 0.1 M
trifluoroacetic acid and heated at 80 °C for 30 min. After removal
of any particulate material by brief centrifugation, the hydrolysates
(designated as the lipid- and protein-bound Sia fractions,
respectively) were derivatized with DMB. The cytosolic fractions were
ultrafiltered through Microcon 10 (Amicon) to remove high molecular
weight materials (Mr > 10,000), and the
filtrates (designated the "free Sia fraction") were derivatized
with DMB.2 The DMB
derivatization of Sia and their analysis on the reverse phase HPLC (the
DMB fluorometric HPLC method) were carried out as described previously
(5). In brief, the sample (20 µl) in 40 mM
trifluoroacetic acid was mixed with 20 µl of 7 mM DMB in 5 mM trifluoroacetic acid, 1 M
2-mercaptoethanol, and 18 mM sodium hydrosulfite and
incubated at 50 °C for 2 h. Ten-µl portions of the reaction
mixture were applied to a TSK gel ODS-120T (4.6 × 250 mm) column
and eluted with acetonitrile:methanol:water (6:4:90, v/v/v), on a JASCO
LC-900 HPLC system equipped with a JASCO FP-920 fluorescence detector
(excitation, 373 nm; emission, 448 nm) operating isocratically at 1.0 ml/min at a column temperature of 26 °C. DMB derivatives of KDN and
Neu5Ac were well separated by HPLC and quantitatively determined in the
pmol-nmol range.
Quantitative Determination of Man, ManNAc, and GlcNAc in the
Cytosol of Trout Ovary and Testis
The free Sia fractions described above were analyzed. This
fraction (derived from 10 mg of tissue) was subjected to
pyridylamination (40) using GlycoTAG (Takara, Japan) according to
instructions provided by the manufacturer. The pyridylaminated sugars
were separated by a HPLC on PALPAK Type A column (4.6 × 150 mm;
Takara, Japan) and quantitated with an integrator connected to a
spectrofluorometer (821-FP, JASCO, Japan) (excitation, 310 nm;
emission, 380 nm). The column was eluted with 0.7 M
potassium borate, pH 9.0:acetonitrile (9:1, v/v) at 65 °C at 0.4 ml/min for 200 min.
Synthesis of [14C]Man-6-P and a Reference Standard
of [14C]KDN-9-P
[14C]Man-6-P was prepared as described previously
(36). From [14C]Man (0.925 MBq, about 0.45 µmol) and
ATP (2.5 µmol), 0.43 µmol of [14C]Man-6-P was
prepared. A reference standard of [14C]KDN-9-P was
synthesized by incubating [14C]Man-6-P (9.25 KBq, 4.5 nmol) and sodium pyruvate (1 µmol) with 0.5 unit of
N-acetylneuraminic acid aldolase in 100 µl of 50 mM MES, pH 7.0, containing 0.01% NaN3 at
37 °C for 100 h. The reaction mixture, which contained both
[14C]KDN-9-P and unreacted [14C]Man-6-P,
was ultrafiltered through Microcon 10 and used as a reference standard
for TLC analysis. The yield of [14C]KDN-9-P was about
10%.
Preparative Synthesis of KDN-9-P and ManNAc-6-P
A large scale preparation of KDN-9-P was synthesized by
incubating Man-6-P (5 µmol) and sodium pyruvate (25 µmol) with 1 unit of N-acetylneuraminic acid aldolase in 250 µl of 25 mM phosphate buffer, pH 7.2, containing 0.01%
NaN3 at 37 °C for 144 h. The reaction mixture was
ultrafiltered through Microcon 10, and the filtrate was applied to a
Mono-Q HR 5/5 column connected to an HPLC system (Irica, Japan). The
column was eluted with a linear NaCl gradient (0-0.25 M)
in 10 mM monoethanolamine-HCl, pH 8.5, at a flow rate of 1 ml/min. Fractions positive for thiobarbituric acid method (41), eluting
around 130 mM NaCl, were collected and desalted by
chromatography on a Sephadex G-10 column (1.0 × 40 cm). The yield
was about 10%. ManNAc-6-P was synthesized as described previously by
Jourdian and Roseman (42). From 0.1 mmol of mannosamine hydrochloride,
0.03 mmol of Man NAc-6-P was prepared through phosphorylation by
hexokinase using 0.1 mmol of ATP and the subsequent acetylation.
Detection of Different Enzyme Activities in the Cytosolic
Fractions of Trout Ovary and Testis
Three g of trout ovaries and testes were separately homogenized
in 3 ml of homogenization buffer (50 mM MES, pH 7.0, containing 100 mM NaCl, 1 mM DTT, and 2 µg/ml
each of aprotinin, leupeptin, and pepstatin A) in a Polytron
homogenizer and centrifuged at 100,000 × g for 60 min.
