(Received for publication, October 12, 1994)
From the
Among sulfated polysaccharides, those in the tunic of ascidians
are unique: their major constituent sugar is galactose, which occurs
exclusively in the L-enantiomeric form. Incorporation of D-[C]glucose into tunic slices in
vitro revealed that the cells epimerize D-glucose into L-galactose during biosynthesis of the sulfated
polysaccharides. The interconversion of these two sugars involves
exchange of hydrogen atoms at the epimerization sites with protons of
the medium. Tunic cells also synthesize trehalose, although this
disaccharide is not a prominent constituent of the tissue. Pulse-chase
experiments using D-[
C]glucose reveal
that incorporation of label into trehalose precedes the synthesis of
the sulfated L-galactan. In addition, the loss of label from
trehalose coincides with the appearance of label in the sulfated L-galactan. Based on these results, we speculate that
trehalose in the ascidian tunic may be a precursor of the sulfated L-galactan.
Sulfated polysaccharides from different invertebrate tissues include several that are not found in vertebrates. Novel sulfated polysaccharides have been reported in the tunic of ascidians(1, 2, 3, 4, 5, 6, 7) , the body wall of a sea cucumber(8, 9, 10, 11, 12) , and the jelly coat from sea urchin eggs(13) .
The sulfated polysaccharides in the tunic of ascidians (Chordata, Tunicata) occur as fractions that are distinct in molecular weight and chemical composition. A high molecular weight glycan composed mainly of sulfated galactose residues is the preponderant fraction in all ascidians studied so far(2, 3, 4, 5, 6, 7) .
In previous studies, we showed that the galactose from the sulfated
polysaccharides of ascidians occurs entirely in the L-enantiomeric form(3, 7, 14) .
Although L-galactose is a constituent of several
polysaccharides (15, 16, 17, 18) ,
these were the first reports of sulfated polysaccharides containing
high amounts of L-galactose but no D-galactose. In
the high molecular weight fraction, galactose occurs mainly as
-L-galactopyranosides, linked glycosidically through
positions 1-4 and sulfated at position 3 (3, 4, 5, 6, 7) . The small
amounts of glucose units found in these polysaccharides from some
species of ascidians are the D-isomer, primarily in
-D-glucopyranosyl units(3, 4) .
The pathway involved in the incorporation of L-galactose into polysaccharides is not known. The major obstacle for this study is that L-galactose occurs in the presence of large amounts of the D-isomer of this sugar(15, 16, 17, 18) . It occurred to us that the ascidian tunic could be useful for studies on the metabolism of L-galactose.
In a previous study we reported that the biosynthesis of sulfated polysaccharides from radiolabeled precursors is more intense in the region of the ascidian tunic containing epidermal cells and that subsequently the incorporated material is redistributed into more peripheral areas of the tunic(19) . Surprisingly, the most effective precursor was D-glucose(20) .
We now investigate in detail the
epimerization of D-glucose to L-galactose during the
biosynthesis of the sulfated L-galactan in the ascidian tunic.
Our results reveal an unexpected parallel between the biosynthesis of
sulfated L-galactan and the incorporation of D-[C]glucose into trehalose in the
ascidian tissue.
The C-
and
H-labeled compounds were isolated following the method
described previously for fresh tissue(2) . Briefly, the dried
slices (
2.0 mg, dry weight) were suspended in 0.5 ml of 0.1 M sodium acetate buffer (pH 5.0) containing 30 µg of twice
crystallized papain, 5 mM EDTA, and 5 mM cysteine and
incubated at 60 °C for 24 h. The incubation mixture was then
centrifuged (2,000
g for 10 min at room temperature),
and the incubation of the pellet with papain was repeated. The clear
supernatants were combined and mixed with 2 volumes of absolute
ethanol. The precipitate formed after standing at -10 °C for
24 h was collected by centrifugation (2,000
g for 15
min at room temperature), dried at 80 °C for 1 h, and dissolved in
100 µl of distilled water.
To estimate the amount of
radioactivity transferred from C- or
H-labeled
sugars to other molecules of the ascidian tunic, the solution described
above was applied to Whatman 3MM chromatographic paper and developed in
1-butanol/pyridine/water (3:2:1, v/v) for 24 h. The origin, now free of
C- or
H-labeled monosaccharides, was cut out,
added to 5 ml of 0.5% PPO(
)/toluene solution, and counted in
a liquid scintillation counter. Throughout the text we refer to this
result as ``incorporated radioactivity.''
