Kinetic studies on endo-b-galactosidase by a novel colorimetric assayb-glycosides using its transglycosylation activity Takeomi Murata, Takeshi Hattori, Satoshi Amarume, Akiko Koichi and
Trang 1Kinetic studies on endo-b-galactosidase by a novel colorimetric assay
b-glycosides using its transglycosylation activity
Takeomi Murata, Takeshi Hattori, Satoshi Amarume, Akiko Koichi and Taichi Usui
Department of Applied Biological Chemistry, Shizuoka University, Japan
Novel chromogenic substrates for endo-b-galactosidase
were designed on the basis of the structural features of
keratan sulfate
Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (2),which consists of two repeating units of
N-acetyl-lactosamine,was synthesized enzymatically by consecutive
additions of GlcNAc and Gal residues to p-nitrophenyl
b-N-acetyllactosaminide In a similar
manner,Glc-NAcb1-3Galb1-4GlcNAcb-pNP
(1),GlcNAcb1-3Galb1-4Glcb-pNP (3),Galb1-4GlcNAcb1-3Galb1-(1),GlcNAcb1-3Galb1-4Glcb-pNP (4),
3GlcNAcb1-34Glcb-pNP (5),and
Galb1-6GlcNAcb1-3Galb1-4Glcb-pNP (6) were synthesized as
analogues of 2 Endo-b-galactosidases released
GlcNAcb-pNP or Glcb-GlcNAcb-pNP in an endo-manner from each substrate
A colorimetric assay for endo-b-galactosidase was
devel-oped using the synthetic substrates on the basis of the
determination of p-nitrophenol liberated from
GlcNAcb-pNP or Glcb-GlcNAcb-pNP formed by the enzyme through a coupled
reaction involving b-N-acetylhexosaminidase (b-NAHase)
or b-D-glucosidase Kinetic analysis by this method showed
that the value of Vmax/Kmof 2 for Escherichia freundii endo-b-galactosidase was 1.7-times higher than that for keratan sulfate,indicating that 2 is very suitable as a sensitive sub-strate for analytical use in an endo-b-galactosidase assay Compound 1 still acts as a fairly good substrate despite the absence of a Gal group in the terminal position In addition, the hydrolytic action of the enzyme toward 2 was shown to
be remarkably promoted compared to that of 4 by the presence of a 2-acetamide group adjacent to the p-nitro-phenyl group This was the same in the case of a comparison
of 1 and 3 Furthermore,the enzyme also catalysed a transglycosylation on 1 and converted it into GlcNAc b1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (9) and GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (10) as the major products,which have N-acetyllactosamine repeating units
Keywords: endo-b-galactosidase; enzyme assay; kinetics; poly (N-acetyllactosamine); transglycosylation
Endo-b-galactosidases were discovered as keratan
sulfate-degrading enzymes,so-called keratanases,in culture filtrates
of Escherichia freundii (glycoside hydrolase family 16) [1],
Coccobacillussp [2], Pseudomonas sp [3], Flavobacterium
keratolyticus (glycoside hydrolase family 16) [4,5], and
Bacteroides fragilis[6] E freundii keratanase was found to
have hydrolysing activity for a wide range of nonsulfated
oligosaccharides isolated from human milk and
carbohy-drate moieties of glycoproteins and glycolipids [7–10] The
use of endo-b-galactosidase has been expanded to detection
of poly (N-acetyllactosamine) chains in a variety of complex
glycoconjugates in addition to keratan sulfate Bacteroides fragilisendo-b-galactosidase has properties similar to those
of E freundii endo-b-galactosidase [11–13] Therefore,the endo-b-galactosidases from E freundii and B fragilis have been widely used as tools for structural and functional analyses of glycans involved in glycoconjugates
An assay using keratan sulfate as a substrate has been widely used for estimation of endo-b-galactosidase activity However,this method is not always reproducible because
of lack of uniformity of the polymer Methods using low molecular mass substrates have been preferred and recommended for accurate determination of activities of endoglycosidases such as a-amylase [14],lysozyme [15], and endo-b-N-acetylglucosaminidase [16],because the purity of the substrate and the reaction pattern can be determined exactly This led us to develop a substrate suitable for use in the analysis of endo-b-galactosidase A series of chromogenic substances having a partially substi-tuted unit of poly (N-acetyllactosamine) were designed as substrate analogues for this enzyme because systematic kinetic studies on the structural modification of substrates would be helpful in revealing the requirements for binding and catalytic specificity In general,glycosidase can cause transglycosylation as well as hydrolysis as a reverse reaction [17–19] Transglycosylation of endo-type glycosi-dase is now used for the synthesis of N-acetyllactosamine-repeating oligosaccharide
Correspondence to T Murata,Department of Applied Biological
Chemistry,Shizuoka University,836 Ohya,Shizuoka,422-8529,
Japan Fax and Tel.: +81 54 238 4872,
E-mail: actmura@agr.shizuoka.ac.jp
Abbreviations: pNP, p-nitrophenyl; b-NAHase,
b-N-acetylhexos-aminidase; b4GalT, b-1,4-galactosyltransferase; b3GnT,
b-1,3-N-acetylglucosaminyltransferase; HPAEC-PAD,high-performance
anion exchange chromatography-pulsed amperometric detection.
Enzymes: endo-b-galactosidase (EC 3.2.1.103); b- D -galactosidase
(EC 3.2.1.23); b-1,4-galactosyltransferase (EC 2.4.1.22);
b-1,3-N-acetylglucosaminyltransferase (EC 2.4.1.149); b-D-glucosidase
(EC 3.2.1.21); b-N-acetylhexosaminidase (EC 3.2.1.52).