The resulting supernatant was used as the cytosolic fraction. For the
KDN-9-P synthetase assay, 4 µl of the substrate mixture containing 40 nmol each of PEP and Man-6-P and 2 nmol of sodium vanadate (a
phosphatase inhibitor) were added to 16 µl of the cytosolic fractions
and incubated at 25 °C for 24 h. Each incubation mixture
received 5 µl of alkaline phosphatase (30 units of enzyme in 2 µmol
of glycine buffer, pH 10.0, containing 250 nmol of MgCl2),
further incubated at 37 °C for 1 h to dephosphorylate the
reaction products, and analyzed for KDN by the DMB fluorometric HPLC
method (see above). For the assay for hexokinase activity, 4 µl of
substrate containing 40 nmol of [14C]Man (150 Bq/nmol),
100 nmol of ATP, 200 nmol of MgCl2, and 10 nmol of sodium
vanadate were added to 16 µl of the cytosolic fraction and incubated
at 25 °C for 24 h. The reaction mixture was analyzed for
[14C]Man-6-P by using a cellulose TLC as described under
"Quantitative Assay for KDN-9-P Synthetase and Neu5Ac-9-P Synthetase
Activities." For the phosphatase assay, 4 µl of KDN-9-P (20 nmol)
were mixed with 16 µl of the cytosolic fraction and incubated at
25 °C for 24 h. The reaction mixtures were analyzed for KDN by
the DMB fluorometric HPLC method (see above).
Purification of KDN-9-P Synthetase from Rainbow Trout Testis
All procedures were carried out at 4 °C. Five g of mature
rainbow trout testes that had been frozen immediately after collection and stored at 
80 °C were thawed, minced, and homogenized in a Polytron homogenizer in 20 ml of homogenization buffer (50 mM MES, pH 7.0, containing 1 mM DTT, 10%
glycerol, and 1 µg/ml each aprotinin, leupeptin, and pepstatin A).
The homogenate was ultracentrifuged at 100,000 × g for
60 min, and the supernatant was applied to a DEAE-Toyopearl 650M column
(Tosoh, Japan; Cl
form, 2 × 4 cm). The column was
washed with 25 ml of 50 mM MES, pH 7.0, containing 1 mM DTT and 10% glycerol. The flow-through and wash
fractions were combined and brought to 70% saturation with solid
ammonium sulfate, stirred for 30 min, and centrifuged at 20,000 × g for 30 min. Solid ammonium sulfate was added to the
supernatant to 90% saturation, stirred for 30 min, and centrifuged again at 20,000 × g for 30 min. The pellet, designated
AS70-90, was dissolved in 20 mM Tris-HCl buffer, pH 9.0, containing 1 mM DTT and 10% glycerol and desalted on a
minicolumn of Sephadex G-25 (PD-10, Amersham Pharmacia Biotech). The
eluate was applied to a Poros HQ plastic column (Perseptive Biosystems)
and eluted with 20 ml of a linear NaCl gradient (0-0.25 M)
in 20 mM Tris-HCl, pH 9.0, 1 mM DTT, and 10%
glycerol. Fractions that contained the enzyme activity, which eluted
between 125-160 mM NaCl, were collected.
Quantitative Assay for KDN-9-P Synthetase and Neu5Ac-9-P
Synthetase Activities
Standard Quantitative Assay--
For quantitative determination
of the amount of enzyme activity, the following procedure was carried
out. Enzyme fractions were first desalted, and the buffer was exchanged
with 50 mM MES, pH 7.0, containing 1 µg/ml each
aprotinin, leupeptin, and pepstatin A by ultrafiltration using Microcon
10. To 20 µl of the enzyme fraction 5 µl of the substrate were
added, either 25 mM Man-6-P (for KDN-9-P synthetase) or 5 mM ManNAc-6-P (for Neu5Ac-9-P synthetase). Each substrate
also contained 25 mM PEP and 10 mM
MnCl2. After incubation at 25 °C for 20 or 30 min, the
mixture was boiled for 3 min. Five µl of alkaline phosphatase (30 units) in 0.5 M glycine-NaOH buffer, pH 10.0, and 60 mM MgCl2 were added and incubated at 37 °C
for 1 h. With this incubation, quantitative dephosphorylation was
attained. Trifluoroacetic acid was added, and the final concentration of trifluoroacetic acid and sample volume was adjusted to 40 mM and 100 µl, respectively. The sample was ultrafiltered
through Microcon 10, and the filtrate was analyzed for the amount of
KDN and Neu5Ac by the DMB fluorometric HPLC analysis, as described above. One unit of enzyme activity is defined as the amount of enzyme
required to synthesize 1 nmol of KDN-9-P or Neu5Ac-9-P/h at
25 °C.