Figure 5:
Separation of the C-labeled
molecules extracted from the ascidian tunic by anion exchange
chromatography on DEAE-cellulose. Panels A-C, slices of
ascidian tunic (
120 mg, wet weight) were incubated at 20 °C
with 3.5 µCi (5.25 nmol) of D-[U-
C]glucose in 0.5 ml of the
incubation medium (see ``Experimental Procedures'') for 3 (panel A) or 6 h (panel B). In panel C, the
ascidian tissue was incubated with D-[U-
C]glucose for 6 h (as in panel
B), but then the incubation medium was removed, and a new medium
without D-[U-
C]glucose was added to the
tissue slices and incubated for additional 3 h. At the end of the
incubation period, the
C-labeled molecules were extracted
from the tunic slices by papain digestion, precipitated with ethanol,
and dissolved in distilled water, as described under
``Experimental Procedures.'' The solutions of
C-labeled molecules were applied to Whatman 3MM
chromatographic paper and developed in 1-butanol/pyridine/water (3:2:1,
v/v) for 24 h. The origins, now free of
C-labeled
monosaccharides but containing the incorporated radioactivity, were cut
out, and the
C-labeled compounds were eluted with
distilled water, dried in a rotary evaporator, and dissolved in 0.5 ml
of distilled water. This solution was applied to a column (1
1
cm) of DEAE-cellulose preequilibrated with 0.05 M pyridine/acetate buffer (pH 6.0), washed with 15 ml of the same
buffer, and developed with a linear gradient of NaCl, prepared by
mixing 15 ml of 0.05 M pyridine/acetate buffer (pH 6.0) with
15 ml of 1.5 M NaCl in the same buffer, at a flow rate of 12
ml/h. Fractions were collected each 2 min and checked by the
metachromaticity assay (
), the radioactivity was counted in a
liquid scintillation counter (
), and the NaCl
concentration(- - -) was estimated by conductivity.
The fractions containing the sulfated polysaccharides (horizontal
bars) were pooled, dialyzed against distilled water, and
lyophilized. The fractions containing the nonacidic compound were
pooled and concentrated on a rotary evaporator, without previous
dialysis. Panel D shows the absolute amounts of label
incorporated into the nonacidic compound (
) and into the sulfated
polysaccharides (
) during the pulse-chase
experiment.
Figure 7:
Purification of the nonacidic compound.
Slices of ascidian tunic (120 mg, wet weight) were incubated with
3.5 µCi (5.25 nmol) of D-[U-
C]glucose in 0.5 ml of the
incubation medium. The
C-labeled compounds were extracted
and separated from
C-labeled monosaccharides as described
in the legend of Fig. 5and under ``Experimental
Procedures.'' The same procedures were applied to
46 mg (dry
weight) of ascidian tunic, but using nonradioactive glucose. The
origins of the chromatograms of both labeled and unlabeled compounds
were eluted with distilled water, combined, concentrated on a rotary
evaporator, and dissolved in 1.0 ml of distilled water. In panel A this solution was applied to a column (10
1 cm) of
DEAE-cellulose preequilibrated with 0.05 M pyridine/acetate
buffer (pH 6.0), followed by 30 ml of the same buffer, then 30 ml of
2.0 M NaCl (arrow), at a flow rate of 8 ml/h.
Fractions (1.1 ml) were collected and checked by the reaction of Dubois et al. (
) and by the metachromatic assay (
), and
counted (
) in a liquid scintillation counter. In panel
B, the fractions containing the nonacidic compound (pool 1 from panel A) were concentrated on a rotary evaporator,
dissolved in 1 ml of distilled water, and applied to a 87.0
1.5-cm column of Bio-Gel P-4 (400 mesh). Fractions were eluted and
analyzed as described for panel A, and those containing the
nonacidic compound (bracket) were pooled and concentrated on a
rotary evaporator. The column was calibrated using chondroitin
6-sulfate from shark cartilage, cellobiose, and D-glucose (arrows 1-3, respectively).
