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Demonstration of the Pleiotrophin-binding Oligosaccharide
Sequences Isolated from Chondroitin Sulfate/Dermatan
Sulfate Hybrid Chains of Embryonic Pig Brains
*
□
S
Received for publication, July 6, 2005, and in revised form, August 23, 2005 Published, JBC Papers in Press, August 23, 2005, DOI 10.1074/jbc.M507304200
Xingfeng Bao
‡1
, Takashi Muramatsu
§2
, and Kazuyuki Sugahara
‡3
From the
‡
Department of Biochemistry, Kobe Pharmaceutical Univeristy, 4-19-1, Motoyama-kita-machi, Higashinada-ku, Kobe
658-8558 and the
§
Department of Biochemistry, Nagoya University School of Medicine, Nagoya 466-8550, Japan
Mammalian brains contain significant amounts of chondroitin
sulfate (CS), dermatan sulfate (DS), and CS/DS hybrid chains.
CS/DS chains isolated from embryonic pig brains (E-CS/DS) pro-
mote the outgrowth of neurites in embryonic mouse hippocampal
neurons in culture by interacting with pleiotrophin (PTN), a hepa-
rin-binding growth factor. Here, we analyzed oligosaccharides iso-
lated from E-CS/DS, which showed that octasaccharides were the min-
imal size capable of interacting with PTN at a physiological salt
concentration. Five and eight sequences were purified from fluores-
cently labeled PTN-bound and -unbound octasaccharide fractions,
respectively, by enzymatic digestion followed by PTN-affinity chroma-
tography. Their sequences were determined by enzymatic digestion in
conjunction with high performance liquid chromatography, revealing
a critical role for oversulfated D and/or iD disaccharides in the low yet
significant affinity for PTN, which is required for neuritogenesis. The
critical D and iD units are GlcUA(2-O-sulfate)

1–3GalNAc(6-O-sul-
fate) and IdoUA(2-O-sulfate)
␣
1–3GalNAc(6-O-sulfate), respectively,
where IdoUA represents L-iduronic acid. In contrast, high affinity
interactions with PTN required decasaccharides with E units
(GlcUA

1–3GalNAc(4, 6-O-disulfate)), B units (GlcUA(2-O-sul-
fate)

1–3GalNAc(4-O-sulfate)), and/or their IdoUA-containing
counterparts (iE and iB) in addition to D/iD units, although the biolog-
ical significance of such strong interactions remains to be investigated.
Thus, chain size and composition are crucial to the interaction with
PTN, and PTN binds to multiple sequences in E-CS/DS chains with
distinct affinity. Notably, not only heparan sulfate but also CS/DS
hybrid chain structures of mammalian brains contain a high degree of
microheterogeneity with a cluster of oversulfated disaccharides and
appear to play roles in regulating the functions of PTN.
Chondroitin sulfate (CS)
4
and dermatan sulfate (DS), a class of glyco-
saminoglycan (GAG)-type polysaccharides, occur covalently attached
to proteoglycan (PG) core proteins (1). Like heparan sulfate (HS),
another class of GAGs, CS/DS chains are abundant at cell surfaces and
in the extracellular matrices. They regulate cell division, adhesion, and
morphogenesis through direct binding to secreted signaling proteins,
thereby modulating their activities, or through interactions with extra-
cellular matrix molecules (2–6, for reviews, see a Ref. 7). CS and DS
typically have backbones consisting of repeating disaccharide units of
-GlcUA-GalNAc- and -IdoUA-GalNAc-, respectively, where IdoUA
represents L-iduronic acid. Notably, hybrid chains, composed of both
units in varying proportions, also exist (8). These units are modified
during chain elongation by specific sulfotransferases at C-2 of GlcUA/
IdoUA and/or C-4 and/or C-6 of GalNAc in various combinations,
thereby producing characteristic sulfation patterns and enormous
structural diversity. These structural characteristics are strictly regu-
lated, as revealed by the compositional analysis of CS/DS chains from
various organs and by immunohistochemical staining using antibodies
recognizing different CS/DS epitopes.
In the mammalian brain, CS/DS have been implicated in neural
development by regulating neuronal adhesion and migration, neurite
formation, and axonal guidance etc. (9, 10). However, the reported func-
tions of brain CS/DS in neuritogenesis are controversial. CS/DS chains
are generally considered to play inhibitory roles in neurite extension and
axonal growth, a contention supported by studies in vivo showing that
removal of CS chains by chondroitinase ABC treatments permits axonal
regeneration after nigrostriatal tract axotomy and spinal cord injury
(11–13). On the other hand, DSD-1-PG, the mouse homologue of rat
phosphacan, which carries a functional domain (the so-called DSD-1
epitope) in the CS/DS side chains, promoted the outgrowth of neurites
toward embryonic rat hippocampal neurons in culture (14). Further
studies have shown that oversulfated CS and DS chains, and hybrid
CS/DS chains from various marine organisms exhibit neuritogenic
activity (Refs. 15–19, for a review, see Ref. 7). Importantly, we have
recently demonstrated that a unique iD (IdoUA(2-O-sulfate)-Gal-
NAc(6-O-sulfate))-containing epitope is spatiotemporally expressed in
particular regions of the developing mouse brain (20) and appears to be
involved in the formation of neurites in cultured embryonic mouse
*The work performed in Kobe was supported in part by HAITEKU (2004 –2008) from the
Japan Private School Promotion Foundation and Grants-in-Aid for Exploratory
Research 17659020, Scientific Research-B 16390026, and Scientific Research on Prior-
ity Areas 14082207 from the Ministry of Education, Culture, Sports, Science, and Tech-
nology of Japan. The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked “advertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
□
S
The on-line version of this article (available at http://www.jbc.org) contains supple-
mental Table SI.
1
Supported by postdoctoral fellowship HAITEKU (2004 –2008) from the Japan Private
School Promotion Foundation.
2
Present address: Dept. of Health Sciences, Faculty of Psychological and Physical Sci-
ences, Aichi Gakuin University, Nisshin 470-0915, Japan.
3
Supported by the Core Research for Evolutional Science and Technology of the Japan
Science and Technology Agency. To whom correspondence should be addressed:
Tel.: 81-78-441-7570; Fax: 81-78-441-7569; E-mail: k-sugar@kobepharma-u.ac.jp.
4
The abbreviations used are: CS, chondroitin sulfate; DS, dermatan sulfate; HS, heparan
sulfate; GAG, glycosaminoglycan; PG, proteoglycan; IdoUA, L-iduronic acid; 2S, 2-O-
sulfate; 4S, 4-O-sulfate; 6S, 6-O-sulfate; ⌬HexUA, 4-deoxy-L-threo-hex-4-enepyranosy-
luronic acid; E-CS/DS, embryonic pig brain-derived CS/DS chains; PTN, pleiotrophin;
FGF, fibroblast growth factor; 2AB, 2-aminobenzamide; MALDI-TOF-MS, matrix-as-
sisted laser desorption ionization time-of-flight mass spectrometry; CSase, chon-
droitinase; HPLC, high performance liquid chromatography; ⌷, GlcUA

1–3GalNAc;
〈, GlcUA

1–3GalNAc(4S); i〈, IdoUA
␣
1–3GalNAc(4S); 〉, GlcUA(2S)