Routine Assay--
For monitoring the elution profile of the
KDN-9-P synthetase activity in the column chromatography fractions, the
assay was routinely carried out as follows. Enzyme fractions were
prepared as described above. To each enzyme fraction (5 µl) was added
[14C]Man-6-P (330 Bq, 160 pmol), PEP (20 nmol),
MnCl2 (10 nmol), and sodium vanadate (1 nmol). The final
volume was adjusted to 10 µl with water. After incubation at 25 °C
for 20 h, 2 µl of alkaline phosphatase (1.5 to 7.5 units) in 1.2 µmol of glycine-NaOH buffer, pH 10.0, containing 120 nmol of
MgCl2 were added, and the reaction was incubated at
37 °C for 30 to 60 min. Three µl of each sample were spotted onto
plastic-backed cellulose TLC plate (Merck) and developed in 1-propanol,
1 M sodium acetate, pH 5.0, water (7:1:2, v/v/v) for 8 h (43). When the effect of ionic strength was determined, the samples
were spotted on Whatman No. 3MM paper and developed for 12 h in
the same propanol:sodium acetate solvent noted above. Radioactivity was
visualized and quantitatively determined using the Fujix BAS-2000
Imaging Analyzer (Fuji Photo Film, Japan).
Identification of the KDN-9-P Synthetase Reaction Product,
KDN-9-P, by Matrix-assisted Laser Desorption Ionization-Time-of-flight
(MALDI-TOF) Mass Spectrometry
Man-6-P and PEP (2.5 µmol each) were incubated with the
AS70-90 fraction (20 units) in 250 µl of 50 mM MES, pH
7.0, containing 1 mM MnCl2, and 1 µg/ml each
aprotinin, leupeptin, and pepstatin A. After incubation at 25 °C for
24 h, a flocculent precipitate that appeared was removed by
centrifugation, and the supernatant was ultrafiltered through Microcon
10. The filtrate was applied to a Mono-Q HR 5/5 HPLC, and the
thiobarbituric acid method-positive material was recovered and
purified, as described under "Preparative Synthesis of KDN-9-P and
ManNAc-6-P." This compound and its alkaline phosphatase-treated
derivative were analyzed by the DMB fluorometric HPLC method. The
alkaline phosphatase-treated derivative was prepared by the Mono-Q HPLC
of the compound after incubation at 37 °C for 3 h with the
enzyme (10 units) in 30 µl of 0.1 M glycine buffer, pH
10.0, containing 10 mM MgCl2. The purified
product and its dephosphorylated derivative were also analyzed by
MALDI-TOF mass spectrometry. The product (10 pmol) was mixed with
20 µl of 2,5-dihydrobenzoic acid (2,5-DHB) in water, and 1 µl of
the sample was applied onto a gold target. After drying, the sample was
analyzed on a Voyager Elite MALDI-TOF mass spectrometry system
(PerSeptive Biosystems) with a nitrogen laser (337 nm) in
negative ion mode at an accelerating potential of 12 kV.
Molecular Weight Estimation of KDN-9-P Synthetase
The apparent molecular weight of KDN-9-P synthetase
was estimated by Sephacryl S-300 chromatography of the AS70-90 fraction (10 units). The column (1.4 × 100 cm) was eluted with 50 mM HEPES buffer, pH 8.5, containing 100 mM
NaCl, 1 mM DTT, 10% glycerol and calibrated using the gel
filtration molecular weight standard kit (Bio-Rad). Fractions (2.4 ml)
were collected and assayed for KDN-9-P synthetase activity by the
routine assay, as described above.
Characterization of the KDN-9-P Synthetase Activity
For determining the following properties of KDN-9-P
synthetase, the enzyme activity was measured according to the routine assay (described above) using the AS70-90 enzyme fraction (0.3 unit),
with varied conditions.
Effect of pH--
The enzyme fraction was desalted by
ultrafiltration using Microcon 10 and dissolved in 2 µg/ml each
aprotinin, leupeptin, and pepstatin A. This was mixed immediately with
an equal volume of 100 mM Tris-HCl buffer, pH 7.2 to 9.0, or 100 mM MES, pH 5.5-7.0. The ionic strength of each
buffer was adjusted to 0.1 by the addition of NaCl.
Effect of Ionic Strength--
To determine the effect of the
ionic strength on KDN-9-P synthetase activity, the concentration of
NaCl in the different incubation mixtures was varied from 0 to 1.0 M.
Effect of Divalent Cations--
Divalent cations were depleted
from the enzyme fraction by incubation on ice for 60 min with 5 mM EDTA in 100 mM MES, pH 7.0, containing 1 µg/ml each aprotinin, leupeptin, and pepstatin A. The mixture was
ultrafiltered to remove EDTA and resuspended in the above buffer devoid
of EDTA. Enzyme activity assays were carried out after addition of the
following salts (1 mM) and preincubation for 30 min on ice:
MgCl2, CaCl2, MnCl2,
FeSO4, CoCl2, NiCl2,
CuCl2, and ZnCl2.
