The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate,to a lesser extent on heparan sulfate and heparin.. The biochemical properties o
Trang 1Purification and characterization of novel salt-active acharan sulfate
Sung-Woon Hong1, Ho-Young Shin1, Yeong Shik Kim2and Dong-Hyun Kim1
1
College of Pharmacy, Kyung Hee University, Seoul, Korea;2Natural Products Research Institute, Seoul National University, Seoul, Korea
Salt-active acharan sulfate lyase (no EC number) has been
purified from Bacteroides stercoris HJ-15,which was
iso-lated from human intestinal bacteria with GAG degrading
enzymes The enzyme was purified to apparent homogeneity
by a combination of QAE-cellulose,diethylaminoethyl
(DEAE)-cellulose,CM-Sephadex C-50,HA ultrogel and
phosphocellulose column chromatography with the final
specific activity of 81.33 lmolÆmin)1Æmg)1 The purified
salt-active acharan sulfate lyase was activated to 5.3-fold by salts
(KCl and NaCl) The molecular weight of salt-active
acha-ran sulfate lyase was 94 kDa by SDS/PAGE and gel
filtra-tion The salt-active acharan sulfate lyase showed optimal
activity at pH 7.2 and 40C Salt-active acharan sulfate
lyase activity was potently inhibited by Cu2+,Ni2+ and
Zn2+ This enzyme was inhibited by some agents,butanediol and p-chloromercuric sulfonic acid,which modify arginine and cysteine residues The purified Bacteroidal salt-active acharan sulfate lyase acted to the greatest extent on acharan sulfate,to a lesser extent on heparan sulfate and heparin The biochemical properties of the purified salt-active acharan sulfate lyase are different from those of the previously puri-fied heparin lyases However,these findings suggest that the purified salt-active acharan sulfate lyase may belong to heparin lyase II
Keywords: Bacteroides stercoris HJ-15; salt-active acharan sulfate lyase; acharan sulfate lyase; acharan sulfate; heparin
Heparin,heparan sulfate and acharan sulfate
glycosamino-glycans (GAGs) are comprised of alternating 1–4-linked
glucosamine and uronic acid residues Heparan sulfate is
composed primarily of monosulfated disaccharides of
N-acetyl-D-glucosamine andD-glucuronic acid while
hep-arin is composed mainly of trisulfated disaccharides of
N-sulfonyl-D-glucosamine andL-iduronic acid [1,2]
Acha-ran sulfate,isolated from the giant African snail Achatina
fulica,has a structure closely related to heparin and heparan
sulfate,with a uniform repeating disaccharide structure
of fi4)-a-D-GlcNAc(1fi4)-a-L-IdoA2S (1fi[3] Acharan
sulfate exclusively contains N-acetyl-D-glucosamine instead
of N-sulfonyl-D-glucosamine in GAGs
Related to the degradation of these GAGs,some heparin
lyases that can eliminatively cleave polysaccharides (heparin
or heparan sulfate GAGs) have been reported [4–6] These
enzymes are classified as: (a) heparin lyase I (heparinase I,
EC 4.2.2.7),acting primarily at the fi 4)-a-D-GlcNS(6S or
OH)(1fi4)-a-L-IdoA2S(1fi linkages present in heparin; (b)
heparin lyase II (heparinase II or heparitinase II),acting at
thefi 4)-a-D-GlcNS(6S or OH)(1fi 4)-a-L-IdoA(2S or OH)
or -b-D-GlcA(1fi linkages present in both heparin and heparan sulfate; and (c) heparin lyase III (heparinase III or heparitinase,EC 4.2.2.8),acting on the fi 4)-a-D-GlcNS(or Ac) (1fi 4)-b-D-GlcA (or IdoA) (1fi linkages found exclu-sively in heparan sulfate The heparin lyases have become increasingly important in understanding the biological roles and structure of the glycoaminoglycans (and proteoglycan), which are involved in the well known anticoagulant activity [7] and the regulation of various cellular processes such as the potentiation of angiogenesis [8] and the modulation of cellular proliferation [9] Several heparin lyases of bacterial origin have been purified and characterized from various species including Flavobacterium heparinum [4,10], Bacillus