1–3GalNAc(4S);
i〉, IdoUA(2S)
␣
1–3GalNAc(4S); C, GlcUA

1–3GalNAc(6S); iC, IdoUA
␣
1–3GalNAc(6S);
D, GlcUA(2S)

1–3GalNAc(6S); iD, IdoUA(2S)
␣
1–3GalNAc(6S); E, GlcUA

1–
3GalNAc(4S,6S); i⌭, IdoUA
␣
1–3GalNAc(4S,6S); ⌬Di-0S or ⌬O, ⌬HexUA
␣
1–3GalNAc;
⌬Di-6S or ⌬C, ⌬HexUA
␣
1–3GalNAc(6S); ⌬Di-4S or ⌬A, ⌬HexUA
␣
1–3GalNAc(4S); ⌬Di-
diS
D
or ⌬D, ⌬HexUA(2S)
␣
1–3GalNAc(6S); ⌬Di-diS
B
or ⌬B, ⌬HexUA(2S)
␣
1–
3GalNAc(4S); ⌬Di-diS
E
or ⌬E, ⌬HexUA
␣
1–3GalNAc(4S,6S); ⌬Di-TriS or ⌬T,
⌬HexUA(2S)
␣
1–3GalNAc(4S,6S); F5-ub, flow-through subfraction of F5; F5-b, bound
subfraction of F5.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 42, pp. 35318–35328, October 21, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
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hippocampal neurons. In that study, we used a monoclonal antibody,
2A12, which was raised against DS from ascidian. Thus, it is reasonable
to assume that the inhibitory and promoting activity of CS/DS chains is
probably defined by specific carbohydrate sequences with characteristic
sulfation patterns in a particular microenvironment.
Structural studies have demonstrated that the disaccharide compo-
sition and the GlcUA/IdoUA ratio of the brain CS/DS chains change
during development (21, 22), and that certain CS/DS epitopes are found
only in specific regions of the mammalian brain (23, 24). These findings
suggest that subpopulations of CS/DS chains play distinct roles during
development. Most interestingly, CS/DS hybrid chains (E-CS/DS) iso-
lated from embryonic pig brain were found to promote the outgrowth of
neurites toward embryonic mouse hippocampal neurons in culture
(22). Further investigation (25) has demonstrated that a small propor-
tion of E-CS/DS subpopulations bind a heparin-binding growth factor
pleiotrophin (PTN). These chains show neuritogenic activity by inter-
acting with endogenously expressed PTN and presenting it to the neu-
ronal cell surface, thereby facilitating the neuritogenic function of this
growth factor. Preliminary results (25), regarding the structure-func-
tion relationship, have suggested important roles for oversulfated di-
saccharides such as the D/iD unit (HexUA(2S)-IdoUA(6S)), the E/iE
unit (HexUA-GalNAc(4S,6S)) and IdoUA-containing structures in the
interaction of E-CS/DS with PTN, where HexUA, 2S, 4S and 6S repre-
sent hexuronic acid, and 2-O-, 4-O-, and 6-O-sulfate, respectively.
In the present study, we isolated and characterized a series of PTN-
bound and -unbound oligosaccharides from E-CS/DS with novel struc-
tures. The results demonstrated that PTN binds with distinct affinity to
multiple sequences in E-CS/DS chains, and that both the size and com-
position of sugar chains are crucial for the interaction. In addition, we
show the first direct evidence that CS/DS chains of the mammalian
brain are characterized by a high degree of microheterogeneity and
highly sulfated domains containing a cluster of oversulfated
disaccharides.
EXPERIMENTAL PROCEDURES
Materials—The E-CS/DS chains were purified from a phosphate-
buffered saline homogenate of embryonic pig brains as described pre-
viously (22, 25). Hyaluronidase SD from Streptococcus dysgalactiae,
chondroitinases ABC (conventional and protease-free preparations),
AC-I, AC-II, and B, and heparitinase were purchased from Seikagaku
Corp. (Tokyo, Japan). Recombinant human PTN for interaction assays
was obtained from RELIA Tech GmbH (Braunschweig, Germany).
⌬Hexuronate-2-O-sulfatase (abbreviated as ⌬hexuronate-2-sulfatase)
was a gift from K. Yoshida, Seikagaku Corp. A PTN-bound affinity col-
umn was prepared by coupling 0.5 mg of recombinant human PTN,
which was produced in yeast, to a HiTrap N-hydroxysuccinimide-acti-
vated column (1 ml) as reported previously (25). Prepacked PD-10 col-
umns and a Superdex
TM
Peptide HR column (10 ⫻300 mm) were
obtained from Amersham Biosciences. An amine-bound silica PA-03
column (4.6 ⫻250 mm) was purchased from YMC Co. (Kyoto, Japan).
All other chemicals and reagents were of the highest quality available.
Inhibition Assays Using a BIAcore System—The inhibitory activity of
sugar chains against the interaction of PTN with immobilized E-CS/DS
chains was examined using a BIAcore system (BIAcore AB, Uppsala,
Sweden). Biotinylated E-CS/DS chains were immobilized onto the
streptavidin-derivatized surface of a sensor chip as described (22). To
investigate the effects of treatments with GAG-degrading enzymes on
the inhibitory activity of E-CS/DS, equal amounts of E-CS/DS (1.35
g)
were individually incubated with chondroitinase ABC (5 international
milliunits), AC-I (2 international milliunits), or B (2 international milli-
units), hyaluronidase SD (2.5 international milliunits), or heparitinase (1
international milliunit), in an appropriate buffer at 37 °C for 1 h, and the
resulting digest was then mixed with 200 ng of PTN in a volume of 130
l and co-injected onto the surface of the sensor chip. The same amount
of PTN and the intact E-CS/DS was run as a control. Response curves
were recorded, and the maximal response of each reaction was used for
calculation. Inhibitory activity was expressed as a percentage relative to
the response obtained from an injection of PTN (200 ng) without sugar
chains.
In the case where oligosaccharides, generated from E-CS/DS, were
used as inhibitors, certain amounts of oligosaccharides (8–80 pmol)
were mixed with 50 ng of PTN and co-injected into the BIAcore system.
The same amount of PTN only was run as a control. The inhibitory
efficiency was calculated as a percentage relative to the response
obtained from the control.
Fragmentation of E-CS/DS—An exhaustive digestion with chon-
droitinase B was used to selectively dissect the E-CS/DS chains. The
digestion of E-CS/DS (100
g) was initiated with 50 international mil-
liunits of chondroitinase B in 100 mMTris-HCl buffer, pH 8.0 (26), in a
total volume of 30
l at 30 °C and run for 4 h. Thereafter the same
amount of the enzyme was added, and the incubation continued for
12 h. After boiling at 100 °C for 1 min to stop the reaction, the digest was
lyophilized and derivatized with a fluorophore 2-aminobenzamide
(2AB), and then the excess 2AB was removed by paper chromatography
(27). The 2AB derivatives were subjected to gel filtration on a column
(10 ⫻300 mm) of Superdex Peptide using 0.2 MNH
4
HCO
3
as an eluant
at a flow rate of 0.3 ml/min (22). Each resolved fraction as indicated in
Fig. 2Awas collected, rechromatographed under the same conditions,
and desalted by repeated lyophilization. Size determination of the com-
pounds in each fraction was achieved by comparison of the elution
positions with those of size-defined oligosaccharide standards derived
from CS-C or CS-D and by a mass spectrometric (MS) analysis as
described below.
Fractionation of E-CS/DS Oligosaccharides on a PTN Affinity
Column—The PTN affinity column (1 ml) contained 0.35 mg of the
protein. Before sample application, the column was washed with 3 ml of
10 mMTris-HCl buffer, pH 7.4 (Buffer A), containing 2.0 MNaCl, and
then equilibrated with 5 ml of 0.15 MNaCl-containing Buffer A as above.
2AB-derivatized oligosaccharide fractions were each dissolved in 250
l
of 0.15 MNaCl-containing Buffer A and applied to the PTN column. To
maximize the absorbance, loading was repeated six times by recycling
each unbound fraction. Oligosaccharides (typically 50 pmol) were sub-
jected to the affinity chromatography, and the column was washed step-
wise with 3 ml of Buffer A containing 0.15, 0.2, 0.3, 0.4, 0.5, 0.7, or 2.0 M
NaCl for analytical runs. For preparative purposes, up to 2500 pmol of
oligosaccharides were applied to the affinity column, which was then
washed with Buffer A containing 0.15 MNaCl and 0.7 MNaCl succes-
sively. The 0.15 MNaCl-eluted fraction was again subjected to affinity
fractionation under the same conditions as described above. The 0.15 M
NaCl-eluted subfraction obtained from the second fractionation was
considered the unbound subfraction, whereas the combined 0.7 M
NaCl-eluted subfraction was considered the bound subfraction of the
parent fraction.
For desalting and quantification, subfractions were subjected to gel
filtration on a Superdex Peptide column as described above. Calculation
of the relative abundance among subfractions of a certain fraction was
achieved by comparing the overall fluorescence intensity of the oligo-
saccharide peak(s) detected in each subfraction.
Anion-exchange Chromatography—Separation of the PTN-unbound
(400 pmol) and -bound (230 pmol) oligosaccharides in the chondroiti-
Pleiotrophin-binding Sulfated Oligosaccharides
OCTOBER 21, 2005•VOLUME 280•NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 35319
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nase B-resistant octasaccharide fraction, and analysis of the enzymatic
digests with bacterial chondroitinases were carried out by anion-ex-
change high performance liquid chromatography (HPLC) with an
amine-bound silica PA-03 column. The identification and quantifica-
tion of the unsaturated di- and tetrasaccharides generated by chon-
droitinases were achieved by making comparisons with the authentic
unsaturated di- and tetrasaccharides from CS and DS chains:
⌬HexUA
␣
1–3GalNAc (⌬Di-0S or ⌬O unit), ⌬HexUA
␣
1–3GalNAc-
(6S) (⌬Di-6S or ⌬C unit), ⌬HexUA
␣
1–3GalNAc(4S) (⌬Di-4S or
⌬A unit), ⌬HexUA(2S)
␣
1–3GalNAc(6S) (⌬Di-diS
D
or ⌬D unit),
⌬HexUA(2S)
␣
1–3GalNAc(4S) (⌬Di-diS
B
or ⌬B unit), ⌬HexUA
␣
1–
3GalNAc(4S,6S) (⌬Di-diS
E
or ⌬E unit), ⌬HexUA(2S)
␣
1–3GalNAc-
(4S,6S) (⌬Di-TriS or ⌬T unit) (28), ⌬HexUA
␣
1–3GalNAc(6S)