Determination of Kinetic Parameters and Substrate
Competition Experiments
Kinetic Parameters--
Twenty µl of the AS70-90
enzyme fraction (1.1 unit), dissolved in 50 mM MES buffer,
pH 7.0, containing 1 µg/ml each aprotinin, leupeptin, and pepstatin A
were mixed with 5 µl of substrate containing 50 nmol of
MnCl2, 125 nmol of PEP, and 25 to 125 nmol of Man-6-P or
2.5 to 50 nmol of ManNAc-6-P. After incubation at 25 °C for 20 min,
the reaction was terminated by heating at 100 °C for 3 min. Five
µl of alkaline phosphatase (30 units) in 2.4 µmol of glycine
buffer, pH 10.0, containing 300 nmol of MgCl2 were then added and incubated at 37 °C for 1 h. Trifluoroacetic acid was added to terminate each reaction. The final concentration of
trifluoroacetic acid and sample volume was adjusted to 40 mM and 100 µl, respectively. The samples were
ultrafiltered through Microcon 10, and the amount of KDN or Neu5Ac in
the filtrates was quantitatively determined by the DMB fluorometric
HPLC analysis, as described above. Kinetic parameters were determined
by double-reciprocal Lineweaver-Burk plots (44).
Substrate Competition Experiments--
The assays were carried
out as described above for determination of kinetic parameters, except
that the incubation mixtures contained both Man-6-P and ManNAc-6-P. In
the competition experiments, the concentration of one of the two sugar
phosphates was held constant, whereas that of the other was varied
(Man-6-P, 5 mM, and ManNAc-6-P, 0.1-0.5 mM or
ManNAc-6-P, 1 mM, and Man-6-P, 1-5 mM).
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RESULTS |
Quantitative Determination of Free and Lipid- and
Protein-bound KDN, Neu5Ac, and Neu5Gc in Trout Ovary and
Testis--
The quantities of free and lipid- and protein-bound Sia in
the ovaries or testes a month before ovulation or spermiation, when
active synthesis of KDN-containing glycoconjugates occurs, are shown in
Table I. The total amount of free and
lipid- and protein-bound KDN, Neu5Ac, and Neu5Gc/g of tissue in ovary
was 19-fold greater than that of testis (5,000 nmol versus
262 nmol). However, the total amount of KDN/g of ovary tissue was about
one-third that of testis (60.5 nmol versus 161 nmol). These
results suggest that although the overall metabolism of the Sia is more
active in ovary3 than in
testis, KDN metabolism is considerably more active in testis. The molar
ratios of free KDN:free Neu5Acyl (Neu5Ac + Neu5Gc) were similar to
those of bound Sia (1:88 versus 1:73 for ovary; 1:0.66
versus 1:0.57 for testis). The cytosolic concentrations of
free KDN and Neu5Acyl are suggested to mirror the overall expression of
bound KDN and Neu5Acyl residues on glycoconjugates in both trout ovary
and testis.
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Table I
Quantities of free and bound sialic acids in trout ovary and testis
Free and lipid- and protein-bound sialic acids were prepared from trout
ovary and testis removed from fish about 1 month before
ovulation/spermiation (September). The Sia were derivatized with DMB
and quantitatively determined by fluorometric HPLC, as described under
"Experimental Procedures."
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Quantitative Determination of Sugar Precursors Required for KDN and
Neu5Ac Synthesis in Rainbow Trout Ovary and Testis--
To assess the
availability of metabolic sugar precursors required for KDN and Neu5Ac
synthesis in rainbow trout ovary and testis, the level of free Man
(precursor for KDN) and ManNAc (precursor for Neu5Ac) were
quantitatively determined. The level of ManNAc in ovary was about
10-fold higher than Man (716 versus 76.4 nmol/g of tissue),
whereas comparable levels of the two precursors were present in testis
(36 versus 42 nmol/g of tissue). These results suggest that
concentrations of the precursors, free Man and ManNAc, were reflected
in the expression of free KDN and Neu5Acyl, respectively, in both trout
ovary and testis (see above). No significant change in the amount of
the free sugars was observed before and after treatment with alkaline
phosphatase, indicating that the pool size of the free phosphorylated
sugars was relatively small.