To
assess the -D- and
-D-glucosidase
activities, the p-nitrophenol liberated from p-nitrophenyl
-D-glucopyranoside and from p-nitrophenyl
-D-glucopyranoside, respectively,
was quantified. The enzymes (0.2 unit) were incubated at 37 °C for
30 min in 50 µl of 50 mM sodium acetate buffer (pH 5.0 for
-D-glucosidase and pH 6.8 for
-D-glucosidase). The reaction was terminated with 1 ml of
0.25 M glycine buffer (pH 10.0), and the absorbance was read
at 540 nm. To assess trehalase activity, trehalose (50 µg) was
incubated with the enzyme (0.1 unit) at 37 °C for 4 h in 50 µl
of 20 mM potassium acetate buffer (pH 6.5). The mixture
incubated with trehalase and the appropriate control solution were
applied on Whatman No. 1 paper and developed by descending
chromatography in isobutyric acid, 1 N NH
OH (5:3,
v/v) for 24 h. The sugars were visualized by silver nitrate staining.
Figure 1:
Labeling of the ascidian
polysaccharides with D-[U-C]glucose. Panel A, time course of incorporation of D-[U-
C]glucose. Slices of ascidian
tunic (
15 mg, wet weight) were incubated at 20 °C with 0.7
µCi (1.05 nmol) of D-[U-
C]glucose
in 100 µl of the incubation medium (see ``Experimental
Procedures''). At different times, the medium was decanted, and
the slices were washed five times with the incubation medium, then
immersed in 2 ml of acetone, kept for 24 h at 4 °C, and dried. The
C-labeled compounds were extracted by papain digestion,
and the incorporated radioactivity was quantitated as described under
``Experimental Procedures.'' Panel B, agarose gel
electrophoresis of the
C-labeled polysaccharides. Twenty
microliters of the ascidian polysaccharides, labeled with D-[U-
C]glucose for different periods of
times, was applied to a 0.5% agarose gel, and electrophoresis was
carried out in 0.05 M 1,3-diaminopropane/acetate buffer (pH
9.0) for 1 h at 120 V. The sulfated polysaccharides from the gel were
fixed with 0.1% N-cetyl-N,N,N-trimethylammonium
bromide in water for 12 h and stained with 0.1% toluidine blue in
acetic acid/ethanol/water (0.1:5:5, v/v). The radioactive bands
corresponding to the
C-labeled polysaccharides were
detected by autoradiography of the fixed and stained gel. The
electrophoretic migration of standard F-1 and F-2 fractions are
indicated at the right. Panel C, identification of
the
C-labeled sugars. The acid hydrolysates of the labeled
compounds, before (
) and after (
) incubation with D-galactose oxidase, were applied to Whatman No. 1 paper and
chromatographed in 1-butanol/pyridine/water (3:2:1, v/v) for 48 h. The
chromatogram was cut into 3-mm-wide strips and counted in a liquid
scintillation counter. The relative amounts (percentage) of label
appearing in each of the sugars, on the basis of the area of each peak
compared with total area, are shown beside each peak. The upper portion of the panel (a and b) shows a strip
from a chromatogram developed with silver nitrate where a standard
solution was applied, containing 10 µg each of D-galactose (lane 1), D-glucose (lane 2), and D-mannose (lane 3), before (a) and after (b) a 4-h incubation with D-galactose oxidase (origin
at right). Incubation conditions and the hydrolysates are
described under ``Experimental
Procedures.''
Identification of the C-labeled sugars in the
acid hydrolysate of the ascidian polysaccharides shows a high
proportion of label in the galactose residues (closed circles in Fig. 1C). The
[
C]galactose is totally resistant to oxidation
by D-galactose oxidase (open circles in Fig. 1C), indicating that this sugar is entirely in the L-enantiomeric form, as already observed for the nonlabeled
polysaccharides(3, 7, 14) . The measurement
of the oxidation by D-galactose oxidase included a control,
which shows the total oxidation of the D-galactose standard (b in Fig. 1C).
Overall, these results
demonstrate that cells in the ascidian tunic incorporate D-[C]glucose and epimerize this sugar
into L-[
C]galactose during the
biosynthesis of their constituent sulfated polysaccharides.