1–
4GlcUA

1–3GalNAc(6S) (⌬C-C) (29), ⌬HexUA
␣
1–3GalNAc(6S)

1–
4GlcUA

1–3GalNAc(4S) (⌬C-A) (29), ⌬HexUA
␣
1–3GalNAc(4S)-

1–4GlcUA

1–3GalNAc(6S) (⌬A-C) from a chondroitinase ABC di-
gest of 2AB-labeled ⌬HexUA
␣
1–3GalNAc(6S)

1–4GlcUA

1–3Gal-
NAc-(6S)

1–4GlcUA

1–3GalNAc(4S)

1–4GlcUA

1–3GalNAc(6S)
(⌬C-C-A-C),
5
⌬HexUA
␣
1–3GalNAc(4S)

1–4GlcUA

1–3GalNAc-
(4S) (⌬A-A) (29), ⌬HexUA(2S)
␣
1–3GalNAc(6S)

1–4GlcUA

1–
3GalNAc(6S) (⌬D-C) (29), ⌬HexUA(2S)
␣
1–3GalNAc(6S)

1–
4GlcUA

1–3GalNAc(4S) (⌬D-A) (29), ⌬HexUA
␣
1–3GalNAc(4S)

1–
4GlcUA(2S)

1–3GalNAc(6S) (⌬A-D) from a chondroitinase ABC
digest of 2AB-labeled ⌬HexUA
␣
1–3GalNAc(4S)

1–4GlcUA

1–
3GalNAc(4S)

1–4GlcUA(2S)

1–3GalNAc(6S) (⌬A-A-D) (30), and
⌬HexUA
␣
1–3GalNAc(4S,6S)