Identification of Enzyme Activities Required for KDN Synthesis in
the Cytosol of Trout Ovary and Testis--
Incubation of homogenates
or cytosolic fractions prepared from trout ovaries and testes with
Neu5Ac and CMP-Neu5Ac did not result in the synthesis of
KDN.4 In contrast, incubation
with Man-6-P and PEP resulted in a marked increase in the amount of KDN
formed. These findings indicated that the cytosol contained a KDN-9-P
synthetase activity. High levels of activities of hexokinase and
phosphatase, both presumed to be involved in the de novo
synthesis of KDN, were also present in the cytosolic fraction (data not
shown). On the basis of these results, we conclude that KDN is
synthesized by the condensation of Man-6-P and PEP and is not derived
from Neu5Ac or CMP-Neu5Ac.
Purification of KDN-9-P Synthetase from Trout Testis--
KDN-9-P
synthetase was purified about 50-fold from rainbow trout testis, as
summarized in Table II. After
purification on a Poros HQ column, the enzyme fraction contained
several proteins when analyzed by SDS-polyacrylamide gel
electrophoresis and silver staining (data not shown). The
50-fold-purified KDN-9-P synthetase was unstable, preventing further
purification of this enzyme activity. Accordingly, the enzyme was
characterized using the ammonium sulfate-precipitated fraction,
AS70-90. The AS70-90 was devoid of phosphatase activity that converted
Man-6-P and KDN-9-P to Man and KDN, respectively. This fraction was
stable for at least 1 month when stored at 80 °C.
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Table II
Summary of the purification of rainbow trout testis KDN-9-P
synthetase
The enzyme activity was estimated by the standard quantitative assay as
described under "Experimental Procedures." One unit of enzyme
activity is defined as the amount of enzyme required to synthesize 1 nmol of KDN-9-P/h at 25 °C. The amount of protein was estimated by
the bicinchoninic acid method (BCA, Pierce) using bovine serum albumin
as standard.
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Identification of KDN-9-P as the Product of KDN-9-P
Synthetase--
Man-6-P and PEP were incubated with the KDN-9-P
synthetase AS70-90 fraction, and the product was analyzed on the DMB
fluorometric HPLC (Fig. 1). The DMB
derivative of the reaction product and its phosphatase-treated
derivative had the same retention times as the DMB derivative of
authentic KDN-9-P (2.26 min) and KDN (8.56 min), respectively. The
reaction product and its phosphatase-treated derivative were also
analyzed by MALDI-TOF mass spectrometry. The molecular ions,
(M-H), and their Na+ adducts, (M + Na 2H), were observed at m/z 346.5 and
368.7, respectively, for the presumed KDN-9-P product and at
m/z 266.8 and 288.2, respectively, for the
phosphatase-treated product. Thus, these results are consistent with
the identification of the product as KDN-9-P.
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Fig. 1.
HPLC elution profiles of DMB derivatives of
the reaction product catalyzed by KDN-9-P synthetase. Man-6-P (10 mM) and PEP (10 mM) were incubated with the
AS70-90 fraction (20 units) in 50 mM MES, pH 7.0, 1 mM MnCl2, 0.1 mM sodium vanadate,
and 1 µg/ml each protease inhibitors at 25 °C for 24 h, and
the product was purified and derivatized with DMB as described under
"Experimental Procedures." The DMB derivative of KDN-9-P synthetase
reaction product (A) and its alkaline phosphatase-treated
derivative (B) were applied to a TSK-gel ODS-120T (4.6 × 250 mm) column and eluted with acetonitrile/methanol/water (9: 7:
84, v/v/v) at 1.0 ml/min at a column temperature of 26 °C. The
elution profiles were monitored with a fluorescence detector
(excitation, 373 nm; emission, 448 nm). The retention times of
authentic KDN-9-P and KDN were 2.26 min and 8.56 min, respectively. The
retention times for peaks 1 and 2 were the same
as authentic KDN-9-P and KDN, respectively.
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Characterization of KDN-9-P Synthetase Activity in Rainbow Trout
Testis--
The optimum pH for KDN-9-P synthetase activity was between
pH 7 and 8. The enzyme was active in the absence of exogenously added
divalent cations but lost all activity when incubated before assay with
5 mM EDTA. The lost enzyme activity was restored to 2.4-3
times higher levels of the original one by the addition of 1 mM MnCl2, CoCl2, or
NiCl2 and to one-third the original activity by 1 mM MgCl2. The other cations (Ca2+,
Fe2+, Cu2+, and Zn2+) did not
restore enzyme activity. The enzyme activity was significantly inhibited in a concentration-dependent manner by NaCl. The
activity was reduced by about 50% in the presence of 150 mM NaCl compared with the activity without added NaCl.