(Received 20 December 2002,revised 11 July 2003,
accepted 17 July 2003)
Trang 2In this paper,we describe the enzymatic synthesis of a
novel substrate 2 and its analogues for use a colorimetric
assay of endo-b-galactosidase activity and the usefulness of
the resulting chromogenic substrates for kinetic studies on
the enzyme In the latter part of this paper,synthesis of
N-acetyllactosamine-repeating oligosaccharide b-glycoside
utilizing endo-b-galactosidase-mediated transglycosylation
is described
Materials and methods
Materials
Endo-b-galactosidases from E freundii and B fragilis were
from Seikagaku Corporation (Tokyo,Japan) and Wako
Pure Chemical Industries,Ltd (Osaka,Japan),respectively
b-D-Galactosidase from Bacillus circulans ATCC31382 [20]
was a kind gift from Meiji Milk Products Co Ltd (Tokyo,
Japan) b-D-Glucosidase from almonds was from Sigma
Chemicals b-N-acetylhexosaminidase (b-NAHase) from
Amycolatopsis orientalisIFO 12806T was purified by 80%
saturated ammonium sulfate precipitation followed by
GlcNAc-cellulofine affinity chromatography Bovine milk
b-1,4-galactosyltransferase (b4GalT) was from Calbiochem
(CA,USA) Crude b-1,3-N-acetylglucosaminyltransferase
(b3GnT) from bovine serum was prepared as follows
Bovine serum was brought to 80% saturation with solid
ammonium sulfate and left standing overnight at 4C The
precipitate was collected by centrifugation at 5000 g for
30 min,dissolved in 50 mMTris/HCl buffer (pH 8.0),and
dialysed against distilled water overnight at 4C The
enzyme solution was lyophilized and then used for synthesis
of oligosaccharides without further purification The crude
enzyme preparation catalysed the transfer of a GlcNAc
residue from UDP-GlcNAc to the OH-3¢ positions of
Galb1-4Glc,Galb1-4GlcNAc,Galb1-4Glcb-pNP and
4GlcNAcb-pNP The specific activity for
Galb1-4GlcNAcb-pNP as an acceptor substrate was 69 lUÆmg)1
Galb1-4GlcNAc,GlcNAcb1-3Galb1-4GlcNAc,Galb1-4Glcb-pNP,Galb1-4GlcNAcb-pNP,and
GlcNAcb1-6Galb1-4Glcb-pNP were synthesized by our previously
described methods [21–24] UDP-GlcNAc and UDP-Gal
were kind gifts from Yamasa Corporation (Choshi,
Japan) All other chemicals were obtained from commercial
sources
Enzyme assay
b-D-Galactosidase, b-D-glucosidase and b-NAHase
activit-ies were assayed as follows A mixture containing 2 mM
substrate solution (Galb-pNP,Glcb-pNP,and
GlcNAcb-pNP) in 0.4 mL 50 mM sodium phosphate buffer pH 6.0
and an appropriate amount of enzyme in a total volume of
0.1 mL was incubated for 10 min at 40C One hundred
microlitres of the reaction mixture were then added to
0.1 mL 1.0MNa2CO3on a microplate at 2-min intervals
during to stop the reaction,and the amount of liberated
p-nitrophenol was determined by measuring absorbance at
405 nm using a microplate reader (Biolumin
960,Amer-sham Pharmacia) One unit of enzyme was defined as the
amount releasing 1 lmol p-nitrophenolÆmin)1 The b3GnT
assay was carried out as follows Galb1-4GlcNAcb-pNP
(5 mg) and UDP-GlcNAc (3.6 mg) were dissolved in 0.5 mL 50 mM Tris/HCl buffer pH 8.0 containing 1.6 mg MnCl2 and 0.5 mg ATP,followed by addition of an appropriate amount of b3GnT preparation The reaction mixture was incubated at 37C for 96 h Fifty microlitres of the reaction mixture was taken out at 24-h intervals during the reaction and boiled for 5 min Resulting GlcNAcb1-3Galb1-4GlcNAcb-pNP was measured by HPLC as des-cribed in the Analytical methods
Analytical methods HPLC was carried out using a Mightysil RP18ODS column (4.6· 150 mm,Kanto Chemical Co Ltd,Tokyo,Japan) in
a Hitachi 6000-series liquid chromatograph with an L-4000 ultraviolet detector (absorbance at 300 nm) Elution of the column was performed with H2O/CH3OH (95 : 5,v/v) The flow rate was 1.0 mLÆmin)1 at 40C HPAEC-PAD analysis was conducted on a DX-300 Bio-LC system equipped with a pulsed amperometric detector (Dionex, Sunnyvale,USA) Oligosaccharides were analysed by a CarboPac P-1 column (Dionex,4· 250 mm) at a flow rate
of 1 mLÆmin)1 at room temperature The elution was effected with 100 mM NaOH for 40 min For NMR analysis,an appropriate oligosaccharide sample was dis-solved in 200 lL of D2O and filtrated with a Millipore filter (0.22 lm) and then put into a sample tube (i.d 3 mm)
1H- and 13C-NMR spectra were recorded on a JEOL JNM-LA 500 spectrometer at 25C Chemical shifts are expressed in d relative to sodium 3-(trimethylsilyl) propio-nate as an external standard ESI-MS analysis was carried out in the positive-ion mode on a JEOL MS-700 (JEOL Ltd,Akishima,Japan) using 2,5-dihydroxy benzoic acid as the matrix Determination of the amount of protein was carried out using a Bio-Rad protein assay kit Determin-ation of total carbohydrate was carried out as follows One hundred microliters of sample was put into a test tube (1· 10 cm),and 100 lL of 5% (w/v) phenol and 0.5 mL concentrated sulfuric acid were immediately added The sample mixture was vortexed and then kept for 20 min at room temperature,and absorbance was read at 490 nm
Preparation of GlcNAcb1-3Galb1-4GlcNAcb-pNP (1) and GlcNAcb1-3Galb1-4GlcNAcb-pNP (3)
Galb1-4GlcNAcb-pNP (504 mg,1 mmol) and UDP-Glc-NAc (651 mg,1 mmol) were dissolved in 25 mL 50 mM
Tris/HCl buffer pH 8.0 containing 99 mg MnCl2,followed
by addition of 850 mU of crude b3GnT preparation from bovine serum The mixture was incubated for 172 h
at 37C,and the reaction was terminated by boiling for
5 min The precipitate was removed by centrifugation (8 000 g,20 min),and the supernatant was loaded onto a Toyopearl HW-40S column (5· 100 cm) equilibrated with 25% methanol at a flow rate of 2 mLÆmin)1 After the chromatography,the eluate was monitored by measuring the absorbance at 300 nm (p-nitrophenyl group) and at
490 nm (phenol-sulfuric acid method) by a spectrometer Both chromatograms showed two peaks (F-1,tubes 24–33; F-2,tubes 49–60) F-1 was combined,concentrated,and lyophilized to produce 1 (24.1 mg) in a 3.4% total yield based on the acceptor added F-2 was recovered as
Trang 3Galb1-4GlcNAcb-pNP (0.35 g) In the same
way,com-pound 3 was prepared from Galb1-4Glcb-pNP (0.5 g) and
UDP-GlcNAc (450 mg) by use of bovine serum b3GnT in
a 4% total yield based on the acceptor added 1H and
13C-NMR data of 1 and 3 were almost identical to data
reported previously [24]
Preparation of GlcNAcb1-3Galb1-4GlcNAcb-pNP (2)
and GlcNAcb1-3Galb1-4GlcNAcb-pNP (4)
Compound 1 (24.1 mg,34.1 lmol) and UDP-Gal (42.6 mg,
68.2 lmol) were dissolved in 3.4 mL 100 mM sodium
cacodylate buffer (pH 6.8) containing 67.3 mg MnCl2
followed by addition of 0.2 U b4GalT from bovine milk
The mixture was incubated for 6 h at 37C and separated