1–4GlcUA

1–3GalNAc(4S) (⌬E-A)
(30).
Delayed Extraction Matrix-assisted Laser Desorption Ionization
Time-of-flight Mass Spectrometry—Dried oligosaccharides (2–4 pmol
of individual oligosaccharides, and 10–20 pmol of mixed oligosaccha-
ride fractions) were first mixed with 1–2
l of (Arg-Gly)
15
(5–20 pmol)
and then 1
l of gentisic acid (1 mg/ml) (31, 32). Each mixture was
spotted on a plate for MS analysis using a mixture of (Arg-Gly)
15
and
gentisic acid as a control. The analysis was run in a positive mode using
the HCD1001 method according to the manufacturer’s instructions.
The MS spectra were recorded on a Voyager DE-RP-Pro (PerSeptive
Biosystems, Framingham, MA) using the linear mode.
Enzymatic Treatments—For the analysis of disaccharide composi-
tion, oligosaccharides (20–100 pmol as disaccharides) were incubated
with 5 international milliunits of chondroitinase ABC for 2 h and then
chondroitinase AC-II (1 international milliunit) for another h. The
resultant digests were derivatized with 2AB, and the excess 2AB was
removed by extraction with CHCl
3
(33). To identify di- and tetrasac-
charide structures at the reducing ends of the separated octasacchar-
ides, aliquots (0.5–2 pmol) of each 2AB-labeled oligosaccharide sub-
fraction were incubated with chondroitinase AC-II (1 international
milliunit) or ABC (a conventional preparation, 1 international milli-
unit), respectively. To characterize the disaccharides at the non-reduc-
ing ends of the isolated oligosaccharides, the PTN-unbound and -bound
subfractions (1–3 pmol) were subjected to digestions with a protease-
free preparation of chondroitinase ABC (1 international milliunit) for
15 min or with chondroitinase AC-II (1 international milliunit) for 60
min, respectively, followed by a derivatization with 2AB. In one case, the
disaccharide at the reducing end was examined by incubation of the
oligosaccharide subfraction (⬃2 pmol) with ⌬hexuronate-2-sulfatase (4
IU) as described (15, 34). All the enzymatic reactions were carried out
at 37 °C if not specified, and heating at 100 °C for 1 min was used to
terminate the reactions. Each digest was subjected to analyses by anion-
exchange HPLC and/or gel filtration as described above.
RESULTS
Chondroitinase B-resistant Structures Contain the Major PTN-bind-
ing Epitopes of E-CS/DS—The E-CS/DS chains had a disaccharide com-
position of ⌬O, ⌬C, ⌬A, ⌬D, ⌬B, and ⌬E at a molar percentage of
14.4:32.9:60.1:1.7:⬍0.1:0.9, as evaluated by chondroitinase ABC diges-
tion, and contained ⬃9% IdoUA-containing disaccharides, which were
cleavable by chondroitinase B and largely scattered along the chains (22,
25). Here, to characterize the epitopes in E-CS/DS for binding to PTN,
we first assayed the effects of various treatments with chondroitinases
or hyaluronidase on the inhibitory activity of E-CS/DS against the inter-
action between PTN and immobilized E-CS/DS in the BIAcore system.
As shown in Fig. 1, E-CS/DS at a concentration of 10
g/ml inhibited
75% of the interaction. However, treatment with chondroitinase ABC or
AC-I abolished the inhibitory activity of E-CS/DS almost completely,
indicating that both treatments destroyed most of the PTN-binding
epitopes in the parent polymer. In contrast, neither hyaluronidase SD
nor heparitinase treatment affected the inhibitory activity of E-CS/DS.
The results of the hyaluronidase digestion suggested that long and con-
secutive sequences of non-sulfated disaccharides are not the structural
elements in E-CS/DS critical for the binding to PTN. The results of the
heparitinase digestion confirmed that the E-CS/DS preparation used in
this study was free of contamination by HS. To our surprise, a chon-
droitinase B digest of E-CS/DS exhibited 65% of the inhibitory activity of
the intact E-CS/DS, indicating that the chondroitinase B-resistant
structures of E-CS/DS contained the majority of the PTN-binding
epitopes.
Gel filtration analysis of the digests of E-CS/DS obtained with chon-
droitinase B or hyaluronidase SD after labeling with 2AB revealed that
chondroitinase B digestion generated a series of oligosaccharides rang-
ing from di- to ⬎20-mer oligosaccharides (Fig. 2A). In contrast, the
hyaluronidase SD digest contained disaccharides and non-separable
5
S. S. Deepa, S. Fukui, and K. Sugahara, manuscript submitted.
FIGURE 1. Effects of enzymatic treatments on the inhibitory activity of E-CS/DS
against the binding of PTN to immobilized E-CS/DS. The E-CS/DS chains (1.35
g)
were incubated with chondroitinase (CSase) ABC (5 international milliunits), chondroiti-
nase AC-I (2 international milliunits), chondroitinase B (2 international milliunits), hyalu-
ronidase SD (2.5 international milliunits), or heparitinase (1 international milliunit), in an
appropriate buffer at 37 °C for 1 h, and each digest was mixed with PTN (200 ng) and
co-injected onto the surface of an E-CS/DS-immobilized sensor chip in a BIAcore system.
Co-injection of PTN (200 ng) and E-CS/DS (1.35
g) without enzymatic treatment was run
as a control. The inhibitory activity of each digest was calculated from the reduced max-
imal response relative to the value obtained from an injection of PTN (200 ng) without
sugar chains.
Pleiotrophin-binding Sulfated Oligosaccharides
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large oligosaccharides (⬎12-mer) in addition to small amounts of tetra-
and hexasaccharides (data not shown).
Isolation of Minimal Oligosaccharides for PTN Binding from a Chon-
droitinase B Digest of E-CS/DS—The parent E-CS/DS chains were dis-
sected using chondroitinase B for the isolation of PTN-binding struc-
tures, taking advantage of the specificity of the enzyme, which not only
retained the major PTN-binding structures in the digest but also pro-
vided structural information on the type of uronic acid (IdoUA) at the
cleavage site. E-CS/DS was exhaustively digested with chondroitinase B,
derivatized with 2AB, and fractionated by gel filtration. The effluent
fractions (F1 to F8) were collected as indicated in Fig. 2A, and the molec-
ular size of the major component in each fraction was determined by
MALDI-MS analysis.
To investigate which fraction contained the minimal structures
required for the binding to PTN, each fraction was subjected to affinity
chromatography on a PTN column, which was eluted with a salt gradi-
ent. Oligosaccharides in the effluent of each parent fraction (F3 to F8)
were determined based on their fluorescence intensity, which corre-
sponds to their relative molar percentage. As illustrated in Fig. 2B,
essentially all the fluorescent materials (2AB-derivatized oligosaccha-
rides) (99.6%) in F4 were detected in the flow-through subfraction (F3
also, data not shown), whereas small yet significant proportions (7.0–
16.5%) of the fluorescent materials were found in the subfractions (F5 to
F7) eluted with higher concentrations of salt (ⱖ0.2 MNaCl) (F8 also,
data not shown). The results indicated that an octasaccharide fraction,
F5, appeared to contain the minimal structures needed for binding to
immobilized PTN at a physiological concentration of salt.