sp BH 100 [11], Prevotella heparinolyticus [12],and Bacter-oides stercorisHJ-15 [13,14]
Bacteroides stercoris HJ-15 has been recently isolated from human intestine and it contains several kinds of GAG degrading enzymes including heparin,heparan sulfate, acharan sulfate and chondroitin sulfate [13–16] We purified two kinds of novel heparin lyases,heparin lyase II-1 (acharan sulfate lyase 1),and heparin lyase II-2 (acharan sulfate lyase 2) and III,from this B stercoris HJ-15 [15,16] The Bacteroidal heparin lyase III cleaved heparin as well as heparan sulfate,but did not cleave acharan sulfate The Bacteroidal acharan sulfate lyase,which potently cleaved acharan sulfate as well as heparin,are highly specific to acharan sulfate compared to the previously reported heparin lyases [16] The purified acharan sulfate lyases 1 and 2 (no EC number) were not activated by salts such as KCl However,when Bacteroidal acharan sulfate lyase-active fraction isolated from B stercoris HJ-15 was incubated with salts (KCl),the enzyme fraction was
Correspondence to D.-H Kim,College of Pharmacy,Kyung Hee
University,1 Hoegi-dong,Dongdaemun-ku,Seoul 130–701,
South Korea Fax: + 82 2 957 5030,Tel.: + 82 2 961 0374,
E-mail: dhkim@khu.ac.kr
Abbreviations: CM,carboxymethyl; DEAE,diethylaminoethyl;
DUA,4-deoxy-a- L -threo-hex-4-enopyranosyl uronic acid;
GAG,glycosaminoglycan; GlcA,glucuronic acid; GlcN,glucosamine;
HA,hydroxyapatite; IdoA,iduronic acid; IEF,isoelectric focusing;
QAE,quaternary amino ethyl; PCMS,p-chloromercurisulfonic acid.
(Received 3 April 2003,revised 15 May 2003,
accepted 30 May 2003)
Trang 2activated by salts Therefore,we tried to purify salt-active
acharan sulfate lyase from B sterocoris HJ-15 that acts
predominantly on acharan sulfate
Materials and methods
Materials
Heparin (porcine intestinal mucosa),heparan sulfate
(por-cine intestinal mucosa),chondroitin sulfate A (bovine
trachea),chondroitin sufate B (porcine intestinal mucosa),
chondroitin sufate C (shark cartilage),thioglycolic acid
(sodium salt),QAE cellulose Fastflow,and HA Ultrogel
(microcrystalline hydroxyapatite,4% beaded in agarose)
were supplied by Sigma Chemical Co Sodium dodecyl
sulfate,CM-Sephadex C-50,phosphocellulose,Sephacryl
S-300 HR resins and molecular weight markers for gel
filtration and protein electrophoresis were obtained from
Pharmacia Biotech Co (Uppsala,Sweden)
Diethylamino-ethyl (DEAE)-cellulose resin was purchased from Wako
Pure Chemical Industries (Tokyo,Japan) Protein assay kit
and Coomassie Brilliant Blue R-250 were from Bio-Rad
(Hercules,CA,USA) Tryptic soy broth was provided by
Difco Co Acharan sulfate was prepared as described by
Kim et al [3] All other chemicals were of the highest grade
available
Bacterial strains and purification of salt-active acharan
sulfate lyase
B stercorisHJ-15 was isolated and cultivated as described
previously [14,16] It was cultured anaerobically under an
atmosphere of 90% (v/v) nitrogen and 10% (v/v) carbon
dioxide at 37C in 100 L of tryptic soy broth (pH 7.2)
containing heparin (0.15 gÆL)1) instead of glucose,0.01%
(w/v) sodium thioglycolate and 0.1% (w/v) ascorbic acid
The cultured cells were harvested in the late exponential
phase (11–12 h) by centrifugation at 4000 g
4C and the resulting cell pellet was washed twice with
saline containing 50 mMsodium phosphate (pH 7.0) The
cell pellet was suspended in 600 mL of Buffer A (50 mM
sodium phosphate buffer,pH 7.0) Cell suspension (30 mL
at a time) was placed into a 50-mL centrifuge tube and
disrupted by 30-min periods of sonication at 1-s intervals on
an ultrasonic processor (Eyela Co.) at an 80% output with