by a Toyopearl HW-40S column (2.5· 80 cm) as described
above Compound 2 was obtained in an 82% total yield
(24.3 mg) based on the acceptor added In the same
way,compound 4 was obtained in a 71% total yield
(13.2 mg) based on the acceptor from 3 and UDP-Gal
1H and 13C-NMR data of 2 and 4 are summarized in
Table 1
Preparation of GlcNAcb1-3Galb1-4GlcNAcb-pNP (5) and its positional isomer GlcNAcb1-3Galb1-4GlcNAcb-pNP (6) Compound 3 (85 mg,128 lmol) and o-nitrophenyl b-D -galactopyranoside (Galb-oNP,238 mg,790 lmol) were dissolved in 5.8 mL 40 mM sodium acetate buffer pH 5.5 followed by addition of 98 mU of b-D-galactosidase from Bacillus circulansATCC31382 The mixture was incubated for 10 h at 50C and was loaded onto an ODS DM1020T column (5· 100 cm) equilibrated with 5% methanol at
a flow rate of 5 mLÆmin)1 in order to remove the o-nitrophenol liberated during the reaction The fractions showing absorbance at 300 nm were concentrated and loaded onto a Toyopearl HW-40S column as above The chromatogram showed two peaks (F-1,1340–1520 mL; F-2,1640–1880 mL) F-2 contained 3 (43 mg) used as the acceptor substrate F-1 was further separated by a Shodex Asahipak NH2P-50 column (21.5· 300 mm) equilibrated with 80% acetonitrile at a flow rate of 5 mLÆmin)1at 40C Eluate was monitored on-line by measuring the absorbance
at 300 nm The chromatogram showed two peaks (F-1a, 327–390 mL; F-1b,396–450 mL) These peaks were
Table 1.1H- and13C-chemical shifts of transfer products in D 2 O solution J 1,2 ,coupling constants are given in Hz.
Compounds
Chemical Shifts (d) C-1 C-2 C-3 C-4 C-5 C-6 NHCO COCH 3 o-ph m-ph p-ph C-O H-1 J 1,2 CH 3
Trang 4combined,concentrated,and lyophilized to produce 5
(5.5 mg) and 6 (7.2 mg) in 5.2 and 6.8% yields based on the
acceptor added,respectively.1H- and13C-NMR data of 5
and 6 are summarized in Table 1
Preparation of GlcNAcb1-3Galb1-4GlcNAcb-pNP (7)
and GlcNAcb1-3Galb1-4GlcNAcb-pNP (8)
Galb-pNP (390 mg,1.29 mmol) and N,
N¢-diacetylchito-biose (GlcANc2,531 mg,1.25 mmol) were dissolved in
7.5 mL 20 mMsodium acetate buffer pH 5.0 followed by
addition of 8.7 U of A orientalis
b-N-acetylhexosamini-dase The mixture was incubated for 100 h at 40C,and the
reaction was terminated by boiling for 5 min The
precipi-tate was removed by centrifugation (8 000 g,15 min),and
the supernatant was loaded onto a Toyopearl HW-40S
column (5· 100 cm) as above Eluate was monitored by
measuring the absorbance at 300 nm (p-nitrophenyl group)
and at 490 nm (phenol-sulfuric acid method) The
chroma-togram showed four peaks (F-1,135–150 mL; F-2,190–
225 mL; F-3,275–295 mL; F-4,420–470 mL) F-2 and F-3
were combined,concentrated,and lyophilized to produce 8
(33.8 mg) and 7 (8.6 mg),respectively,in a 6.5% total yield
based on the donor added.1H- and13C-NMR data of these
disaccharides are summarized in Table 1
Hydrolytic actions of endo-b-galactosidase
onp-nitrophenyl b-glycosides
The hydrolytic actions of endo-b-galactosidase on
p-nitro-phenyl oligosaccharide b-glycosides and a reducing
oligo-saccharide listed in Table 2 were investigated by incubating
a mixture (50 lL) containing 1 mMof substrates in 50 mM
sodium acetate buffer pH 5.8 with 1 mU of the enzymes at
37C for 20 min The enzyme hydrolysates were analysed
by HPLC or HPAEC-PAD as described in the Analytical
method section The reaction was linear from 5 to 15 min
The rate of attack on 2 was arbitrarily set at 100
Colorimetric assay of endo-b-galactosidase activity
A mixture containing 0.5 mMof each p-nitrophenyl oligo-saccharide b-glycoside and 50 mU of b-D-glucosidase or
25 mU of b-N-acetyllactosaminide in 500 mL 50 mM
sodium acetate buffer pH 5.8 and an appropriate amount
of the enzyme was incubated at 37C Samples (each
50 lL) were taken at intervals (0,5,10,15 and 20 min) during the incubation and inactivated by adding 50 lL 1.0MNa2CO3 The amount of liberated p-nitrophenol was determined by measuring absorbance at 405 nm using a microplate reader One unit of the enzyme was defined as the amount hydrolysing 1 lmol of 2 per min The initial rates of the enzymatic reaction were evaluated from kinetic curves of product accumulation as described above The parameters of Michaelis–Menten-type kinetics were evalu-ated by 1/v–1/[S] plots and the least-squares method The substrate concentration ranges used for compounds 1,2,3,
4 and 5 were 0.05–0.4,0.02–0.75,0.1–0.8,0.25–2.0 and 0.25– 1.5 mM,respectively
Assay of endo-b-galactosidase activity by HPLC The standard assay was carried out as follows A reaction mixture (500 lL) containing an appropriate substrate and endo-b-galactosidase in 10 mM sodium acetate buffer (pH 5.8) was incubated at 37C,and samples (each
50 lL) were taken at 3-min intervals during incubation After inactivation of each sample by adding 150 mL of 1M
acetic acid,the amount of liberated GlcNAcb-pNP was determined by HPLC as described in Analytical methods
Transglycosylation reaction of endo-b-galactosidase fromE freundii
Compound 1 (16 mg,23 lmol) was dissolved in 1.9 mL
20 mMsodium acetate buffer pH 5.8 followed by addition
of 2.3 mU endo-b-galactosidase from E freundii The mixture was incubated for 30 days at 37C and was loaded onto a Sep-pak accel QMA column (2· 4 cm) equilibrated with H2O at a flow rate of 1 mLÆmin)1 Eluate was collected
in 2-mL fractions and monitored by measuring the absorb-ance at 300 nm using a spectrometer The fractions showing absorbance at 300 nm were combined,concentrated and loaded onto a Shodex Asahipak GS-220FP column (7.6· 250 mm) equilibrated with H2O at a flow rate of 0.6 mLÆmin)1at 40C Eluate was monitored on-line by measuring the absorbance at 300 nm The chromatogram showed four peaks (Fig 1A) Peak B,which was presumed
to be a transglycosylation product,was concentrated and lyophilized to produce 9 (0.8 mg) in 3.3% yield based on the substrate added.1H- and13C-NMR data of 9 are summar-ized in Table 1
Results
Preparation of colorimetric substances
A series of chromogenic substances were designed as substrates of endo-b-galactosidase based on the structural features of keratan sulfate,which is an alternating polymer
of N-acetyllactosamine units jointed to each other by a
Table 2 Relative hydrolytic rates of endo-b-galactosidases on
p-nitro-phenyl oligosaccharide b-glycosides The hydrolytic actions of
endo-b-galactosidase on different substrates were investigated as described in
Materials and methods The vertical arrow indicates the point of
cleavage One mM of each substrate was used for the determination of
relative hydrolytic rates –,not hydrolyzed even in the presence of 10
mU of the enzyme.