The results from the affinity fractionation were verified by inhibition
assays. The flow-through subfraction (F5-ub) and bound subfraction
(F5-b) of F5, which were eluted with 0.15 and 0.7 MNaCl, respectively,
were used as inhibitors against the binding of PTN to immobilized
E-CS/DS. A smaller amount (8 pmol) of F5-b inhibited ⬃53% of the
interaction, whereas 80 pmol of F5-ub failed to show significant inhibi-
tion (Fig. 2C). These results confirmed that the high (0.7 M) and low
(0.15 M) salt-eluted subfractions of F5 from the PTN column enriched
the PTN-bound and -unbound oligosaccharides in the parent fraction,
respectively. Similar results were also observed for the bound and
unbound subfractions of F6 (data not shown). In addition, the good
separation of the PTN-bound and -unbound oligosaccharides by affin-
ity fractionation suggested that the 2AB tag at the reducing end of each
oligosaccharide had little influence on the interaction with PTN at a
physiological salt concentration.
Disaccharide compositions of the chondroitinase B-resistant frac-
tions and their subfractions obtained from the affinity fractionation are
summarized in TABLE ONE. Fractions F1 to F4, which did not bind to
the PTN column, contained non-, 6-O-, and/or 4-O-sulfated disaccha-
rides in different proportions, but no detectable oversulfated disaccha-
rides. In contrast, the bound subfractions of both F5 and F6 (F5-b and
F6-b) contained considerable amounts of oversulfated disaccharides
such as D/iD (26.9–20.4%) and E/iE (6.7–8.6%), whereas no or small
amounts (0 –2.6%) of such disaccharides were found in the correspond-
ing unbound subfractions. Notably, much higher (2.9- to 4.3-fold) pro-
portions of non-sulfated disaccharides were detected for the unbound
subfractions than the bound subfractions of F5, F6, and F7. Apparently,
these results revealed a strong correlation between the charge density
and disaccharide composition of oligosaccharides and their PTN-bind-
ing activity.
To investigate and compare the structures of PTN-unbound and
-bound oligosaccharides in detail, both F5-ub and F5-b were separated
by anion-exchange HPLC. Eight subfractions (F5-ub-a to -h) were
FIGURE 2. Chondroitinase B-resistant octasaccharides of E-CS/DS contain the mini-
mal structure for binding to a PTN-immobilized column. A, a gel filtration chromato-
gram of the chondroitinase B digest of E-CS/DS. E-CS/DS (100
g) was extensively
digested with chondroitinase B (100 international milliunits) and fractionated by gel
filtration on a column of Superdex peptide (10 ⫻300 mm) at a flow rate of 0.3 ml/min
using 0.2 MNH
4
HCO
3
as effluent. Fractions were collected as indicated. The elution posi-
tions of CS oligosaccharides from 2- to 18-mer are indicated at the top by arrows. The V
t
is around 78 min. B, affinity profiles of fractions, F4 to F7, on a PTN column. Each fraction
(⬃50 pmol) resolved by gel filtration shown in Awas subjected to affinity chromatogra-
phy on a PTN-Sepharose column, eluted stepwise with 10 mMTris-HCl buffer containing
0.15 (equilibrating buffer), 0.2, 0.3, 0.4, 0.5, 0.7, and 2.0 MNaCl. The affinity subfractions
were then subjected to gel filtration on a column of Superdex peptide, and the eluant
was monitored with a fluorescence detector. The fluorescence intensity of oligosaccha-
rides in each subfraction was used for calculation of the distribution of oligosaccharides
among subfractions of a parent fraction. The values represent means ⫾S.D. from three
independent experiments. The 0.15 MNaCl-eluted subfractions (flow-through fraction,
FL) and subfractions eluted with higher concentrations of salts (0.2– 0.5 MNaCl) were
considered to contain PTN-unbound and -bound oligosaccharides, respectively. Like-
wise, F3 and F8 were also analyzed (data not shown). Note that the octasaccharide frac-
tion F5 was the smallest fraction, which contained a significant proportion (7%) of PTN-
bound materials. C, inhibitory activity of PTN-unbound (F5-ub) and -bound (F5-b)
subfractions against the interaction of PTN with immobilized E-CS/DS. The parent frac-
tion F5 and its subfractions, F5-ub and F5-b, at the amounts indicated were mixed with PTN
(50 ng) and co-injected onto the surface of an E-CS/DS-immobilized chip in a BIAcore system.
The inhibitory activity was calculated from the reduced response relative to the value
obtained from an injection of PTN (50 ng). The values represent means ⫾S.D. from three
independent experiments. Note that a lesser amount (8 pmol) of the bound subfraction
(F5-b)ofF5 on a PTN column inhibited 53% of this interaction, whereas a greater amount (80
pmol) of the unbound subfraction (F5-ub)ofF5 showed no detectable inhibitory activity.
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resolved for F5-ub (Fig. 3A), whereas six main well separated bound
subfractions (F5-b-I to -VI) in addition to seven minor subfractions
(marked as F5-b-1 to -7) were isolated from F5-b (Fig. 3B). Almost all
oligosaccharides in F5-ub were eluted before 50 min, whereas the main
ones in F5-b came out after 50 min, suggesting that the PTN-bound
oligosaccharides had a higher charge density than the unbound coun-
terparts. Notably, the presence of the small peaks between 35 to 48 min
(peaks 1–5) in Fig. 3Bwas due to an insufficient removal of the unbound
compounds during the washing step in the preparative run as their
elution times were identical (Fig. 3B) to those of the peaks in Fig. 3A.
Structural Characterization of the Isolated PTN-unbound Octasac-
charide Subfractions F5-ub-a to F5-ub-h—The oligosaccharides, iso-
lated by anion-exchange HPLC, were characterized by MALDI-
TOF-MS analysis and enzymatic digestions in conjunction with HPLC.
The MS analysis indicated that three subfractions, F5-ub-a, -b, and -c,
contained trisulfated octasaccharides, and that the main components in
the other five subfractions, F5-ub-d, -e, -f, -g, and -h, were tetrasulfated
octasaccharides (supplementary Table IS). Each subfraction was sub-
jected to digestions with chondroitinase AC-II, chondroitinase ABC (a
conventional or protease-free preparation), or a mixture of chondroiti-
nases ABC (unless specified, chondroitinase ABC means a conventional
preparation), and AC-II as described under “Experimental Procedures.”
All components in these subfractions were digested completely by
chondroitinase AC-II, indicating that all the internal uronic acid resi-
dues of each component are GlcUA.
The results obtained from the enzymatic characterization of F5-ub-a
to -h are summarized in TABLE TWO. Except F5-ub-g, which con-
tained two components in comparable amounts, these subfractions
mainly contained a single predominant component. The enzymatic
analysis of subfraction F5-ub-h is described below as an example. A
sequential digestion with chondroitinases ABC and AC-II gave rise to
⌬C and ⌬A units in a molar ratio of ⬃1: 3 (Fig. 4A), suggesting that the
predominant component in F5-ub-h is composed of one C and three A
units. It is known that treatment with chondroitinase ABC of an oligo-
saccharide (ⱖ6-mer) with a 2AB tag at the reducing end results in an