The apparent molecular weight of KDN-9-P synthetase was estimated to be
about 80,000 by Sephacryl S-300 gel filtration chromatography. KDN-9-P
synthetase had an obligatory requirement for Man-6-P and PEP, as
determined by the standard quantitative assay method, as described
under "Experimental Procedures." Man could not replace Man-6-P, nor
could pyruvate replace PEP in the condensation reaction. No inhibition
of the KDN-9-P synthetase activity was observed upon incubation of
Man-6-P and PEP with either KDN (up to 1 mM), Neu5Ac (up to
1 mM), CMP-KDN (up to 0.5 mM), or CMP-Neu5Ac
(up to 0.5 mM).
Kinetic Properties of KDN-9-P Synthetase in Rainbow Trout
Testis--
Synthesis of KDN-9-P from Man-6-P and PEP was found to
increase linearly with the amount of enzyme, up to 2.2 units in 25 µl
of incubation mixtures. The kinetics of KDN-9-P formation was also
shown to increase linearly with time for up to at least 8 h. At 5 mM Man-6-P, KDN-9-P synthesis increased as the
concentration of PEP increased and approached saturation at 5 mM. Therefore, the kinetic constants for the enzyme were
determined by varying the concentrations of Man-6-P in the presence of
5 mM PEP after incubation with 1.1 units of enzyme for 20 min in 25-µl incubation mixtures. Michaelis-Menten and
Lineweaver-Burk plots for the synthesis of KDN-9-P are shown in Fig.
2, A and B. The
kinetic constants for Man-6-P were Km, 3.66 mM, and Vmax, 0.201 mM/h.
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Fig. 2.
Kinetic analysis for the synthesis of KDN-9-P
and Neu5Ac-9-P. PEP (5 mM) and varying concentrations
of Man-6-P or ManNAc-6-P were incubated with the AS70-90 enzyme
fraction (1.1 units) at 25 °C for 20 min. The initial velocities
were measured by quantitating the products by the DMB fluorometric HPLC
method. Michaelis-Menten plots for the synthesis of KDN-9-P
(A) and Neu5Ac-9-P (C) against varying
concentrations of Man-6-P and ManNAc-6-P, respectively, and the
Lineweaver-Burk plots for the synthesis of KDN-9-P (B) and
Neu5Ac-9-P (D) are shown.
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The AS70-90 enzyme fraction contained both KDN-9-P synthetase and
Neu5Ac-9-P synthetase activities. These two activities could be
differentiated, however, based on their substrate specificities and
kinetic constants. Similar to the synthesis of KDN-9-P, synthesis of
Neu5Ac-9-P from ManNAc-6-P and PEP was found to increase linearly with
time for at least 4 h. At 5 mM ManNAc-6-P, synthesis
of Neu5Ac-9-P also increased as the concentration of PEP increased and
approached saturation at 5 mM. Therefore, the kinetic
constants for Neu5Ac-9-P synthetase were determined by varying the
concentration of ManNAc-6-P with 5 mM PEP using the same
amount of enzyme described above. As shown in Fig. 2, C and
D, the kinetic constants for ManNAc-6-P were determined to
be Km, 0.245 mM, and
Vmax, 0.301 mM/h.
Separate Enzyme Activities Are Required for Synthesis of KDN-9-P
and Neu5Ac-9-P--
To determine if KDN-9-P synthetase and Neu5Ac-9-P
synthetase activities in the AS70-90 fraction were due to the same
active site on a single enzyme or to two different enzymes, the mixed substrate method of Dixon and Webb (45) was employed. As shown in Fig.
3, the experimentally determined results
significantly matched more closely the theoretical curve predicted for
the two-enzyme rather than the one-enzyme theory, suggesting that the
two enzyme activities are due to two separate catalytic sites. Slight
deviation of the observed vt from the theoretical
vt, calculated on the two-enzyme assumption, is
interpreted to result from cross-inhibition of each enzyme by substrate
competition (i.e. ManNAc-6-P for KDN-9-P synthetase and
Man-6-P for Neu5Ac-9-P synthetase), as discussed more fully by
Chevillard et al. (46). Thus, each substrate may bind to the
catalytic site on the respective enzyme but not serve as a
substrate.
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Fig. 3.
Separate synthetases are required for
synthesis of KDN-9-P and Neu5Ac-9-P. Substrate competition
experiments. Incubation mixtures containing the partially purified
KDN-9-P and Neu5Ac-9-P synthetases (AS70-90 fraction, 1.1 units) were
set up that contained fixed concentrations of either Man-6-P
(A) or ManNAc-6-P (B) and varying concentrations
of ManNAc-6-P (A) or Man-6-P (B), respectively.
Incubation was carried out at 25 °C for 20 min. The kinetic
constants for Man-6-P and ManNAc-6-P were determined as described under
"Experimental Procedures." The theoretical total velocities,
vt, calculated assuming one enzyme (open
circle) or two enzymes (open circle with cross) and the
observed vt (closed circle) are shown. In
both cases, the observed vt more closely
approximates that expected for two enzyme activities rather than
one.