cooling Cell debris was removed by centrifugation at
25 000 g
2 for 60 min at 4C All operations were carried
out at 4C unless otherwise noted The cell extract
(600 mL) was passed through a QAE cellulose column
(5· 40 cm) which had been pre-equilibrated with Buffer A
The column was washed with the same buffer until no
acharan sulfate lyase activity was detectable in the effluent
The fractions which passed through the column were
applied to a DEAE-cellulose column (5· 30 cm)
equili-brated with Buffer A The column was then eluted with the
same buffer until any ASL activity could not be detected
The noninteracting fluid passed through the column was
collected The total volume of the flow through was
1800 mL The eluate was loaded onto a CM-Sephadex
C-50 column (3· 30 cm) previously equilibrated with Buffer
A The column was washed with 1000 mL (1 L) of the same
buffer and then eluted with a 2-L linear gradient of KCl
from 0 to 0.6Min Buffer A at a flow rate of 105 mLÆh)1 All fractions obtained were assayed for heparin lyase and acharan sulfate lyase activities Four fractions (Fr-s,Fr-a, Fr-b and Fr-c) containing the activity of these enzymes were collected separately and assayed for the activities degrading acharan sulfate and heparan sulfate Fr-s had acharan sulfate lyase activity,which was activated by salts,was dialyzed against Buffer A for the further purification The dialyzed enzyme preparation (210 mL) was applied
to a HA Ultrogel column (2.5· 9 cm) equilibrated with Buffer A Being washed with 500 mL of the same buffer, the column was eluted with a 800-mL linear gradient,from
50 mM sodium phosphate buffer (pH 7.0) to 400 mM sodium phosphate buffer (pH 7.0) at a flow rate of
120 mLÆh)1 The active fractions were pooled and dialyzed twice against 2 L of Buffer A The dialyzed enzyme (78 mL) was loaded onto a phosphocellulose column (3· 25 cm) equilibrated with Buffer A After washing the nonabsorbed proteins with 300 mL of the same buffer,the column was eluted with an 800-mL linear gradient,from
50 mM sodium phosphate buffer (pH 7.0) to 400 mM sodium phosphate buffer (pH 7.0) at a flow rate of
100 mLÆh)1 The active fractions (salt-active acharan sulfate lyase,fraction numbers 17–25) were investigated for homogeneity by SDS/PAGE
Enzyme activity assay The activity of acharan sulfate lyase was measured accord-ing to the previously published procedure [17] The activity was calculated from the change of absorbance per minute using an extinction coefficient of 3800M )1 for products (1 U¼ 1 micromole of DUA containing product formed per minute) [17] The specific activity was calculated by dividing the micromoles of product produced per minute by the milligrams of protein in the cuvette Protein concentra-tion was measured by a Bradford assay using bovine serum albumin as a standard [18]
Characterization of salt-active acharan sulfate lyase SDS/PAGE was performed for the determination of molecular mass according to Laemmli’s procedure [19] The gel was stained with Coomassie Brilliant Blue R-250 solution and further stained with silver The pI value of heparin lyase was determined by IEF electrophoresis using Model 111 Mini IEF Cell (from Bio-Rad) according to the manufacturer’s instructions The molecular weight of the native enzyme was estimated by gel filtration using Seph-acryl S-300 HR column (1.6· 70 cm) calibrated with gel filtration low molecular weight calibration kit (from Sigma Co.) and high molecular calibration kit (from Amersham Pharmacia Biotech) The pH optimum of acharan sulfate lyase was determined using 50 mMsodium phosphate buffer (pH 6.0–8.5) Temperature dependency of the enzyme was investigated by measuring enzyme activity at different temperatures (25–60C) To investigate the effect of divalent metal ions and KCl (or NaCl) on the lyase activity, divalent metal ion (final concentration,100 lM),chemical modifying agents (50 lM) and KCl (0–500 mM) were added into the reaction mixture Kinetic constant of acharan sulfate lyase was determined by measuring the initial rates at