The interconversion of sugars is a relatively common reaction in
carbohydrate metabolism. It can proceed as in the enzymatic conversion
of UDP-D-glucose to UDP-D-galactose. The
interconverting enzyme and all other such epimerases exhibit an
absolute requirement for a tightly bound molecule of NAD or NADP
. Oxidation is followed by reduction with
inversion of the configuration at the epimerization site, and a proton
is exchanged with the enzyme-bound NAD
or
NADP
(25) . In contrast, in the epimerization
of D-mannose to D-glucose, which involves the action
of D-mannose phosphate isomerase, the hydrogen atom at carbon
2 of D-mannose is exchanged with the protons of
water(26) . A similar mechanism occurs in several other
epimerization reactions, such as the double epimerization in the
synthesis of UDP-L-rhamnose from UDP-D-glucose (27) or the conversion of polysaccharide-bound
-D-glucuronic acid to
-L-iduronic acid in
the course of heparin or dermatan sulfate
biosynthesis(28, 29) .
These two classes of sugar
epimerization reactions can be distinguished experimentally. The second
class of reactions is accompanied by the incorporation of H
from
H-labeled water into the sugar (29, 30) and by the exchange of
[
H]hydrogen atoms from the epimerization sites of
the hexosyl moieties with protons of the medium(31) . When the
reaction involves NAD
or NADP
-bound
enzymes, these exchanges of
H atoms will not occur.
To
distinguish between these two possibilities, slices of tunic were
incubated with D-glucose labeled with tritium at C-2, C-3,
C-5, or C-6. The [H]hydrogen atoms at C-2, C-3,
and C-5 are exchanged with protons of the medium and do not remain in
the hexosyl moieties after epimerization to L-galactose (Fig. 2, A and B). In contrast, the
[
H]hydrogen atom at C-6 of D-glucose
appears in the L-galactose (Fig. 2C). This
result shows that the C-6 position is not inverted during the
conversion of D-glucose to L-galactose. (
)
Figure 2:
Identification of the H-labeled sugars in the acid hydrolysates. Slices of
ascidian tunic (
15 mg, wet weight) were incubated with 20 µCi
(0.66 nmol) of D-[3-
H]glucose (panel
A), D-[5-
H]glucose (panel
B), and D-[6-
H]glucose (panel
C) for 6 h at 20 °C. The
H-labeled compounds were
extracted from the tissue slices as described under ``Experimental
Procedures'' and in the legend of Fig. 1. The acid
hydrolysates of the
H-labeled compounds were applied to
Whatman No. 1 paper and chromatographed in 1-butanol/pyridine/water
(3:2:1, v/v) for 48 h. The chromatograms were cut into 3-mm-wide strips
and counted in a liquid scintillation counter. The upper part
of the figure shows the chromatographic migration of D-galactose (1), D-glucose (2), and D-mannose (3) standards. Distribution of the
H-labeled sugars after labeling the tunic with D-[2-
H]glucose is similar to that shown
in panel A.
These results indicate that conversion of D-glucose to L-galactose involves exchanges of hydrogen atoms at the epimerization sites with protons of the medium.
Figure 3:
Identification of the C-labeled sugars in the acid hydrolysates of the molecules
from the ascidian tunic labeled for different periods of time. Slices
of the ascidian tunic were incubated with D-[U-
C]glucose for 1 (panel
A), 2 (panel B), 3 (panel C), 4 (panel
D), 6 (panel E), and 8 h (panel F). The
C-labeled molecules were extracted as described under
``Experimental Procedures,'' and their acid hydrolysates were
separated and quantitated by paper chromatography as described in the
legend of Fig. 1C. The relative amounts (percentage) of
each of the
C-labeled sugars, on the basis of the area of
each peak compared with the total area, are shown beside each peak. Panels G and H show the absolute amounts of label
incorporated into each sugar; D-glucose (
), L-galactose (
), and D-mannose (
). The
data of panel G were obtained from the experiment shown in panels A-F, and those of panel H were from a
similar experiment not shown in the
figure.
An explanation for the experiment of Fig. 3is
that epimerization of D-glucose to L-galactose occurs
after incorporation of D-glucose into the polymer, as
described previously for the conversion of polysaccharide-bound
-D-glucuronic acid to
-L-iduronic acid
during biosynthesis of heparin and dermatan
sulfate(28, 29) . The experiment of Fig. 4supports this possibility. When the D-[
C]glucose is removed from the
incubation medium after 6 h, the content of radioactivity appearing as L-[
C]galactose increases in the absence
of free D-[
C]glucose (Fig. 4, A-C, D-F, and closed circles in G).