Trang 5b-(1-3) linkage Tetrasaccharide 2 containing two
N-acetyll-actosamine repeats and its analogues were synthesized by
the alternative addition of 3) linked GlcNAc and
b-(1-4) linked Gal to Galb1-4GlcNAcb-pNP and
Galb1-4Glcb-pNP,respectively,using two kinds of glycosyltransferases
Thus,compounds 1 and 3 were first prepared by the
regioselective transfer of GlcNAc residue from
UDP-GlcNAc to Galb1-4UDP-GlcNAcb-pNP and Galb1-4Glcb-pNP
by b3GnT from bovine serum They were further converted
into 2 and 4 utilizing b4GalT from bovine milk (Fig 2A,B)
The enzyme efficiently catalysed the transfer of a Gal moiety
to the OH-4¢ position of the acceptors in high yields (82 and 71%) depending on the acceptor The positional isomers 5 and 6 were prepared simultaneously by Gal transfer from Galb-oNP to the OH-3¢ and OH-6¢ positions of 3 using
B circulans b-D-galactosidase-mediated transglycosylation (Fig 2B) The resulting products were obtained in a molar ratio of 1 : 1.3 and in a 12% overall yield based on the acceptor added Compound 7 and its isomer 8 were prepared from Galb-pNP using b-NAHase-mediated trans-glycosylation (Fig 2C)
Hydrolytic actions of endo-b-galactosidases The hydrolytic actions of endo-b-galactosidases on synthetic chromogenic substances (each 1 mM) were investigated by using enzyme preparations from E freundii and B fragilis Each enzyme splits compounds 1–6 into the corresponding reducing oligosaccharides and chromogenic substances, GlcNAcb-pNP/Glcb-pNP For example,compound 2 was completely hydrolysed in an endo-manner into Galb1-4GlcNAcb1-3Galb and GlcNAcb-pNP The relative hydro-lytic rates of 1 and 4 compared with that of 2 (set at 100) were 47 and 10,i.e 2- and 10-fold differences,respectively The hydrolytic activities toward 3, 5 and 6 were not detected under the experimental conditions as described in the Materials and methods Furthermore,the hydrolytic rate of reducing trisaccharide GlcNAcb1-3Galb1-4GlcNAc was compared with that of its glycoside 1 in order to examine how the p-nitrophenyl group participates in the hydrolytic action There was little progression of hydrolysis of the reducing trisaccharide under the experimental conditions,
Fig 1 HPLC analysis of the reaction mixture obtained by
endo-b-galactosidase-mediated transglycosylation and time courses of the
pro-duction of transglycosylation products 9 and 10 from 1 and degradation
of 1 (A) HPLC analysis was performed as described in Materials and
methods (B) A reaction mixture (50 lL) containing 11.5 m M
com-pound 1,0.1% BSA and 1.5 mU E freundii endo-b-galactosidase in
20 m M sodium acetate buffer,pH 5.8,was incubated at 37 C The
amount of each product formed from the initial substrate was
deter-mined by HPLC s,Peak B (compound 9); d,peak A (compound 10);
h,GlcNAcb-pNP; j,compound 1.
Fig 2 Summary of enzymatic synthesis of p-nitrophenyl oligosaccha-ride b-glycosides used in this work (A) Consecutive additions of Glc-NAc and Gal to Galb1-4GlcGlc-NAcb-pNP by b3GnT and b4GalT (B) Consecutive additions of GlcNAc and Gal to Galb1-4Glcb-pNP by b3GnT and b- D -galactosidase or b4GalT (C) N-acetylglucosaminy-lation of Galb-pNP by b-N-acetylhexosaminidase-mediated transgly-cosylation.