Trang 3various substrate concentrations (200,400,600,1000,2000,
3000 lg) under the standard reaction conditions
These lyase activities on other sulfated polysaccharides
were also measured One milligram of each substrate was
added to the reaction mixture Because of their low
solubility,100 lg of acharan sulfate were used in this assay
The internal amino acid sequence of purified salt-active
acharan sulfate lyase was analyzed by an Applied Biosystem
protein sequencer model 492 in Korea Basic Science
Institute
Results
Purification of salt-active acharan sulfate lyase
Bacteroides stercoris HJ-15,which degrades a variety of
GAGs including heparin,heparan sulfate,acharan sulfate
and chondroitin sulfates [13],constitutively produced
hep-arin lyase and acharan sulfate lyase activities However,
when induced with acharan sulfate or heparin,total acharan
sulfate activity increased by about 3.5-fold (data not
shown) Furthermore,total acharan sulfate lyase activity
was activated 5.7-fold by salts,KCl and NaCl However,the previously purified enzymes,acharan sulfate lyases and heparinase III,from B sterocoris were not activated by salts Therefore,to purify salt-active heparin lyase,B stercoris HJ-15 cells were disrupted by ultrasonic,and the super-natant,the crude extract,was subjected to a combination
of QAE-cellulose and DEAE-cellulose column chromato-graphy to remove interacting proteins Acharan sulfate lyase activity passed through these columns without binding
to the matrices The effluent was applied to CM-Sephadex C-50 column chromatography (Fig 1) The salt-active acharan sulfate lyase activity fraction was then further purified to homogeneity by a series of hydroxyapatite Ultrogel chromatography and finally phosphocelluose col-umn chromatography (Fig 2) The specific activity and total activity at each purification step are summarized in Table 1
The specific activity of the purified acharan sulfate lyase activity had 80.33 UÆmg)1protein with a yield of 7.4% The purified acharan sulfate lyase was apparently homogeneous
by SDS/PAGE and its molecular mass was identically estimated to be 94 000 Da (Fig 2)
Characterization of salt-active acharan sulfate lyase The molecular weight of salt-active acharan sulfate lyase under nondenaturing conditions was determined by gel filtration (data not shown) Acharan sulfate lyase was estimated to be about 94 000 Da It suggests that acharan sulfate lyase is composed of one subunit The optimal pH
of acharan sulfate lyase was determined to be 7.2–7.3 for acharan sulfate,heparin and heparan sulfate (Fig 3),and the optimum temperature for the maximal activity was shown at 40C (Fig 4)
Fig 1 Elution profile of CM-Sephadex C-50 ion exchange (A),
hydroxyapatite ultrogel (B)and phosphocellulose (C)column
chromato-graphies s,acharan sulfate lyase activity without KCl; m,acharan
sulfate lyase activity with 50 m M KCl;
8 n,heparin lyase activity; simple
line,absorbance at 280 nm.
Fig 2 SDS/PAGE of the purified salt-active acharan sulfate lyase at various steps of purification Lanes 1 and 8,marker; lane 2,preparation after crude extract; lane 3,preparation after QAE column tography; lane 4,preparation after DEAE-cellulose column chroma-tography; lane 5,preparation after CM-Sephadex C-25 column chromatography; lane 6,preparation after hydroxyapatite ultragel column chromatography; and lane 7,purified salt-active heparin lyase
II after phosphocellulose column chromatography.