unsaturated tetrasaccharide tagged with 2AB in addition to free disac-
charide(s), irrespective of the structure of the parent oligosaccharide
(27). As shown in Fig. 4B, chondroitinase ABC generated a predominant
fluorescent peak corresponding to ⌬A-A-2AB, suggesting that the pre-
dominant component in F5-ub-h has an A-A structure at the reducing
end. This was verified by the observation that ⌬A-2AB was the only
fluorescent peak detected in a chondroitinase AC-II digest (Fig. 4C)of
this subfraction, suggesting that the reducing end of the predominant
oligosaccharide in F5-ub-h is an A unit. Moreover, the disaccharide at
the non-reducing end was clarified by a partial digestion of F5-ub-h with
FIGURE 3. Separation of F5-ub and F5-b by anion-exchange chromatography. F5-ub
(⬃400 pmol) and F5-b (⬃230 pmol) were separated by anion-exchange HPLC on an
amine-bound silica PA-03 column using a linear gradient of NaH
2
PO
4
as indicated, at a
flow rate of 1 ml/min. Eight subfractions, F5-ub-a to F5-ub-h, were isolated from F5-ub (A),
whereas six main well separated subfractions (F5-b-I to -VI) in addition to seven minor
subfractions (marked as F5-b-1 to -7) were obtained from F5-b (B). Note that all the main
subfractions of F5-b were eluted later than F5-ub subfractions.
TABLE ONE
Disaccharide compositions of chondroitinase B-resistant oligosaccharide fractions of E-CS/DS
Each fraction derived from E-CS/DS was digested with chondroitinase ABC and chondroitinase AC-II, and the products were identified and quantified by
anion-exchange HPLC on an amine-bound silica column as described in “Experimental Procedures.”
Oligosaccharide fractions
a
Content
b
Disaccharides Sulfation degree
d
⌬O
c
⌬C⌬A⌬D⌬B⌬E⌬T
% mol%
F1 6.3 — — 100 —
e
— — — 1.00
F2 10.7 35.4 27.1 37.6 — — — — 0.65
F3 13.6 12.6 61.2 26.2 — — — — 0.87
F4 21.8 22.2 37.1 40.7 — — — — 0.78
F5-ub 17.5 25.7 26.1 48.2 — — — — 0.74
F5-b 1.3 8.9 24.3 33.2 26.9 — 6.7 — 1.25
F6-ub 10.0 20.4 33.9 43.0 1.9 — 0.7 — 0.82
F6-b 2.2 4.7 20.6 39.2 20.4 1.8 8.6 4.7 1.35
F7-ub 13.9 28.4 29.0 41.5 0.7 — 0.4 — 0.73
F7-b 2.2 8.2 31.5 51.0 6.0 — 3.3 — 1.01
F8 0.5 ND
f
ND ND ND ND ND ND NC
g
a
The names refer to the peaks and fractions designated in Fig. 2 (Aand B). “ub” and “b” denote the unbound and bound subfractions, respectively.
b
The relative percentage was calculated based on the fluorescence intensity of each fraction shown in Fig. 2 (Aand B).
c
For abbreviations including ⌬A(⌬Di-4S), ⌬B(⌬Di-diS
B
), ⌬C(⌬Di-6S), ⌬D(⌬Di-diS
D
), ⌬E(⌬Di-diS
E
), ⌬O(⌬Di-0S), and ⌬T(⌬Di-TriS) for unsaturated disaccharide units, see
“Experimental Procedures.”
d
Sulfation degree was calculated as the average number of sulfate groups/disaccharide unit.
e
“—”, not detected.
f
ND, not done due to the limited amounts obtained.
g
NC, not calculated.
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a protease-free preparation of chondroitinase ABC, taking advantage of
strong tendency of the protease-free preparation (15, 30) to exert an
exolytic effect when acting on oligosaccharides such as hexa- and
octasaccharides. Fig. 4Dshows that ⌬C was the major disaccharide
generated by a controlled digestion of F5-ub-h with chondroitinase
ABC (a protease-free preparation), suggesting that a ⌬C unit is located
at the non-reducing end of the predominant oligosaccharide of this
subfraction. Based on these results, it was concluded that the subfrac-
tion F5-ub-h contained a predominant component with the sequence
⌬C-A-A-A.
Structural analysis of all the other PTN-unbound octasaccharides
revealed that they are composed of different combinations of O, C,
and A but no di- or trisulfated disaccharide, consistent with the
results obtained from an analysis of the disaccharide composition of
F5-ub (TABLE ONE). None of these octasaccharides bound to the
PTN column at a physiological salt concentration, implying impor-
tant roles for oversulfated (di- or trisulfated) disaccharides in the
binding of oligosaccharides to PTN. Notably, except for ⌬C-C-C-C
(F5-ub-d) and ⌬C-C-A-C (F5-ub-f), which have been isolated from
shark cartilage CS-C
5
and CS-D (15) or CS-C,
5
respectively, all the
other sequences were isolated for the first time from natural CS/DS
chains. It is also noteworthy that these octasaccharides are derived
from the corresponding parent decasaccharide sequences flanked
by two IdoUA residues in E-CS/DS (TABLE TWO) and that iC
units were discovered in some of these sequences. Although sulfo-
transferase that can produce iC units have been detected in bovine
serum (35), iC units was never demonstrated previously in native
DS chains.
Structural Characterization of the Isolated PTN-bound Octasaccha-
ride Subfractions F5-b-I to F5-b-VI—A similar strategy was employed to
characterize the structures of the main subfractions (F5-b-I to -VI) of
F5-b, which were separated as shown in Fig. 3B. They were considered
to contain the smallest oligosaccharides, which could interact with
PTN. Among these six subfractions, five were determined by MS to
share a common octasaccharide core but with different numbers of
sulfate groups (supplementary Table IS). The MS analysis of F5-b-VI
was not successful probably due to its greater molecular size and higher
degree of sulfation as evidenced by the disaccharide composition anal-
ysis (TABLE THREE). Hence, an enzymatic analysis was carried out for
the other five subfractions (F5-b-I to -V), and the results are summa-
rized in TABLE THREE.
TABLE TWO
Enzymatic analyses of subfractions of the PTN-unbound octasaccharides
Each subfraction was subjected to digestion with chondroitinase (CSase) ABC (a conventional or protease-free preparation) and/or CSase AC-II, and the products
were identified and quantified by anion-exchange HPLC either before or after labeling with 2AB.
Subfractions Purity CSase ABC/AC-II followed
by 2AB labeling
a
CSase ABC CSase AC-II Non-
reducing
Sequences
end units
b
Parent
structures
d
mol% molar ratio
F5-ub-a 84.0 ⌬O⫹⌬C (1:3) ⌬C-C ⌬C⌬C⌬C-O-C-C
e
iC-O-C-C-iX
e
F5-ub-b 75.4 ⌬O⫹⌬C⫹⌬A (1:1:2) ⌬A-C ⌬C⌬A⌬A-O-A-C
e
iA-O-A-C-iX
e
F5-ub-c 94.8 ⌬O⫹⌬A (1:3) ⌬A-A ⌬A⌬A⌬A-O-A-A
e
iA-O-A-A-iX
e
F5-ub-d 86.5 ⌬C(⬃95%) ⌬C-C ⌬CND
c
⌬C-C-C-C iC-C-C-C-iX
e
F5-ub-e 85.8 ⌬C⫹⌬A (3:1) ⌬C-C ⌬C⌬A⌬A-C-C-C
e
iA-C-C-C-iX
e
F5-ub-f 93.0 ⌬C⫹⌬A (3:1) ⌬A-C ⌬C⌬C⌬C-C-A-C iC-C-A-C-iX
e
F5-ub-g 60, 40 ⌬C⫹⌬A (1:1) ⌬A-C ⫹⌬C-A
(3:2, mol/mol)
⌬C⫹⌬A
(1:1, mol/mol)
⌬A⌬A-C-A-C (60%)
e
iA-C-A-C-iX
e
⌬A-C-C-A (40%)
e
iA-C-C-A-iX
e
F5-ub-h 96.0 ⌬C⫹⌬A (1:3) ⌬A-A ⌬A⌬C⌬C-A-A-A
e
iC-A-A-A-iX
e
a
The unsaturated disaccharides were detected in the 2AB-derivatized digest of each subfraction with CSases ABC and AC-II. O, C, and A stand for GlcUA-GalNAc, GlcUA-
GalNAc(6S), and GlcUA-GalNAc(4S), respectively, whereas the symbol ⌬denotes the unsaturation of the GlcUA residue at the non-reducing end.
b
The non-reducing end units refer to the major disaccharide generated from each 2AB-tagged octasaccharide subfraction by a controlled treatment with a protease-free
preparation of chondroitinase ABC, which exhibits an exolytic action (15, 30), as shown in Fig. 4D.
