Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

Article abstract of DOI:10.1134/S1068162006010067

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the sulfate group in position C4 of xylose

Full text of DOI:10.1134/S1068162006010067

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 1, pp. 63–70. © Pleiades Publishing, Inc., 2006.
Original Russian Text © M.I. Kusaykin, M.S. Pesentseva, A.S. Sil’chenko, S.A. Avilov, V.V. Sova, T.N. Zvyagintseva, V.A. Stonik, 2006, published in Bioorganicheskaya Khimiya,
2
006, Vol. 32, No. 1, pp. 71–79.
Aryl Sulfatase of Unusual Specificity from the Liver
of Marine Mollusk Littorina kurila
1
M. I. Kusaykin, M. S. Pesentseva, A. S. Sil’chenko, S. A. Avilov, V. V. Sova,
T. N. Zvyagintseva, and V. A. Stonik
Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences,
pr. 100-letiya Vladivostoka 159, Vladivostok, 690022 Russia
Received April 18, 2005; in final form, July 1, 2005
Abstract—An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina
kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the
sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glyco-
sides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous
according to electrophoresis and has M 45 ± 1 kDa. The semiinactivation time of the enzyme at 60°ë is 20 min,
and its K value for the hydrolysis of p-nitrophenyl sulfate is 8.7 ± 1 mM. It was shown that natural sulfated
m
polyhydroxysteroids inhibit activity of the sulfatase; their I values depend on their structures and are within
5
0
–
3
–5
the range from 10 to 10 M.
Key words: aryl sulfatase, specificity, inhibition; marine mollusk Littorina kurila; sulfated polyhydroxysteroids,
triterpene glycosides
DOI: 10.1134/S1068162006010067
1
INTRODUCTION
free from an aryl sulfatase activity, whereas the homo-
geneous aryl sulfatases exhibited traces of the steroid
sulfatase activity [16]. Many naturally occurring sul-
fated sugars appear to be substrates for aryl sulfatases
but not for glycosulfatases, etc.
Sulfatases (EC 3.1.6) form a large class of enzymes
that catalyze the hydrolytic splitting off of sulfate from
various substrates. They are found in both prokaryotes
[
1, 2] and eukaryotes: invertebrates [3–5] and mammals
6–8]. Bacterial sulfatases predominantly split off sul-
At present, the attention of researchers is focused on
sulfatases that can simplify the structure of sulfated
polysaccharides and thereby facilitate the structural
analysis of these complex molecules. Reports on
polysaccharide sulfatases and, in particular, fucoidan
sulfatases are practically absent. It has earlier been
shown by Japanese investigators that a partially purified
sulfatase preparation from the marine mollusk Charo-
nia lampas effectively desulfated only cellulose sulfate
and weakly, sulfates of amylose, glycogen, sulfated
polysaccharides of algae, sea urchin ovicells, etc. [17].
Later, in some species of marine mollusks collected at
shore of England, a sulfatase was identified that exhib-
ited activity toward sulfated carbohydrates of both low
and high molecular masses, including fucoidan [3].
[
fate from synthetic substrates, such as 4-nitrophenyl
sulfate and 4-nitrocatechol sulfate [9, 10]. Mammalian
sulfatases are involved in transformations of muco-
polysaccharides, sulfolipids, and sulfosteroids [6–8]. In
invertebrates, arylsulfatases, steroidsulfatases, and gly-
cosulfatases of different specificity have been identi-
fied, which affect either sugar sulfates or sulfated
polysaccharides [3–5].
Comparative studies of structures of the sulfatases
showed that they are evolutionarily conservative
enzymes [11]. The active site of sulfatases contains an
α
unusual ë -formylglycine residue, which is formed
during the posttranslational modification from cysteine
in eukaryotic enzymes and from serine in prokaryotic
enzymes [11, 12]. A structural similarity between the
known aryl sulfatases and phosphatases was reported. It
was hypothesized that these enzyme groups are evolu-
tionarily related [13, 14].