Trang 4The purified acharan sulfate lyase was activated 5.3-fold
by the strength of salts,such as KCl and NaCl (Fig 5) However,divalent cations CaCl2,and MgCl2,did not activate this enzyme compared to KCl and NaCl The salt-active acharan sulfate lyase activity was slightly increased by addition of Mn2+,whereas they were severely inhibited
by Cu2+,Ni2+and Zn2+(Table 2) The purified enzyme was inhibited by PCMS
PCMS potently inhibited salt-active acharan sulfate lyase
but little inhibited by the other agents that modify histidine and cysteine residues (Table 3)
Amino acid composition analysis revealed that the salt-active acharan sulfate lyase contains a large proportion of lysine (data not shown) The pI value of the purified salt-active acharan sulfate lyase was 8.5,but slightly lower than those of the previously purified Flavobacterial heparin lyases range from 8.9 to 10.1 We analyzed the internal sequences of a peptide obtained by digestion of each enzyme with trypsin (Table 4) The internal sequence of the salt-active acharan sulfate lyase showed homology of 50% to Flavobacterial heparin lyases I and II previously reported, but did not showed homology to Flavobacterial heparin lyase III [10,20]
Fig 3 Effect of pH on the activity of salt-active acharan sulfate lyase.
The enzyme activity was assayed in 50 m M sodium phosphate buffer at
the indicated pH d,activity for acharan sulfate; j,activity for
hep-aran sulfate; m,activity for heparin.
Fig 4 Effect of temperature on the activity of salt-active acharan
sul-fate lyase The enzyme activity was assayed in 50 m M sodium
phos-phate.
Fig 5 Effect of KCl on the activity of salt-active acharan sulfate lyase d,salt-active acharan sulfate lyase II purified from Fr-s in Fig 1; m, heparin lyase II-1 (acharan sulfate lyase 1) purified from Fr-a in Fig 1 [16]; j,heparin lyase II-2 (acharan sulfate lyase 2) purified from Fr-a
in Fig 1 [16].
Table 1 Purification summary of salt-active acharan sulfate lyase One
unit (U) is the activity forming 1 lmol disaccharides from acharan
sulfate per minute Numbers in parentheses indicate the activities for
heparin used as a substrate.
Stage
Total activity (U)
Total protein (mg)
Specific activity (UÆmg)1) Crude extract 165.4 5250.12 0.03
QAE cellulose column
chromatography
65.2 1407.18 0.05 DEAE-cellulose column
chromatography
90.3 747.04 0.12
CM Sephadex C-50 column
chromatography
37.2 13.67 2.72 Hydroxyapatite ultrogel
column chromatography
34.5 1.82 18.96 Phosphocellulose column
chromatography
12.2 (1.3) 0.15 81.33 (8.67)
Table 2 Effect of divalent metal ions on salt-active acharan sulfate lyase activity.
Metal iona Residual activityb(%)
a Final concentration,1 m M b 0.03 U of homogenously purified enzyme activity was taken as 100%.
5
Trang 5Substrate specificity of purified salt-active acharan
sulfate lyase
The purified salt-active acharan sulfate lyase degraded
heparin and heparan sulfate as well as acharan sulfate
(Table 5) The salt-active acharan sulfate lyase was the most
active to acharan sulfate When the salt-active acharan
sulfate lyase activity for heparan was taken as 100%,the
enzyme activities for acharan sulfate and heparan sulfate
were 952.3 and 149.5%,respectively However,all types of
chondroitin sulfates were not also substrates for the enzyme
Kinetic constants of purified salt-active acharan
sulfate lyase
Michaelis–Menten constants were determined under the
optimum reaction conditions in experiments designed to
calculate reaction velocities at each substrate concentration (Table 6) Kmand Vmaxof acharan sulfate on salt-active acharan sulfate lyase were estimated at 65.4 lgÆmL)1and 131.2 lmolÆmin)1Æmg)1,respectively As for heparin on salt-active acharan sulfate lyase, Kmand Vmaxvalues of heparin and heparan sulfate were calculated at 18.5 lgÆmL)1, 8.7 lmolÆmin)1Æmg)1 and 40.7 lgÆmL)1,13.1 lmolÆ min)1Æmg)1,respectively
Discussion
In the present report,we have purified salt-active acharan sulfate lyase specifically acting on acharan sulfate from Fr-s fraction of CM-Sephadex C-50 chromatography,which efficiently resolved GAGs degrading lyases of B stercoris HJ-15 As Fr-b and Fr-c fractions showed a higher specificity to heparan sulfate and heparin,they were considered to be similar to heparin lyase III and heparin
Table 3 Effect of divalent metal ions on salt-active acharan sulfate lyase
activity Homogenously purified enzyme activity (0.03 U) was taken as
100%.