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The Ratio of KDN-9-P to Neu5Ac-9-P Synthetase Activity Differ in
Each Step During Purification--
To confirm that the KDN-9-P and
Neu5Ac-9-P synthetase activities resulted from separate enzymes, the
ratio of both activities was determined in each fraction obtained
during purification. If a single enzyme was responsible for synthesis
of both KDN-9-P and Neu5Ac-9-P, then the ratio of the two activities
would be predicted to remain unchanged during purification. In
contrast, if more than one enzyme were responsible, then a differential change in the ratio of activities would be predicted. As shown in Table
III, the ratio of KDN-9-P to Neu5Ac-9-P
synthetase activities differed significantly from each other during
purification. The lower value (0.48) for the AS70-90 fraction compared
with that for the DEAE-Toyopearl fraction (0.69) was likely due to the
instability of KDN-9-P synthetase relative to Neu5Ac-9-P synthetase in
the subsequent purification steps. Taken together with the results of
the substrate competition experiments, these results strongly support
our conclusion that synthesis of KDN-9-P is catalyzed by a specific
KDN-9-P synthetase that is a different enzyme than the Neu5Ac-9-P
synthetase, which catalyzes synthesis of Neu5Ac-9-P.
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Table III
Specific activities of KDN-9-P and Neu5Ac-9-P synthetases in rainbow
trout testis and liver
Each fraction was prepared in exactly the same way from both tissues.
The KDN-9-P and Neu5Ac-9-P synthetase activities of each fraction
containing the same protein concentration were estimated by the
standard quantitative assay as described under "Experimental
Procedures." One unit of enzyme activity is defined as the amount of
enzyme required to synthesize 1 nmol of KDN-9-P or Neu5Ac-9-P/h at
25 °C.
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Rainbow Trout Liver Contains Higher Levels of Neu5Ac-9-P Synthetase
than KDN-9-P Synthetase Activity--
The enzyme fractions, Ucfg sup,
DEAE-Toyopearl, and AS70-90, were prepared from the trout liver by the
same procedures as those described for testis and assayed for KDN-9-P
and Neu5Ac-9-P synthetase activities. As shown in Table III, the level
of KDN-9-P synthetase was extremely low in all fractions compared with
that of Neu5Ac-9-P synthetase activity. For example, in the AS70-90 fraction, the ratio of specific activities of KDN-9-P to Neu5Ac-9-P synthetase in liver was 0.031, whereas in testis it was 0.49. This
difference in the level of expression of the two synthetase activities
in testis and liver is in accord with our conclusion that the
Neu5Ac-9-P and KDN-9-P synthetases are distinct enzymes and that the
KDN-9-P synthetase is differentially enriched in trout testis compared
with liver.
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DISCUSSION |
Synthesis of N-acylneuraminic acids in eukaryotes has
been studied extensively for the last 40 years (35, 47-54, and
reviewed in Refs. 55 and 56). Recently, many of the enzymes involved in
Neu5Ac metabolism have been purified (57-59) and cloned (60-63). These include GlcNAc 2-epimerase (60), UDP-GlcNAc 2-epimerase (ManNAc
kinase) (57, 61), CMP-Neu5Ac synthetase (58, 59, 62), CMP-Neu5Ac
transporter (63), and several different monosialyltransferases (reviewed in Ref. 64). Although CMP-KDN synthetase (25) and KDN
transferases (26, 28), two key enzymes involved in the synthesis of
KDN-containing glycoconjugates, have been identified, there have been
no published reports as to how the KDN monomer is synthesized. The
significance of the new information provided in this study is that the
pathway for KDN synthesis in rainbow trout ovary and testis has been
shown to involve a condensation of PEP and Man-6-P as a key reaction:
Man-6-P + PEP 
KDN-9-P + Pi. This reaction is catalyzed
by the KDN-9-P synthetase. Hexokinase activity catalyzing formation of
Man-6-P and phosphatase activity catalyzing dephosphorylation of
KDN-9-P were also detected in the cytosol of trout ovary and testis.
These activities may be due to the ubiquitous hexokinase and
phosphatases known to be present in the cytosol of various cells (36,
37), although we cannot exclude the possibility that there may be
hexokinase and phosphatase activities specific for Man and KDN-9-P, respectively.