We continue to investigate the enzymes from the
liver of a Gastropoda mollusk Littorina kurila, an easily
accessible source of hydrolytic enzymes. The goal of
this study is the characterization of properties and spec-
ificity of the aryl sulfatase from L. kurila.
Some authors believe that the existing nomenclature
of sulfatases does not correspond to their specificity
[
15]. For example, none of shellfish and mammalian
RESULTS AND DISCUSSION
steroid sulfatases could be obtained thus far in the state
We previously identified two forms of aryl sulfatase
in the liver of L. kurila that differ in their molecular
masses [18]. In this study, we isolated aryl sulfatase by
1
Corresponding author; phone: +7 (4232) 31-0705; fax: +7 (4232)
1-4050; e-mail: mik@piboc.dvo.ru
3
6
3

6
4
KUSAYKIN et al.
A420
the enzyme specificity (50 µg/ml) can inhibit activity of
the sulfatase.
1
1
1
0
0
0
0
.4
.2
.0
.8
.6
.4
.2
0
The effect of sulfated polyhydroxysteroids on the
activity of sulfatase from L. kurila was tested using
p-nitrophenyl sulfate as a substrate. The polyhydroxy-
steroids studied can be tentatively divided into three
structural groups: (A) sulfated polyhydroxysteroids
from brittle stars (family Ophiuroidea), (B) sulfated
aglycons of asterosaponins, and (C) sulfated glycosides
of starfish [not glycosylated compound (4, C)]
(
Table 1). Compounds 1–5 of group A exert the stron-
–5
gest inhibitory effect on sulfatase (I ~ 10 M). Com-
50
pounds 4 and 5 of group B had the least inhibitory
–
3
activity (I ~ 10 M), and substances of group C exhib-
50
–
4
ited the intermediate values of I (~10 M).
5
0
Interestingly, the introduction of a hydroxy group
into the hydrophobic side chain of compound (5, A)
considerably increased the inhibitory activity compared
with compound (6, A). Altered positions of substituents
and an introduction of a double bond into the steroid
nucleus had no effect on the inhibitory capacity of com-
pounds (6, A) and (7, A). An analysis of the data on the
inhibitory effect of compounds (1, C) and (3, C) sug-
gests that the sulfatase has affinity for the xylose that
has a sulfate at C4, since the inhibitory activity of com-
pound (1, C) is two times higher than that of compound
3
00
350
400
450
Volume, ml
Gel filtration of arylsulfatase on a Sepharose CL 6B column
2.5 × 90 cm) under elution with 0.025 M acetate buffer,
pH 5.4; activity monitoring.
(
a scheme that altered from that described in [18] in that
it did involve the stage of hydrophobic chromatogra-
phy. This modification allowed us to substantially
reduce the isolation time and to obtain the most active
form of the enzyme by gel filtration on Sepharose CL
(
3, C) that contains a residue of glucose sulfated at C6
in place of xylose residue (Table 1). The desulfatation
of holostane triterpene glycosides by the L. kurila sul-
fatase was detected by TLC. The compounds affected
by sulfatase had tetra- and pentasaccharide carbohy-
drate chains in which xylose attached to the triterpene
aglycon contained a sulfate group at C4 [22].
6
B (figure). The resulting sulfatase was homogeneous
by SDS-PAGE and effectively catalyzed the cleavage of
p-nitrophenyl sulfate.
The properties of enzyme were examined at pH 5.4.