Trang 6although the reducing trisaccharide was hydrolysed very
slowly to form GlcNAcb1-3Gal and GlcNAc when a
10-fold amount of enzyme was added (Table 2) This
indicates that the p-nitrophenyl group is critical for the
hydrolytic action of endo-b-galactosidase p-Nitrophenyl
disaccharide b-glycosides 7, 8,Galb1-4GlcNAcb-pNP,
and 6Galb1-4GlcNAcb-pNP and
GlcNAcb1-6Galb1-4Glcb-pNP did not act as substrates for the enzyme
Colorimetric assay of endo-b-galactosidase activity
As shown in Fig 3,a novel method for
endo-b-galactosi-dase assay using 2 was designed on the basis of the results
described above This assay involves the colorimetric
determination of p-nitrophenol liberated from the substrate
by the action of the enzyme through a coupled reaction
involving b-NAHase Thus,the enzyme exclusively
produ-ces GlcNAcb-pNP from 2 and then b-NAHase hydrolyses
GlcNAcb-pNP to free p-nitrophenol In this
case,com-pound 1 is not suitable for this assay because the terminal
GlcNAc residue is subjected to hydrolysis by b-NAHase
Compound 2 was incubated with E freundii
endo-b-galac-tosidase in the presence and absence of b-NAHase
(Fig 4A) The rate of hydrolysis was first-order with respect
to the enzyme throughout the course of the determination
The reaction proceeded linearly for at least 20 min under
the experimental conditions used (Fig 4A) In this case,
only 10 ng of the endo-b-galactosidase could be determined
by this assay method The dose–response plot of the coupled
enzyme vs the colour intensity of the enzyme was found to
be a plateau in the range of 20–40 mU for 15 min The
addition of 20 mU of coupled enzyme to the assay system
was sufficient to obtain the maximal activity of
endo-b-galactosidase (Fig 4B) In a similar manner,compound 4
was applied to determination of endo-b-galactosidase
activity with b-D-glucosidase as a coupled enzyme (Fig 4C)
When 4 was used as a substrate,100 ng of the enzyme was
required for determination of the activity by this assay
method The addition of 50 mU of coupled enzyme to the
assay system was sufficient to obtain the maximal activity of
endo-b-galactosidase (Fig 4D)
Kinetics of endo-b-galactosidase
In order to elucidate in more detail the substrate specificity
of endo-b-galactosidase,parameters of
Michaelis–Menten-type kinetics for 1–5 were evaluated by a 1/v–1/[S] plot
(Fig 5) Compound 2 was assayed with A orientalis b-NAHase as a coupled enzyme and 3–5 with almond b-D-glucosidase using the newly developed colorimetric assay described above Compound 1 was assayed by HPLC The kinetic parameters are summarized in Table 3 Com-pound 2 was the best among the synthetic substrates with a
kcat/Kmvalue of 911ÆmM )1Æs)1for E freundii endo-b-galac-tosidase Compound 1 still acts as a fairly good substrate despite the absence of a Gal group at the terminal position, because the kcat/Km value of 1 was 31% of that of 2 However,the kcat/Km value of 4,in which Glc had been replaced with GlcNAc,was only 0.9% of that of 2 The same tendency was seen in a comparison of 1 and 3 This was also the case for B fragilis endo-b-galactosidase as shown in Table 3
Transglycosylation reaction of endo-b-galactosidase fromE freundii
In order to examine the transglycosylation activity of endo-b-galactosidase, E freundii endo-b-galactosidase was
Fig 3 Principle of the colorimetric assay method for
endo-b-galac-tosidase using compounds 2 and 4 through a coupled reaction involving
b-NAHase or b- -glucosidase.
Fig 4 Effects of coupled enzymes on the release of p-nitrophenol from synthetic substrates (A) Effect of b-NAHase on the production of p-nitrophenol from 2 Compound 2 (125 nmol) was incubated with
E freundii endo-b-galactosidase (9.3 ng) in 0.5 mL 10 m M sodium acetate buffer,pH 5.8,in the presence or absence of b-NAHase (25 mU) The reaction was stopped by the addition of 1.0 M Na 2 CO 3 , and then liberated p-nitrophenol was determined spectrophotometri-cally at 405 nm d,Endo-b-galactosidase and b-NAHase; s,endo-b-galactosidase; m, b-NAHase (B) Time course of the production of p-nitrophenol formed from 2 through a coupled reaction involving b-NAHase (C) Effect of b- D -glucosidase on the production of p-nitrophenol from 4 Compound 4 (250 nmol) was incubated with
E freundii endo-b-galactosidase (93 ng) in the presence or absence of b- D -glucosidase (50 mU) as a coupled enzyme d,Endo-b-galactosi-dase and b- D -glucosidase; s,endo-b-galactosidase; m, b- D -glucosi-dase (D) Time course of the production of p-nitrophenol from
4 through a coupled reaction involving b- D -glucosidase.
Trang 7incubated with a high substrate concentration of 1 (8.4%).
After the incubation,the reaction mixture was analysed by
HPLC As shown in Fig 1A,two peaks,A and B,which
were presumed to be transglycosylation products,appeared
at retention times of 20 and 25 min,respectively Based on
the results of ESI-MS analysis,the molecular mass of peak
B was estimated to be 1072,which coincides with the value
of GlcNAc-Gal-GlcNAc-Gal-GlcNAc-pNP Peak B was
collected and lyophilized to produce 9 (3.3% actual yield
base on the donor substrate 1) The1H- and 13C-NMR
signals of 9 were easily assigned by correlation with the
spectra of 1 and 2 (Table 1) The most direct evidence that
the GlcNAcb1-3Gal unit is bound to the C-4¢ position of
GlcNAc was obtained from an 8-p.p.m downfield shift of
C-4¢ and the HMBC spectrum (Fig 6) The results of NMR and ESI-MS analyses revealed that 9 is a pentasaccharide b-glycoside,GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4Glc-NAcb-pNP Peak A was also subjected to ESI-MS analysis The molecular mass of peak A was estimated to be 1437.5, which coincides with the theoretical value of GlcNAc-Gal-GlcNAc-Gal-GlcNAc-Gal-GlcNAc-pNP This result shows that the GlcNAcb1-3Gal residue from 1 was also trans-ferred to 9 by the enzyme Taking into account the regioselective synthesis of 9 from 1,peak A was estimated
to be a heptasaccharide b-glycoside,GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP (10) The time course of the production of transfer products and the degradation of 1 was observed by HPLC analysis as shown in Fig 1B The maximum production of 9 and 10 was reached at 4 h,and their amounts were gradually decreased during the subsequent reaction,finally causing complete hydrolysis
Discussion
Endo-b-galactosidases have been used widely as analytical tools in the field of glycobiology and glycotechnology However,there have been few studies on kinetics of the enzyme because a simple and sensitive assay method has not been developed The conventional assay for endo-b-galac-tosidase activity is based on the method of Park and Johnson using keratan sulfate as a substrate However,this method is not always reproducible because of lack of uniformity of the polysaccharide Therefore,we synthesized p-nitrophenyl di,tri,and tetrasaccharide b-glycosides 1–8 as substrates for endo-b-galactosidase by combining a glycos-idase-catalysed transglycosylation and a glycosyltrans-ferase-catalysed reaction Endo-b-galactosidases from
E freundii and B fragilis hydrolysed p-nitrophenyl oligo-saccharide b-glycosides 1–6 as shown in Table 2 Based on the hydrolytic action toward the enzyme, 2 was found to be the best substrate among the synthetic substances Com-pound 1,in which terminal Gal was trimmed from 2,still acts as a fairly good substrate The present assay system has better reproducibility and is simpler than the method of Park and Johnson From a practical viewpoint, 2,a well-defined substrate,was shown to be very useful for a routine submicrogram assay of endo-b-galactosidase in biological materials
Further kinetic studies on endo-b-galactosidases were carried out using a series of modified substrates mentioned above Kinetic parameters of activities of E freundii and
B fragilis endo-b-galactosidases toward 1–5 are summar-ized in Table 3 The catalytic efficiencies of 2 for both enzymes were the highest among the synthetic substrates The value of Vmax/Kmof 2 for the E freundii enzyme was 1.7-times higher than that for keratan sulfate as shown in Table 3,indicating that it is very useful as a substrate instead of keratan sulfate for analytical use in the endo-b-galactosidase assay In addition,this similarity in the values of Vmax/Kmsuggests that the sulfate group on the 6-position on GlcNAc of keratan sulfate is not always essential for the hydrolytic action of the enzyme Replace-ment of Glc by internal GlcNAc of 2 resulted in a remarkable reduction in the catalytic efficiency on 4 This was also true for a comparison of 1 and 3 The increase in
Fig 5 Effects of concentrations of substrate on endo-b-galactosidase
activity using compounds 2 (A) and 4 (B) The results shown in (A) were
obtained from an experiment in which the substrate concentration was
varied over a range of 0.02–0.75 m M ,and the results shown in (B) were
obtained from an experiment in which the substrate concentration was
varied over a range of 0.25–2.0 m M Insets show the Linweaver–Burk
plots.