Chemical modifying agent IC 50 (m M )
N-Tosyl- L -phenylalanine chloromethyl ketone >1
Phenylmethylsulfonyl fluoride >1
Sodium-p-tosyl- L -lysine chloromethyl ketone >1
p-Chloromercuric sulfonic acid 0.02
Table 4 Internal amino acid sequence of salt-active acharan sulfate lyase from Bacteroides stercoris HJ-15.
Enzyme
Internal amino acid sequence
Homology (%) Salt-active acharan sulfate lyase …GTIQYG…
Flavobacterial heparin lyase Ia 213 50
…GKITYV…
Flavobacterial heparin lyase II a 157 50
…GAIVYD…
Flavobacterial heparin lyase IIIa 567 33
…LMIQSL…
a Data from [10,20].
Table 5 Substrate specificity of acharan sulfate lyase and the previously reported heparin lyases Activity on heparin (or heparan sulfate in Flavobacterial heparin lyase III) as the substrate was set at 100%.
Substrate
Activity (%)
Salt-active Hep II Hep II-1 (ASL1)b HepII-2b(ASL2) Hep IIIb Hep I Hep II Hep III
Heparan sulfate (porcine) 149.5 128 121.7 610 30 172 100
a Data from [1,4,5,13,21] b Data from [15,16] c Unpublished data.
Table 6.
7 K m and V max values of salt-active acharan sulfate lyase.
Enzyme
K m (lgÆmL)1) V max (UÆmg protein)1) Acharan sulfate Heparin Heparin sulfate Acharan sulfate Heparin Heparin sulfate Salt-active acharan sulfate lyase 65.4 18.5 40.7 131.2 8.7 13.1
Acharan sulfate lyase 1a 28.1 8.8 7.5 65.0 11.6 14.3
Acharan sulfate lyase 2 a 42.2 20.6 16.4 107.6 22.9 31.3
a
Data from [16].
Trang 6lyase I prepared from F heparinum,respectively [15,16].
Two acharan sulfate lyases previously purified from Fr-a
fraction showed the different substrate specificity compared
to those of Fr-b,Fr-c and the previously reported heparin
lyases [16] These enzymes were highly specific to acharan
sulfate compared to heparin and heparan sulfate The
salt-active acharan sulfate lyase purified from Fr-s fraction
showed a different substrate compared heparin lyase I and
III,but exhibited a similar substrate specificity of two
acharan sulfate lyase previously purified from Fr-a
Acha-ran sulfate was the best substrate for the purified present
enzyme Particularly,the present enzyme was significantly
activated by salts,KCl and NaCl,although two acharan
sulfate lyases were not activated by salts Several attempts at
N-terminal analysis failed to yield sequence information
suggesting the N-terminus to be blocked Therefore,we
analyzed the internal sequences of a peptide obtained by
digestion with trypsin The internal sequence of the peptide
showed poor homology to Flavobacterial heparin lyases I
and II Although the substrate specificities of heparin lyase I
and III are well understood,the structural requirement for
the cleavage of heparin and heparan sulfate by heparin lyase
II is not well characterized This could be due to a structural
complexity of heparin and heparan sulfate and a lack of
homogeneous polysaccharide A recently characterized
GAG,acharan sulfate,from African giant snail Achatina
fulicacan provide the criteria to classify the heparin lyase II
among the specificity of heparin lyases
In conclusion,the substrate specificity as well as the
characterization of salt-active acharan sulfate lyase are
different from those of the previous reported heparin lyases
(heparin lyase I and III prepared from F heparinum),but is
similar to those of Flavobacterial heparin lyase II
There-fore,we suggest that the salt-active acharan sulfate lyase
may belong to heparin lyase II
Acknowledgements
This work was supported by KOSEF grant 1999-2-209-010-5.
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