The enzyme activity at 50°ë was found to retain com-
pletely over a period of 20 min at 50°ë, whereas at
Triterpene glycosides also could inhibit the sulfa-
–
4
6
0°C, the enzyme lost 50% of its activity for the same tase with I values of ~10 M (Table 2). Therefore, the
50
time. The molecular mass of sulfatase, as indicated by enzymatic desulfatation of these compounds was car-
PAGE, was 45 kDa, and the K value for p-nitrophenyl ried out in solutions where their concentration was less
m
sulfate was 8.7 ± 1 mM.
than I (<80 µg/ml). Under these conditions, sulfatase
50
desulfated the 4-sulfate group in the xylose residues
within the triterpene glycosides of sea cucumbers. The
reaction led to the formation of desulfated derivatives
of frondoside A, pseudostichoposide A, and cucumari-
The specificity of sulfatase from L. kurila was exam-
ined using sulfated polyhydroxysteroids of different
structure as substrates (Table 1). After incubation with
the enzyme, the chromatographic mobility of sulfated
polyhydroxy steroids remained unchanged, indicating
that no desulfatation of natural substances took place.
oside G in about 50% yields. The products were iso-
1
lated by chromatography on a hydrophobic sorbent
Polychrom-1 and silica gel. The desulfatation was
monitored by TLC, and structures of the products were
It has earlier been shown that sulfated polyhydroxy-
steroids are the inhibitors of 1
3-β-D-glycanases
1
3
13
determined by C NMR spectra. It is known that, in C
NMR spectra, the α- and β-effects of sulfate groups
makes the carbon atom of the xylose residue to which
the sulfate group is attached to resonate at a weaker
field relative to the nonsulfated carbon atoms of this
residue, whereas the signals of adjacent carbon atoms
are shifted by 2–3 ppm toward a stronger field.
from marine mollusks [19, 20]. It was found with the
use of natural sulfated polyhydroxysteroids and their
synthetic derivatives that the inhibitory effect of these
steroids is largely determined by their biophilicity,
which is created by polar substituents in the polycyclic
nucleus and the presence of hydrophobic side chain.
Spectral investigations showed that chalistanol sulfate
–6
13
(
a natural sulfated polyoxysteroid, I 10 M) induces
A comparison of C NMR spectra of native and sul-
50
conformational changes in the 1
3-β-D-glycanase fatase-treated triterpene glycosides showed the splitting
molecule [21]. It was presumed that sulfated polyhy- off of the sulfate group, which in native compounds is
droxysteroids at the concentration used in the study of attached to C4 of the xylose residue (Table 3).
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Table 2. Inhibitory effect of holostane glycosides on sulfatase
Compound
I , M
50
O
OH
O
O
CH OH
2
NaO SO
3
O
O
OMe
O
CH
O
3 O
O
OAc
O
HO
HO
OH
× 10–4
4
OH
OH
O
O
OH
HO
OH
Frondoside A
O
OH
O
O
CH OH
2
NaO SO
3
O
O
O
CH3 O
O
OAc
× 10–3
1
OMe
O
OH
O
HO
OH
HO
OH
OH
Cucumarioside G1
O
O
OH
O
O
CH OH
2
NaO SO
3
O
O
O
OMe
CH
1 × 10–3
O
3 O
O
O
HO
OH
HO
OH
OH
OH
Pseudostichoposide A
The sulfatase from L. kurila did not affect the native
Thus, the sulfatase from L. kurila can be considered
fucoidan. Fucoidan from Fucus evanescens and its sul- as aryl sulfatase that does not affect fucoidan but cata-
fatase-treated sample on a DEAE Toyopearl column lyzes the splitting off of the sulfate group at position C4
showed similar elution profiles at chromatography. The of the xylose residue incorporated into the carbohy-
1
3
identity of the C NMR spectra and similar electro- drate chains of molecules of holostane triterpene glyco-
phoretic mobilities of the samples confirmed this con- sides from sea cucumbers.
clusion.