Trang 8catalytic efficiency was clearly due to the N-acetyl group on
C-2 of GlcNAc linked to the aglycon moiety However,
compound 4 still acts as a fairly good substrate: the
Vmax/Kmvalue of 4 is 14.1% of that of
Galb1-4GlcNAcb1-3Galb1-4Glcb-Cer It may be used as a substitute for the
detection of glycosphingolipid-degrading
endo-b-galactosi-dase This concept for the enzyme assay could be applied to
other types of endo-b-galactosidases from Diplococcus
pneumonia[25], Clostridium perfringens [26,27], and mollusk
Painopectensp [28],which hydrolyse internal b-galactosidic
linkages of blood group A and B antigens,Gala1-4Galb1-4GlcNAc and GlcNAca1-4Galb1-4GalNAc,and GlcAb1-3Galb1-3Gal structures,respectively
The structure of the site of cleavage by the enzyme,which was deduced from results of kinetic studies using well-defined synthetic oligosaccharides,is shown in Fig 7A We propose a binding structure so that Galb1-4GlcNAcb1-3Galb1-4GlcNAcb-pNP has a matching shape,which can accommodate a chain of five residues (A,B,C,D,and E) in the active site Synthetic tri- and tetrasaccharide glycosides had only one cleavage site for the endo-b-galactosidase, which splits the glycoside bond between C and D On the other hand,Fukuda and Matsumura reported that endo-b-galactosidases from E freundii hydrolysed corneal
Fig 6 HMBC spectrum of compound 9 with1H and13C spectra on the
sides of the two-dimensional spectrum Only the expanded region of the
HMBC spectrum showing connectivity of GlcNAc C-1¢¢ with Gal C-1¢,
newly formed by endo-b-galactosidase-mediated transglycosylation,is
presented.
Fig 7 Proposed structure of the cleavage site of endo-b-galactosidase The enzyme hydrolyses both N-acetyllactosamine-repeating tetrasac-charide b-glycoside (A) and keratan sulfate (B) Arrows show the cleavage site of the glycosidic linkages of each substrate.
Table 3 Kinetic parameters of endo-b-galactosidases from Escherichia freundii and Bacteroides fragilis The parameters of Michaelis–Menten-type kinetics were evaluated by 1/v-1/[S] plots and the least-squares method This summary was compiled from results reported here and from data in the literature The substrate concentration ranges used for compounds 1,2,3,4 and 5 were 0.05–0.4,0.02–0.75,0.1–0.8,0.25–2.0 and 0.25–1.5 mM, respectively K m ,Mean ± SEM (mM); V max ,Mean ± SEM (lmolÆmin)1Æmg)1); k cat ,Mean ± SEM (sec)1); k cat /K m ,sec)1ÆmM)1 –, not determined.
Trang 9keratan sulfate,releasing GlcNAc6SO3b1-3Gal and
GlcNAc6SO3b1-3Gal6SO3b1-4GlcNAc6SO3b1-3Gal as
major products [8] This finding suggests that the enzyme
tolerates C-6 sulfation of the sugar residues A and B,which
have to be partially O-sulfated in keratan sulfate,but not
C-6 sulfation of the sugar residue C The hydrolysates
GlcNAc6SO3b1-3Gal and GlcNAc6SO3
b1-3Gal6-SO3b1-4GlcNAc6SO3b1-3Gal may occupy corresponding
sites B-C and -A-B-C,respectively,as shown in Fig 7B The
sugar residue D at the cleavage site influences the sensitivity
of oligosaccharides to the enzyme,because hydrolytic action
was promoted by the presence of an N-acetyl group on C-2
of GlcNAc corresponding to sugar residue D (Table 3)
Disaccharide b-glycoside 7 did not act as a substrate
Reduction of the reducing-end residue of
Galb1-4Glc-NAcb1-3Galb1-4Glc inhibited the action of
endo-b-gal-actosidases from E freundii [7,29] These results indicate
that a sugar pyranose structure such as GlcpNAc or Glcp on
leaving site D is required for the hydrolytic action of
endo-b-galactosidases Furthermore,the mode of linkage of sugar
residues A to B was strict for the binding locus in the active
site,because conversion of the (1–4) into (1–3)-linkages of
terminus Gal to GlcNAc residues remarkably decreased the
enzyme action (Table 2) These observations indicate that a
tetrasaccharide sequence consisting of two LacNAc
repeat-ing units such as 2 is preferable to the bindrepeat-ing locus in the
active site A series of chromogenic substrates were shown
to be advantageous as probes for substrate recognition at
the active site in the enzyme
Generally,glycosidase has transglycosylation activity as
a reverse reaction of hydrolysis Taking into account the
hydrolytic action of endo-b-galactosidase, 1 was used as a
substrate for transglycosylation reaction The enzyme
produced 9 and 10 as major products through consecutive
transfer of the GlcNAcb1-3Gal unit (Fig 8) Thus,the
enzyme catalysed regioselective transfer to the OH-4¢
position of 1 of the GlcNAcb1-3Gal unit from the same
substrate to produce 9 In this case, 1 serves as both
donor and acceptor substrates in the transglycosylation
Furthermore,the disaccharide elongation reaction
pro-gresses in order and produces heptasaccharide glycoside
10 from 9 However,once 9 and 10 had reached
maximum concentrations,they gradually decreased in a
subsequent reaction and finally caused complete degrada-tion (Fig 1B,C) indicating that 9 and 10 are fairly good substrates for endo-b-galactosidase and reflect an N-acetyllactosamine-repeating structure The analytical yield
of 9 was estimated by HPLC analysis (Fig 1) to be 7.0% based on the donor substrate The large difference between yields in actual and analytical data is though to