c
ND, not done.
d
The “i” stands for an L-iduronate residue, and iX represents any disaccharides with an IdoUA residue including iA, iC, iD, iE, iB, or iT, except for iO (for the abbreviations, see
the “Experimental Procedures”).
e
Novel structures.
FIGURE 4. Chromatograms of the enzymatic characterization of F5-ub-h and F5-b-V.
Oligosaccharide fractions, typically 1–3 pmol, were digested with given enzymes and
analyzed by anion-exchange HPLC on a PA-03 column. In some cases, the enzymatic
digests were derivatized with a fluorophore 2AB before being applied to HPLC. Aand E,
F5-ub-h (A) and F5-b-V (E) were sequentially treated with chondroitinase ABC and chon-
droitinase AC-II, labeled with 2AB, and analyzed by HPLC. Band F, F5-ub-h (B) and F5-b-V
(F) were individually treated with chondroitinase ABC and analyzed by HPLC. Cand G,
F5-ub-h (C) and F5-b-V (G) were individually treated with chondroitinase AC-II, labeled
with or without 2AB, and analyzed by HPLC. D, F5-ub-h was partially digested with chon-
droitinase ABC (a protease-free preparation), labeled with 2AB, and analyzed by HPLC. H,
F5-b-V was digested with chondroitinase AC-II and labeled with 2AB, further digested
with chondroitinase ABC, and then labeled again with 2AB, and analyzed by HPLC. Peaks
eluted before 10 min are attributable to free 2AB and some unknown side reaction
products of the 2AB labeling. The peaks marked by asterisks were derived from impuri-
ties or the buffers used for the enzymatic treatments. The elution position of authentic
2AB-derivatized unsaturated di- and oligosaccharides are indicated by arrows.1,⌬Di-0S
(⌬O); 2,⌬Di-6S (⌬C); 3,⌬Di-4S (⌬A); 4,⌬Di-diS
D
(⌬D); 5,⌬Di-diS
E
(⌬E); 6,⌬Di-TriS (⌬T); 7,
⌬A-A; 8, the predominant component of F5-ub-h (⌬C-A-A-A, see “Results”); 9,⌬A-D; and
10, the hexasaccharide at the reducing end of the predominant component of F5-b-V
(⌬D-A-D or ⌬D-iA-D, see “Results”).
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A sequential digestion with chondroitinase ABC and chondroitinase
AC-II generated different sets of combinations of various disaccharides
containing ⌬A, ⌬C, ⌬D, and ⌬E units for each subfraction. The chro-
matogram for F5-b-V is shown in Fig. 4E. It should be noted that the
2AB-tagged ⌬A-D structure is resistant to treatment with chondroiti-
nase AC-II.
5
Similar results were obtained for other 2AB-tagged CS
tetrasaccharides with a D unit at the reducing end such as ⌬C-D
5
and D
unit-enriched CS hexasaccharides such as ⌬D-D-C.
6
Analysis of the
chondroitinase ABC digests revealed that the predominant compo-
nents of four subfractions (F5-b-I to -IV) contained a common tetrasac-
charide D-C or iD-C at their reducing ends, which resulted in ⌬D-C-
2AB, whereas that of F5-b-V was found to be A-D or iA-D, which
resulted in ⌬A-D-2AB (Fig. 4F). F5-b-I, -II, and -III were completely
degraded by chondroitinase AC-II, and all produced one predominant
peak corresponding to ⌬C-2AB. These findings indicate that all the
internal uronic acid residues of the predominant components of these
three subfractions are GlcUA, and that all these main components have
a C unit at their reducing ends. These results are consistent with those
obtained from the digestion with chondroitinase ABC.
In contrast, chondroitinase AC-II did not degrade F5-b-IV or -V into
di- or tetrasaccharides from the reducing ends (TABLE THREE). How-
ever, 2AB derivatization of the chondroitinase AC-II digest of F5-b-IV
or F5-b-V showed only one predominant disaccharide ⌬C and a hexa-
saccharide or ⌬E and a hexasaccharide (Fig. 4G), respectively. The
results suggested that the predominant components of F5-b-IV and -V
have ⌬C and ⌬E at their non-reducing ends, respectively. Moreover,
sequential digestion with chondroitinase AC-II and then chondroiti-
nase ABC of these two subfractions followed by labeling with 2AB gave
rise to ⌬C and ⌬D disaccharides for F5-b-IV, whereas the same treat-
ment of F5-b-V yielded ⌬D and ⌬E (Fig. 4H) in a molar ratio of ⬃1:1.
These results demonstrated that the tetrasaccharides at non-reducing
ends of the predominant compounds in F5-b-IV and -V have ⌬C-D and
⌬E-D structures, respectively.
In addition, F5-b-III was sensitive to treatment with ⌬hexuronate-2-
sulfatase (data not shown), which removes a sulfate group only from the
C-2 position of a ⌬HexUA located at the non-reducing terminus (34).
The results suggest that the predominant component of F5-b-III has a
⌬D unit at the non-reducing terminus, because this subfraction con-
tained only two kinds of disaccharide units, C and D.
Taking the results obtained from the enzymatic analyses and MS
together, it was concluded that the predominant components in F5-b-I
to -V have the following sequences: ⌬C-C-D-C (F5-b-I), ⌬A-C-D-C or
⌬C-A-D-C (F5-b-II), ⌬D-C-D-C (F5-b-III), ⌬C-D-D-C or ⌬C-D-iD-C
(F5-b-IV), and ⌬E-D-A-D or ⌬E-D-iA-D (F5-b-V) (the “i” in iA and iD
stands for IdoUA) (TABLE THREE).
Thus, all five of the oligosaccharides isolated from the PTN-bound
subfraction F5-b contained one or more disulfated disaccharides such
as D, iD, E, and iE, which is in marked contrast to the oligosaccharides
separated from the PTN-unbound (F5-ub) subfractions, where no dis-
ulfated disaccharide was detected. These D/iD-enriched oligosaccha-
rides were eluted before 0.3 MNaCl from the PTN column (Fig. 2B),
suggesting that these units played an important role in the low yet sig-
nificant affinity for CS/DS-PTN interaction. All the sequences identi-
fied here, except for the ⌬C-A-D-C structure (15), are novel. These
octasaccharides are derived from the corresponding parent decasaccha-
ride sequences flanked by two IdoUA residues in E-CS/DS (TABLE
THREE) as in the case of the PTN-unbound octasaccharides (TABLE
TWO). Notably, we have now verified iD units, whose existence in the
mouse cerebellum was previously suggested based on the immuno-
staining with the iD-specific monoclonal antibody, 2A12, raised against
ascidian DS (20).
Characterization of the PTN-bound and -Unbound Subfractions of
Decasaccharide Fraction F6—A significant proportion (16.5%) of F6 was
retained on the PTN column after a wash with the equilibration buffer:
more than that of F5 (7%) but comparable to that of F7 (15.1%) (Fig. 2B).
More significantly, the amount of materials eluted with high salt (0.4
and 0.5 MNaCl) was significantly larger in F6 (4.1%) than in F5 (less than
0.3%) or F7 (1.6%) (Fig. 2B). These results suggested that F6 contained
6
K. Kalayamamitra, P. Kongtamelert, and K. Sugahara, manuscript in preparation.
TABLE THREE
Enzymatic analyses of subfractions of the PTN-bound octasaccharides
Each subfraction was subjected to digestions with CSase ABC and/or AC-II, and the products were labeled with or without 2AB and then identified and quantified
by anion-exchange HPLC on an amine-bound silica column.
Subfractions Purity CSases ABC/AC-II followed
by 2AB labeling
a
CSase
ABC
CSase
AC-II
CSase AC-II/2AB
labeling
b
Sensitivity to
2-O-sulfatase Sequences Parent
structures
c
mol% mol ratio
F5-b-I 81.7 ⌬C⫹⌬D (2.6:1) ⌬D-C ⌬CND
d
ND ⌬C-C-D-C
e
iC-C-D-C-iX
e