The biological activity of some compounds is
Some characteristics of enzymes similar to sulfatase known to correlate with the content of sulfates. For
from L. kurila have been reported. For example, a sul- example, it was found that the desulfated derivatives of
fatase that hydrolyzes p-nitrophenyl sulfate but not triterpene glycosides from sea cucumbers possess
fucoidan was found in the liver of the marine mollusk stronger antifungal, antitumor, and hemolytic activities
Haliotis sp. [5]. On the other hand, a partially purified than their sulfated analogues [25]. On the other hand,
preparation from the mollusk Patinopecten maximus the anticoagulating activity of fucoidans from F. vesi-
effectively hydrolyzed synthetic substrates p-nitro- culosus and Pelvetia canaliculata increases with the
catechol sulfate and sulfated L-fucose and partially growing content of sulfates [26]. The regioselective sul-
desulfated natural fucoidan [24].
fatation of carbohydrate molecules can be accom-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 1 2006

ARYL SULFATASE OF UNUSUAL SPECIFICITY
69
plished chemically, using specifically protected sugars, Table 3. Chemical shifts (ppm) of carbon atoms (C3, C4,
and C5) of the xylose residue incorporated in natural sulfated
triterpene glycosides and their desulfated derivatives
and enzymatically. In the enzymatic approach, two
types of enzymes are used: sulfotransferases for which
nonsulfated sugars serve as acceptors, and glycosyl-
transferases that use sulfated sugar derivatives as
Compound
C3
C4
C5
acceptors. An original approach to obtaining sulfated Frondoside A
sugar derivatives was recently proposed, which com-
75.8
77.7
75.8
78.1
76.2
78.0
76.1
70.3
75.3
70.7
75.3
70.7
64.4
66.9
64.0
66.7
64.0
66.5
Frondoside A-DS*
bines the chemical and enzymatic methods. Sulfatation
is accomplished chemically, and a selective desulfata-
tion, by means of sulfatases [27]. The sulfatase from
Cucumarioside G1
Cucumarioside G -DS*
L. kurila is promising for the directed desulfatation of Pseudostichoposide A1
1
natural glycosides aimed at design of new biologically
active compounds.
Pseudostichoposide A -DS*
1
*
DS, desulfated derivative.
EXPERIMENTAL
of the substrate (1 mg/ml) was incubated for 30 min at
7°ë, then 100 µl of 1 M Na CO solution was added,
and the absorption of the solution at 420 nm was mea-
sured.
A unit of enzyme activity was defined as the amount
of enzyme that catalyzed the conversion of 1 µmol of
the substrate per min.
Reagents. Inorganic salts, acids, and Polychrom
were commercial preparations from Reakhim (Russia);
carriers for chromatography (DEAE cellulose,
Sepharose CL 6B) and BSA were from Sigma (United
States).
3
2
3
Substrates and inhibitors. p-Nitrophenyl sulfate
was from Sigma (United States). Triterpene glycosides
from sea cucumbers: frondoside A, pseudostichoposide
Determination of thermostability. Aliquots of a
solution of the enzyme in buffer, pH 5.4 (50 µl,
A, cucumarioside G , and sulfated polyoxysteroids
1
−
2
1
(
0 units) were kept for 20 min in a water thermostat
were kindly provided by the Laboratory of the Chemis-
try of Sea Natural Compounds (Pacific Institute of
Bioorganic Chemistry, Far East Division, Russian
Academy of Sciences).
Fucoidan was isolated from a brown alga F. evane-
scens by the procedure described in [28] and addition-
ally purified to remove alginic acid as follows. Glacial
acetic acid (150 ml) was added to 300 ml of a fucoidan
solution (50 mg/ml), and the precipitate was immedi-
ately separated by centrifugation (9000 g, 10 min). The
supernatant was adjusted to pH 7.0 with NaOH. The
resulting salt was removed by ultrafiltration on an
NMWL 1000 membrane (Millipore, United States) by tion mixture containing 50 µl of the enzyme (10 units)
successive dilution. The resulting solution was applied in a buffer, pH 5.4, and 50 µl of a p-nitrophenyl sulfate
onto a column of DEAE cellulose (20 × 30 cm) equili- solution (concentration 0.1–3 mg/ml) was incubated
brated with 0.01 N HCl. Elution with a stepwise gradi- for 20 min at 37°ë, and the activity was determined.
ent of NaCl (0.35, 0.5, 0.75, 1, 1.5, 2, and 3 M) using The Michaelis constant (K ) was calculated using the
l of each solution led to fucoidan, whose yield was
registered by the phenol sulfate method [29]. Sugar-
containing fractions were dialyzed, concentrated by
ultrafiltration on an NMWL 1000 membrane, and lyo-
philized. The fucoidan fraction that was eluted with a
M NaCl solution was used.