Figure 4:
Chase experiment of the C-labeled molecules of the ascidian tunic. Slices of the
ascidian tunic were labeled by incubation with D-[U-
C]glucose for 6 h at 20 °C in
two independent experiments (panels A-F), as described
in the legend of Fig. 1. The incubation medium was removed, a
new medium without D-[
C]glucose was
added, and the tissue slices were incubated for different periods of
time, as indicated in panels B-C and E-F.
The
C-labeled compounds were extracted as described under
``Experimental Procedures.'' Panels A-F,
identification of the
C-labeled sugars in the acid
hydrolysates was performed by paper chromatography, as described in the
legend of Fig. 1C. Panel G shows the absolute
amounts of label incorporated into L-galactose (
) and D-glucose (
) in Experiment 2 (panels D-F). Panel H, distribution of
C between F-1 and F-2
fractions during the chase experiment shown in panels
D-F. The
C-labeled polysaccharides were
analyzed by agarose gel electrophoresis as described in the legend of Fig. 1B. The radioactive bands corresponding to the
electrophoretic migrations of standard F-1 (
) and F-2 (
)
fractions were scraped into 5 ml of 0.5% PPO/toluene solution and
counted in a liquid scintillation
counter.
In the tunic of the ascidian S. plicata, the sulfated polysaccharides occur as three main
fractions, which are markedly distinct in their molecular weight and
chemical composition(2, 3) . The high molecular weight
fraction, denominated F-1, contains a high proportion of L-galactose and minor amounts of D-glucose, whereas
the other two fractions, of low molecular weight, denominated F-2-A and
F-2-B, contain, besides L-galactose, a higher proportion of D-glucose, amino sugars, and small amounts of mannose.
Therefore, the pronounced changes in the proportions of L-[C]galactose and D-[
C]glucose during the chase
experiment (Fig. 4, A-C, D-F, and G) might reflect modification
in the proportions of F-1 and F-2 fractions. However, the experiment of Fig. 4H excludes this possibility. The proportions of
C-labeled fractions F-1 and F-2 remain constant during the
chase experiment.
The distribution of
label between D-glucose and L-galactose residues in
the nonacidic compound and the sulfated polysaccharides in Fig. 5(A-C) was examined following acid
hydrolysis. The nonacidic compound is labeled exclusively in D-[C]glucose residues, and the sulfated
polysaccharides have a high proportion of L-[
C]galactose (Fig. 6, A-C), which is similar to the proportion of L-galactose found in the nonlabeled sulfated polysaccharides
from the ascidian tunic (Fig. 6E). Therefore, it
appears that the
C-labeled sulfated polysaccharides
obtained during the three experimental conditions of Fig. 5(A-C) are the ``final''
biosynthetic product. In addition, most of the L-[
C]galactose units of these polymers
resist periodate oxidation (Fig. 6D), indicating that
these residues are sulfated. In the ascidian polysaccharides, galactose
exists mainly as
-L-galactopyranosides, linked
glycosidically through positions 1-4 and sulfated at position 3 (3, 4, 5, 6, 7) . Thus, the
sulfated residues can be distinguished from the nonsulfated units by
their resistance to periodate oxidation.
Figure 6:
Distribution of C between L-galactose and D-glucose residues in the nonacidic
compound and in the sulfated polysaccharides. Panels
A-C, the nonacidic compounds and the sulfated
polysaccharides from the pulse and chase experiments of Fig. 5, A-C, were subjected to acid hydrolysis (6 N trifluoroacetic acid at 100 °C for 4 h). The mixtures of
C-labeled sugars were applied to Whatman No. 1 paper and
separated by descending chromatography in 1-butanol/pyridine/water
(3:2:1, v/v) for 48 h. The proportions of
C-labeled sugars
were estimated as described in the legend of Fig. 1C. Panel D, another sample of sulfated polysaccharides labeled
for 6 h with D-[U-
C]glucose, which was
submitted to periodate oxidation and borohydride reduction, as
described(6) . The proportions of L-[
C]galactose and D-[
C]glucose units in this chemically
modified polysaccharide were determined as described above. Panel
E, nonlabeled sulfated polysaccharide was submitted to acid
hydrolysis, and the mixture of sugars was separated by paper
chromatography, as described for the
C-labeled compounds.