be caused by a loss of 9 through the process of chromatographic separation procedures
Poly (N-acetyllactosamine) has been shown to be present
on membrane glycoconjugates [30,31] and has been identi-fied as a precursor of Lewis X,sialyl Lewis X,and blood group antigens The amounts of poly (N-acetyllactosamine) chains have been shown to be changed during cellular differentiation [32] and malignant transformation of cells [33,34] Furthermore, poly (N-acetyllactosamine)s are recognized with high affinity by galectins [35] and are involved in apoptosis [36] Recently,Ando et al reported that sialylated poly (N-acetyllactosamine) on the cell surface facilitated apoptotic cell uptake by macrophages [37] On the other hand,it has been reported that Helicobacter pylori selectively interacted with poly (N-acetyllactosamine) of human erythrocytes [38,39] and with Neu5Aca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4Glcb-Cer and NeuAca2-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4GlcNAcb1-3Galb1-4Glcb-Cer isolated from human gastric adenocarcinoma [40] These findings suggest that poly (N-acetyllactosamine) chains involved in glyco-conjugates play important roles in biological events such as metastasis,apoptosis,phagocytosis,and infection Accord-ingly,compounds 9 and 10 should be useful as model compounds for studying biological functions of N-acetyl-lactosamine repeating domains involved in glycoproteins and glycolipids
In this study,the first enzymatic synthesis of poly (N-acetyllactosamine) has been achieved by endo-b-galac-tosidase-mediated transglycosylation This enzyme would
be an excellent tool for producing a poly (N-acetyllactosa-mine) chain as well as for the detection of poly (N-acetyl-lactosamine)s involved in glycoconjugates
Acknowledgements
We thank Yamasa Corporation and Meiji Milk Products for the gift of UDP-GlcNAc and UDP-Gal and the gift of b- D -galactosidase from
B circulans ATCC31382,respectively We also thank JEOL Hightech Ltd (Akishima,Japan) for ESI-MS analysis of the transfer products This work was supported by a Grant-in-Aid for Scientific Research (No 14760047) from the Ministry of Education,Science,Sports, Culture,and Technology of Japan.
References
1 Kitamikado,M.,Ueno,R & Nakamura,T (1970) Enzymic degradation of whale cartilage keratosulfate II Identification of a keratosulfate-degrading bacterium Bull Jap Soc Sci Fish 36, 1174–1176.
2 Hirano,S & Meyer,K (1971) Enzymatic degradation of corneal and cartilaginous keratosulfates Biochem Biophys Res Commun 44,1371–1375.
3 Nakazawa,K & Suzuki,S (1975) Purification of keratan sulfate-endogalactosidase and its action on keratan sulfates of different origin J Biol Chem 250,912–917.
Fig 8 Proposed mechanism of E freundii
endo-b-galactosidase-medi-ated transglycosylation reaction Compound 1 as both a donor and
acceptor substrate was incubated with E freundii
endo-b-galactosi-dase The enzyme catalysed the transfer of the GlcNAcb1-3Gal unit
from 1 to nonreducing-terminal GlcNAc residues of 1 and 9.
Trang 104 Kitamikado,M.,Ito,M & Li,Y.T (1981) Isolation and
char-acterization of a keratan sulfate-degrading endo-b-galactosidase
from Flavobacterium keratolyticus J Biol Chem 256,3906–3909.
5 Ito,M.,Hirabayashi,Y & Yamagata,T (1986) Substrate
speci-ficity of endo-b-galactosidases from Flavobacterium keratolyticus
and Escherichia freundii is different from that of Pseudomonas sp.
J Biochem (Tokyo) 100,773–780.
6 Scudder,P.,Uemura,K.,Dolby,J.,Fukuda,M.N & Feizi,T.
(1983) Isolation and characterization of an endo-b-galactosidase
from Bacteroides fragilis Biochem J 213,485–494.
7 Fukuda,M & Matsumura,G (1975) Endo-b-galactosidase of
Escherichia freundii Hydrolysis of pig colonic mucin and milk
oligosaccharides by endoglycosidic action Biochem Biophys Res.
Commun 64,465–471.
8 Fukuda,M.N & Matsumura,G (1976) Endo-b-galactosidase of
Escherichia freundii Purification and endoglycosidic action on
keratan sulfates,oligosaccharides,and blood group active
glyco-protein J Biol Chem 251,6218–6225.
9 Fukuda,M.N.,Watanabe,K & Hakomori,S.I (1978) Release
of oligosaccharides from various glycosphingolipids by
endo-b-galactosidase J Biol Chem 253,6814–6819.
10 Fukuda,M.N (1981) Purification and characterization of
endo-b-galactosidase from Escherichia freundii induced by hog gastric
mucin J Biol Chem 256,3900–3905.
11 Scudder,P.,Hanfland,P.,Uemura,K & Feizi,T (1984)
Endo-b- D -galactosidases of Bacteroides fragilis and Escherichia
freundii hydrolyze linear but not branched oligosaccharide
domains of glycolipids of the neolacto series J Biol Chem 259,
6586–6592.
12 Scudder,P.,Tang,P.W.,Hounsell,E.F.,Lawson,A.M.,
Mehmet,H & Feizi,T (1986) Isolation and characterization of
sulphated oligosaccharides released from bovine corneal keratan
sulphate by the action of endo-b-galactosidase Eur J Biochem.
157,365–373.
13 Scudder,P.,Lawson,A.M.,Hounsell,E.F.,Carruthers,R.A.,
Childs,R.A & Feizi,T (1987) Characterization of
oligosacchar-ides released from human-blood-group O erythrocyte
glycopep-tides by the endo-b-galactosidase of Bacteroides fragilis A study of
the enzyme susceptibility of branched poly (N-acetyllactosamine)
structures Eur J Biochem 168,585–593.