Purification of sulfatase. Sulfatase was purified by
the scheme described in [18], except that the stage of
hydrophobic chromatography was omitted.
Protein was determined by the Lowry method [30] minated by adding 100 µl of Na CO , and the residual
with BSA as a standard.
U-2, Germany) at a temperature from 20 to 70°ë at an
interval of 5°ë. After cooling, 100 µl of a p-nitrophenyl
sulfate solution (1 mg/ml) was added, and the residual
activity was determined as described above. The stabil-
ity of the enzyme that was kept at 60°ë (300 µl of solu-
tion) was checked by taking 50-µl aliquots every
1
0 min for 1 h.
Molecular mass was determined by 10% PAG–
0
[
.1% SDS electrophoresis by the method of Laemmli
31].
Determination of the Michaelis constant. A reac-
–2
m
3
Lineweaver–Burk transform.
Determination of the inhibitory activity. The
reaction mixture contained 50 µl of the solution of the
–2
enzyme (10 units) in buffer, pH 5.4, and 50 µl of an
aqueous solution of inhibitors of different concentra-
tions (1.5, 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 mg/ml). The
mixture was kept for 10 min, 50 µl of a p-nitrophenyl
sulfate solution (1 mg/ml) was added, and the mixture
was incubated for 20 min at 37°ë. The reaction was ter-
2
2
3
activity of the enzyme was determined. I was taken to
50
Sulfatase activity was registered by the appearance be the concentration of the substance at which a 50%
of p-nitrophenol in the reaction of sulfatase with inhibition of the enzyme activity occurs.
p-nitrophenyl sulfate. A reaction mixture containing
Preparation of products of enzymatic transfor-
–
2
5
0 µl (10 units) of the enzyme solution in 0.05 M suc- mation of triterpene glycosides. Glycosides were
cinate buffer, pH 5.4, and 50 µl of an aqueous solution added to a solution of sulfatase (20 ml, 2 units) in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 1 2006

7
0
KUSAYKIN et al.
buffer, pH 5.4, so that their final concentrations were no 11. Schirmer, A. and Kolter, R., Chem. Biol., 1998, vol. 5,
pp. 181–186.
higher than I , and the mixture was incubated for 72 h.
50
The reaction was quenched by boiling. The reaction 12. Selmer, T., Hallman, A., Schmidt, B., Sumper, M., and
von Figura, K., Eur. J. Biochem., 1996, vol. 238,
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evaporated to dryness. The dry residue was chromato-
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Harrop, S.J., Hopwood, J.J.., and Guss, J.M., Structure,
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by TLC.
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ing frequency of 125.77 MHz.
1
997, vol. 5, pp. 277–289.
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ACKNOWLEDGMENTS
1
8. Kusaikin, M.I., Burtseva, Yu.V., Svetasheva, T.G.,
Sova, V.V., and Zvyagintseva, T.N., Biokhimiya (Mos-
cow), 2003, vol. 68, pp. 384–392.
We thank Dr. Sci. A.A. Kura and Ph.D. E.V. Levina
Pacific Institute of Bioorganic Chemistry, Far East
(
1
2
9. Zvyagintseva, T.N., Makar’eva, T.N., Stonik, V.A., and
Division, RussianAcademy of Sciences) for samples of
polyhydroxysteroids and fruitful discussion.
This work was supported by the Russian Foundation
for Basic Research, project no. 03-04-49534; FKh8
RAS; and a grant to support Leading Scientific
Schools.
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