The nonlabeled sugars were visualized in the chromatogram by silver
nitrate staining and quantitated by densitometry (see (2) for
additional details).
Overall, the experiments of Fig. 5and Fig. 6demonstrate that variations in the
amounts of D-[C]glucose and L-[
C]galactose units observed during
the pulse (Fig. 3) and pulse-chase (Fig. 4) experiments
in fact reflect changes in the amounts of
C-labeled
nonacidic compound and
C-labeled sulfated polysaccharides
in the ascidian tunic under the different experimental conditions.
The purified C-nonacidic compound
migrates as trehalose (
71% of the total radioactivity) on paper
chromatography (Fig. 8A), releases exclusively D-[
C]glucose after acid hydrolysis (Fig. 8B), does not migrate on paper electrophoresis at
pH 5.0 (Fig. 8C), resists
-D-glucosidase (Fig. 8D) and
-D-glucosidase (Fig. 8E), but is degraded by trehalase (Fig. 8F). Upon the action of this specific enzyme it
releases exclusively D-[
C]glucose.
Overall, these experiments demonstrate unequivocally that the nonacidic
compound is mainly the disaccharide trehalose. (
)
Figure 8:
Characterization of the nonacidic
compound as trehalose. The nonacidic compound (2,000 cpm),
previously purified on anion exchange and gel filtration columns (Fig. 7, A and B), was applied to Whatman No.
1 paper, before (panel A) and after incubation with
-D-glucosidase (panel D),
-D-glucosidase (panel E), and trehalase (panel F), and chromatographed in isobutyric acid, 1.0 N NH
OH (5:3, v/v) for 24 h. In panel B, the
acid hydrolysate (6.0 N trifluoroacetic acid at 100 °C for
4 h) of the
C-labeled nonacidic compound was applied to
Whatman No. 1 paper and chromatographed in 1-butanol/pyridine/water
(3:2:1, v/v) for 48 h. In panel C, the
C-nonacidic compound (
2,000 cpm) was applied to
Whatman 3MM chromatographic paper and submitted to electrophoresis in
0.3 M pyridine/acetate buffer (pH 5.0), run for 4 h at 500 V.
The chromatograms and paper electrophoresis were cut into 3-mm-wide
strips and counted in a liquid scintillation counter. The upper portion of the panels shows a strip of the chromatograms
or of the paper electrophoresis where standard solutions were applied,
containing 20 µg each of cellobiose (1), trehalose (2), sucrose (3), D-glucose (4), D-galactose (5), D-mannose (6), D-glucuronic acid (7), and D-glucosamine (8), and the strip was developed with silver nitrate.
Incubation conditions are described under ``Experimental
Procedures.''
Figure 9:
Attempts to isolate intermediates of the D-glucose sulfated L-galactan metabolic route.
Slices of ascidian tunic (
15 mg, wet weight) were incubated at 20
°C with 0.7 µCi (1.05 nmol) of D-[U-
C]glucose in 100 µl of the
incubation medium for 1 min (panels A and D) and 6 h (panels B and E). In panels C and F the ascidian tissue was incubated with D-[U-
C]glucose for 6 h (as in panels B and E), but then the incubation medium was
removed, and a new medium without D-[U-
C]glucose was added to the tissue
slices and incubated for additional 3 h. At the end of the incubation
periods, the medium was decanted, and the slices were washed 10 times
with the incubation medium and then incubated with twice crystallized
papain (30 µg) in 100 µl of 0.1 M sodium acetate
buffer (pH 5.0), containing 5 mM EDTA and 5 mM cysteine, and incubated at 60 °C for 24 h. The clear
supernatants were applied to Whatman No. 1 paper and separated by
descending chromatography in isobutyric acid, 1.0 N NH
OH (5:3, v/v) for 24 h. The chromatogram was cut
transversely into 3-mm-wide strips, which were counted in a liquid
scintillation counter. The upper portion of panel D shows a strip of the chromatogram where a standard solution was
applied, containing 20 µg each of uridine 5`-diphosphate D-glucose (1), D-glucose 6-phosphate (2), and D-glucose (3); the sugar nucleotide
was localized using UV light, and the other sugars were developed with
silver nitrate. In panels D-F, distribution of
radioactivity in the 5-20-cm regions of the paper chromatograms
in panels A-C are shown on an expanded scale (right
ordinate).