The cytosolic trout testis KDN-9-P synthetase had an obligatory
requirement for Man-6-P and PEP and could not condense Man and PEP or
pyruvate. Notably, however, the partially purified KDN-9-P synthetase
activity used in this study could also condense ManNAc-6-P and PEP,
suggesting that the enzyme fractions also contained Neu5Ac-9-P
synthetase activity. These two activities were not separated from each
other by column chromatography on Poros HQ or Cibacron Blue
F3GA-agarose. However, the following three lines of evidence strongly
support our conclusion that KDN-9-P synthetase and Neu5Ac-9-P
synthetase are separate enzymes. First, the specific activity ratio of
KDN-9-P to Neu5Ac-9-P synthetase activity varied among the fractions
obtaining during the different steps in the purification procedure
(Table III). If these activities were on the same enzyme, the ratio
would be expected to be constant in each fraction. Second, the results
of substrate competition experiments using Man-6-P and ManNAc-6-P as
competitors were consistent with the theoretical prediction that the
two activities were distinct and thus likely represent two separate
enzymes. Third, it was shown that the rainbow trout liver contained
predominately Neu5Ac-9-P synthetase and little, if any, KDN-9-P
synthetase activity. In contrast, trout testis contained both enzyme
activities but was enriched in the level of KDN-9-P synthetase
activity. Notably, these findings appear consistent with the fact that
the levels of KDN in liver are significantly less than in testis,
whereas Neu5Ac is abundant in both tissues (21). These results also support our conclusion that KDN-9-P synthetase is actually distinct from Neu5Ac-9-P synthetase in trout testis. Our analyses do not allow
us to determine unequivocally, however, if both activities reside on a
single or separate polypeptide chains. To differentiate between these
two possibilities, purification of either the KDN-9-P or Neu5Ac-9-P
synthetase to homogeneity will be required.
The relative expression level of KDN- to Neu5Acyl-containing
glycoconjugates in trout testis is relatively high (59.5 versus 34 nmol/g tissue; Table I; Refs. 21-23). This is
consistent with the relatively higher level of free KDN to free
Neu5Acyl (101 versus 66.6 nmol/g of tissue; Table I) and the
somewhat higher level of Man to ManNAc (42.3 versus 36 nmol/g of tissue). This latter result is also consistent with our
finding that in trout testis Man and ManNAc are the biosynthetic
precursors for KDN and Neu5Ac, respectively. We found, however, a
3.5-fold higher level of Neu5Ac-9-P synthetase than that of KDN-9-P
synthetase, based on the ratio of specific activities in the cytosolic
Ucfg fraction (Table II). Although this may appear to be inconsistent with the relatively lower abundance of Neu5Acyl than KDN residues in
trout testis (Table I), we believe this finding may simply reflect the
reduced levels of ManNAc-6-P required for the Neu5Ac-9-P synthetase.
Thus, the relatively lower level of expression of Neu5Acyl to KDN
residues in trout testis appears to be positively correlated with
attenuation of enzyme activities required for synthesis of ManNAc.
It is known that Man-6-P can be synthesized by at least two possible
biosynthetic pathways in eukaryotic cells. In the first, Man-6-P is
formed in the cytosol from fructose 6-phosphate by phosphomannoisomerase. Alternatively, Man can be phosphorylated by a
6-O-phosphokinase after transport into the cell via
mannose-specific transporters (65, 66). Most Man residues that are
incorporated into the N-linked oligosaccharide chains in
glycoproteins are derived from the latter pathway (65, 66). Therefore,
the substrate pool of Man-6-P could originate from intracellular free
Man via direct phosphorylation or via phosphomannoisomerase. Our
studies clearly show that Man-6-P can be synthesized in the cell once Man is provided. Interestingly, ManNAc-6-P is reported to be
synthesized from UDP-GlcNAc and ATP by a single bifunctional enzyme,
UDP-GlcNAc 2-epimerase/ManNAc kinase (57). This appears to be a
distinctly different feature for the synthesis Neu5Ac compared with
KDN, because an analogous pathway from UDP-Glc to Man-6-P via Man has not been reported.
Expression of KDN-containing glycoproteins and glycolipids in mammalian
tissues has only recently been reported. These studies have shown that
KDN residues are expressed in a tissue-, developmental-, tumor-, and
protein-specific manner (5, 8, 19, 20). Elucidation of the metabolic
pathway for KDN expression in mammalian cells is difficult to study
because of the small quantities of KDN present (5). The present studies
thus represent an important step in this endeavor because they describe
the pathway for synthesis of the KDN monomer in trout testis, catalyzed
by a KDN-9-P synthetase, a finding that has not been previously
described. The difficulty in purifying the enzyme to homogeneity
highlights the importance of the need to clone, overexpress, and purify
the KDN-9-P synthetase activity from trout testis as one step in
advancing our understanding of how expression of the KDN glycotope may
be regulated (67). Given that the levels of free KDN are 2.4-fold
higher in fetal cord red blood cells compared with matched maternal red
blood cells and is also elevated in ovarian cancer cells, the present study highlights the importance of future studies to elucidate the role
that free KDN and KDN-glycoconjugates may play in normal development
and malignancy.
,
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ABSTRACT
INTRODUCTION