14 Usui,T.,Ogawa,K.,Nagai,H & Matsui,H (1992) Enzymatic
synthesis of p-nitrophenyl 4 5 -O-b- D -galactosyl-a-maltopentaoside
as a substrate for human a-amylase Anal Biochem 202,61–67.
15 Nanjo,F.,Sakai,K & Usui,T (1988) p-Nitrophenyl
penta-N-acetyl-b-chitopentaoside as a novel synthetic substrate for the
colorimetric assay of lysozyme J Biochem (Tokyo) 104,255–
258.
16 Takegawa,K.,Fujita,K.,Fan,J.Q.,Tabuchi,M.,Tanaka,N.,
Kondo,A.,Iwamoto,H.,Kato,I.,Lee,Y.C & Iwahara,S (1998)
Enzymatic synthesis of a neoglycoconjugate by transglycosylation
with Arthrobacter endo-b-N-acetylglucosaminidase: a substrate
for colorimetric detection of endo-b-N-acetylglucosaminidase
activity Anal Biochem 257,218–223.
17 Ichikawa,Y.,Look,G.C & Wong,C.-H (1992)
Enzyme-catalyzed oligosaccharide synthesis Anal Biochem 20,215–238.
18 Shoda,S.,Fujita,M & Kobayashi,S (1998) Glycanase-catalyzed
synthesis of non-natural oligosaccharides Trends Glycosci
Gly-cotech 10,279–289.
19 Murata,T & Usui,T (2000) Enzymatic synthesis of important
oligosaccharide units involved in N- and O-linked glycans Trends
Glycosci Glycotech 12,161–174.
20 Fujimoto,H.,Miyasato,M.,Ito,Y.,Sasaki,T & Ajisaka,K.
(1998) Purification and properties of recombinant b-galactosidase
from Bacillus circulans Glycoconj J 15,155–160.
21 Sakai,K.,Kastumi,R.,Ohi,H.,Usui,T & Ishido,Y (1992)
Enzymatic synthesis of N-acetyllactosamine and
N-acetylallo-lactosamine by the use of b- D -galactosidase J Carbohydr Chem 11,553–565.
22 Usui,T.,Kubota,S & Ohi,H (1993) A convenient synthesis of b- D -galactosyl disaccharide derivatives using the b- D -galactosidase from Bacillus circulans Carbohydr Res 244,315–323.
23 Matahira,Y.,Tashiro,A.,Sato,T.,Kawagishi,H & Usui,T (1995) Enzymic synthesis of lacto-N-triose ll and its positional analogues Glycoconj J 12,664–671.
24 Murata,T.,Tashiro,A.,Itoh,T & Usui,T (1997) Enzymic synthesis of 3¢-O- and 6¢-O-N-acetylglucosaminyl-N-acetyllactos-aminide glycosides catalyzed by b-N-acetyl- D -hexosaminidase from Nocardia orientalis Biochim Biophys Acta 1335,326–334.
25 Takasaki,S & Kobata,A (1976) Purification and characteriza-tion of an endo-b-galactosidase produced by Diplococcus pneu-moniae J Biol Chem 251,3603–3609.
26 Fushuku,N.,Muramatsu,H.,Uezono,M.M & Muramatsu,T (1987) A new endo-b-galactosidase releasing Gala1–3Gal from carbohydrate moieties of glycoproteins and from a glycolipid.
J Biol Chem 262,10086–10092.
27 Ashida,H.,Anderson,K.,Nakayama,J.,Maskos,K.,Chou, C.-W., Cole, R.B., Li, S.-C & Li, Y.-T (2001) A novel endo-b-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAca1–4Gal from glycans specifically expressed
in the gastric gland mucous cell-type mucin J Biol Chem 276, 28226–28232.
28 Takagaki,K.,Nakamura,T.,Takeda,Y.,Daidouji,K & Endo,
M (1992) A new endo-b-galactosidase acting on the Galb1–3Gal linkage of the proteoglycan linkage region J Biol Chem 267, 18558–18563.
29 Nakagawa,H.,Yamada,T.,Chien,J.L.,Gardas,A.,Kitamikado, M.,Li,S.C & Li,Y.T (1980) Isolation and characterization of an endo-b-galactosidase from a new strain of Escherichia freundii.
J Biol Chem 255,5955–5959.
30 Fukuda,M.,Fukuda,M.N & Hakomori,S (1979) Develop-mental change and genetic defect in the carbohydrate structure of band 3 glycoprotein of human erythrocyte membrane J Biol Chem 254,3700–3703.
31 Spooncer,E.,Fukuda,M.,Klock,J.C.,Oates,J.E & Dell,A (1984) Isolation and characterization of polyfucosylated lactosa-minoglycan from human granurocytes J Biol Chem 259,4792– 4801.
32 Muramatsu,T.,Gachelin,G.,Nicolas,J.F.,Condamine,H., Jakob,H & Jacob,F (1978) Carbohydrate structure and cell differentitation: unique properties of fucosylglycopeptides isolated from embryonal carcinoma cells Proc Natl Acad Sci USA 75, 2315–2319.
33 Yamashita,K.,Ohkura,T.,Tachibana,Y.,Takasaki,S & Kobata,A (1984) Comparative study of the oligosaccharides released from baby hamster kidney cells and their polyoma transformant by hydrazinolysis J Biol Chem 259,10834–10840.
34 Pierce,M & Arango,J (1986) Rous sarcoma virus-transformed baby hamster kidney cells express higher levels of aspar-agine-linked tri- and tetraantennary glycopeptides containing [GlcNAcb (1,6) Man-a (1,6) Man] and poly–N–acetyllactosamine sequences than baby hamster kidney cells J Biol Chem 261, 10772–10777.
35 Barondes,S.H.,Cooper,D.N.,Gitt,M.A & Leffler,H (1994) Galectins Structure and function of a large family of animal lec-tins J Biol Chem 269,20807–20810.
36 Perillo,N.L.,Pace,K.E.,Seilhamer,J.J & Baum,L.G (1995) Apoptosis of T cells mediated by galectin-1 Nature 378,736–739.
37 Ando,K.,Hagiwara,T.,Beppu,M & Kikugawa,K (2000) Naturally occurring anti-band 3 antibody binds to apoptotic human T-lymphoid cell line Jurkat through sialylated poly-N-acetyllactosaminyl saccharide chains on the cell surface Biochem Biophys Res Commun 275,412–417.