The
absence of detectable amounts of L-galactose among the D-glucose derivatives isolated from tunic slices, together
with the unequivocal evidence that this sugar appears in the ascidian
polysaccharides, indicates that L-galactose intermediates, if
present, are formed in minimal amounts. An alternative explanation is
that epimerization occurs after incorporation of D-glucose
into the polymer. In fact, galactose exists in the ascidian
polysaccharides mainly as -L-galactopyranosides, linked
glycosidically through positions
1-4(3, 4, 5, 6, 7) .
The small amounts of glucose units occur in these polymers mostly as
4-linked,
-D-glucopyranosyl residues(3) . Thus,
the positions involved in the glycosidic linkage are not inverted
during the conversion of these two types of residues (see Fig. Z1).
Figure Z1: Structure 1
CONCLUSIONS AND FUTURE PERSPECTIVES
We investigated the epimerization of D-glucose to L-galactose during biosynthesis of a sulfated L-galactan in the ascidian tunic. Attempts to prepare
homogenates from this tissue were unsuccessful since the tunic is rigid
and composed of large amounts of fibers and amorphous substance and
very few cells(33) . Here we describe a more successful
alternative using in vitro pulse labeling of tunic slices.
This assay reveals that D-[C]glucose is
incorporated into the ascidian tunic (Fig. 1A) and
epimerizes into L-[
C]galactose (Fig. 1C) during the biosynthesis of sulfated
polysaccharides (Fig. 1B). The interconversion of these
two sugars involves exchanges of hydrogen atoms at the epimerization
sites with protons from the medium (Fig. 2).
The
radioactivity incorporated into molecules of the ascidian tunic as D-[C]glucose units increases rapidly up
to 4 h and thereafter remains constant until the end of the labeling
period (Fig. 3). When the D-[
C]glucose is removed from the
incubation medium after 6 h, the radioactivity incorporated as L-[
C]galactose increases markedly
during the chase period (Fig. 4). These changes in the content
of the two sugars in the ascidian tunic in fact reflect changes in the
proportions of
C-labeled sulfated L-galactan and
a
C-labeled nonacidic compound (Fig. 5D),
which was identified as trehalose (Fig. 8).
Apparently,
accumulation of trehalose in the ascidian tissue is a requirement for
the synthesis of the sulfated L-galactan from D-glucose (Fig. 3). During the pulse-chase experiments,
consumption of trehalose is accompanied by an increase in the synthesis
of sulfated L-galactan ( Fig. 4and 5D). In
addition, trehalose is synthesized rapidly by cells of the tunic (Fig. 3) but does not accumulate in the tissue (Fig. 7A), suggesting that in ascidians, this
disaccharide does not have its usual energy storage function (34) . ()
The results obtained in the present
investigation permit us to speculate that trehalose may be associated
with the biosynthesis of the sulfated L-galactan, perhaps even
acting as a donor of the sugar units. Although this proposal is at odds
with the many published reports that show sugar diphosphate nucleotides
as intermediates in the biosynthesis of glycoconjugates, biosynthetic
pathways involving disaccharides are not unknown. A variety of oral
streptococci and other microorganisms can utilize sucrose as a donor
substrate in the course of dextran synthesis catalyzed by
glucosyltransferase(35) . These reactions are possible since
the G of the
-linkage of sucrose (and possibly
trehalose) is
-6.6 kcal/mol, whereas the
G values for the glycosidic linkages range from -2.0 to
-4.6 kcal/mol(36) . In this case, conversion of D-glucose to L-galactose would occur after
incorporation of D-glucose into the polymer. In fact, attempts
to isolate metabolic intermediates involved in the biosynthesis of the
sulfated L-galactan reveal exclusively compounds that migrate
as uridine 5`-diphosphate D-glucose and D-glucose
6-phosphate, which are precursors of trehalose, and two other compounds
containing D-glucose. No intermediate containing L-galactose was identified (Fig. 9).
We attempted to
incorporate [C]trehalose into the sulfated L-galactan using the in vitro pulse labeling of the
tunic slices (as in Fig. 1but with
[
C]trehalose in place of D-[
C]glucose). No radioactivity was
incorporated into the polysaccharides (not shown). This result may be
because of difficulties in the transport of trehalose through the cell
membrane. We will have to develop a cell-free system, perhaps starting
with cells derived from the tunic and grown in culture, to test this
hypothesis.