F5-b-II 68.9 ⌬A⫹⌬C⫹⌬D (1:1.7:1.1) ⌬D-C ⌬CND ND⌬A-C-D-C
or ⌬C-A-D-C iA-C-D-C-iX
e
iC-A-D-C-iX
e
F5-b-III 79.1 ⌬C⫹⌬D (1:1.2) ⌬D-C ⌬CND ⫹
f
⌬D-C-D-C
e
iD-C-D-C-iX
e
F5-b-IV 75.4 ⌬C⫹⌬D (1.0:1.9) ⌬D-C —
g
⌬CND⌬C-D-D-C
e
or ⌬C-D-iD-C
e
iC-D-D-C-iX
e
iC-D-iD-C-iX
e
F5-b-V 82.7 ⌬D⫹⌬E⫹⌬A-D (1.2:1.0:1.0) ⌬A-D — ⌬END⌬E-D-A-D
e
or ⌬E-D-iA-D
e
iE-D-A-D-iX
e
iE-D-iA-D-iX
e
F5-b-VI 82.7 ⌬A⫹⌬T⫹⌬Tetra (3.0:1.0:1.8)
h
⌬Tetra
h
—ND ND ND ND
a
The unsaturated di- and tetrasaccharides were detected in the 2AB-derivatized digests of individual subfractions with CSases ABC and AC-II. C, A, D, E, and T stand for the
following disaccharide units: C, GlcUA-GalNAc(6S); A, GlcUA-GalNAc(4S); D, GlcUA(2S)-GalNAc(6S); E, GlcUA-GalNAc(4S,6S); T, GlcUA(2S)-GalNAc(4S,6S). The
symbol ⌬denotes the unsaturation of the GlcUA residue at the non-reducing end.
b
The predominant unsaturated disaccharides in the 2AB-derivatized CSase AC-II digest of each subfraction are shown.
c
For the definitions of “i” and “iX”, see footnote d to TABLE TWO.
d
ND, not determined because of the very limited amounts available.
e
Novel sequences.
f
Plus (⫹) indicates sensitivity to treatment with ⌬hexuronate-2-O-sulfatase.
g
No appreciable amount of unsaturated disaccharide was detected in a CSase AC-II digest of the corresponding subfraction.
h
⌬Tetra stands for an unidentified unsaturated tetrasaccharide.
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oligosaccharides with more optimal structures for binding to PTN.
Hence, to investigate these high affinity structures, the PTN-bound sub-
fraction (F6-b) was further characterized with a combination of enzy-
matic treatments in conjunction with anion-exchange HPLC, PTN-af-
finity chromatography, and MS analysis.
A large proportion (58%) of F6-b was resistant to chondroitinase
AC-II (marked by I,II, and III in panel a of Fig. 5A). In strong contrast,
almost all (⬎98%) of the PTN-unbound subfraction (F6-ub) was com-
pletely degraded into disaccharides by this enzyme (panel b in Fig. 5A),
suggesting a co-relation between the chondroitinase AC-II-resistant
structures and the PTN-binding activity. To investigate this structure-
function relation, a chondroitinase AC-II digest of F6-b was subjected to
affinity fractionation on the PTN column. A comparison of the elution
patterns of F6-b and its chondroitinase AC-II digest revealed that the
chondroitinase AC-II-resistant structures in F6-b constituted most of
the materials originally eluted in the high salt-eluted subfractions (0.4
and 0.5 MNaCl-eluted) of F6-b (Fig. 5B), suggesting that the compo-
nents, which strongly interacted with PTN, were abundant in the chon-
droitinase AC-II-resistant structures.
To investigate the structural features of the chondroitinase AC-II-
resistant components of F6-b, a chondroitinase AC-II digest of F6-b was
separated by gel filtration, and four subfractions (F6-b-I, -II, -III, and
-IV) were isolated as indicated in panel a of Fig. 5A. F6-b-I and Fb-b-II
contained mainly deca- and hexasaccharides, respectively, as assessed
by MALDI-TOF-MS analysis (Fig. 6, Aand B), whereas F6-b-III and -IV
consisted of trisulfated tetrasaccharides and monosulfated disacchar-
ides, respectively, as determined by anion-exchange HPLC (TABLE
FOUR). Subfractions of F6-b-I and -II were completely degraded into
di- and tetrasaccharides by treatment with a mixture of chondroitinases
ABC and AC-II (panel d of Figs. 5A,6C, and 6D). Molar ratios of the
resultant disaccharides and tetrasaccharides are summarized in TABLE
FOUR. Intriguingly, only 40% of F6-b-I was resistant to a mixture of
chondroitinases AC-I and AC-II (panel c in Fig. 5A), suggesting that
60% of F6-b-I, which corresponded to 21% of F6-b, was sensitive to
chondroitinase AC-I, and thus might contain IdoUA-containing struc-
tures (see “Discussion”).
Next, the interaction with PTN of these four subfractions resolved by
gel filtration was assessed by affinity chromatography using the PTN
column. Neither F6-b-II, -III, nor -IV bound to the column, although
F6-b-II contained a ⌬D-A-D or ⌬D-iA-D hexasaccharide as the pre-
dominant component (70% in molar percentage) and F6-b-III con-
tained a mixture of ⌬A-D and ⌬E-A tetrasaccharides (TABLE FOUR),
indicating that these hexa- and tetrasaccharides could not bind PTN at
a physiological salt concentration. In contrast, F6-b-I was the only sub-
fraction that bound to the PTN column, and therefore the affinity frac-
tionation pattern of F6-b must represent that of F6-b-I (Fig. 5B). Thus,
F6-b-I most likely contained the high affinity PTN-binding components
of F6-b. Comparison of the disaccharide compositions of F6-b-I
(TABLE FOUR) and F6-b (TABLE ONE) revealed that E or iE and B or
iB, but not D/iD units, in F6-b were selectively concentrated in F6-b-I,
suggesting that these units are involved in the high affinity interaction
between E-CS/DS and PTN.
DISCUSSION
It has been recently demonstrated that a subpopulation of the
E-CS/DS chains promotes the outgrowth of neurites through interac-
tion with PTN and that oversulfated disaccharides, such as D/iD and
E/iE units, in combination with IdoUA-containing structures are
required for the interaction of E-CS/DS with PTN (25). In the present
study, the major PTN-binding epitopes in E-CS/DS were identified. Our
results support the previous findings and provided further insight into
the oligosaccharide-binding specificity of PTN.
Contrary to the treatment with chondroitinase ABC or AC-I, diges-
tion with chondroitinase B reduced the PTN-binding activity of
E-CS/DS only to a small extent (Fig. 1), which allowed us to isolate the
major PTN-binding epitopes in E-CS/DS from a digest with chondroiti-
nase B. The smallest oligosaccharides in the chondroitinase B digest of
E-CS/DS, which bound to PTN at a physiological salt concentration,
were octasaccharides, although stronger interactions were observed
with decasaccharides or larger oligosaccharides. This size dependence is
reminiscent of the interactions of GAG oligosaccharides with other
heparin-binding growth factors such as fibroblast growth factor (FGF) 1
FIGURE 5. Characterization of the PTN-bound and -unbound subfractions of F6. A,
gel filtration chromatograms of the enzymatic digests of PTN-bound (F6-b) and -un-
bound (F6-ub) subfractions. F6-b was subjected to treatments with chondroitinase AC-II
(panel a), chondroitinases AC-II and AC-I (panel c), or a mixture of chondroitinases AC-II
and ABC (panel d), respectively, and then analyzed by gel filtration on a column of Super-
dex Peptide. The gel filtration pattern of the chondroitinase AC-II digest of F5-ub is
shown in bfor comparison. Sizes of resolved peaks were determined by MALDI-TOF-MS
or by HPLC, and are indicated by numbers of constituent monosaccharides: 2–10, di- to
decasaccharide. B, the affinity profiles of F6-b and its chondroitinase AC-II-resistant sub-
fraction on a PTN column. The affinity fractionation was performed as described in the
legend to Fig. 2B. The values for F6-b represent means ⫾S.D. from three independent
experiments, whereas the values for the CSase AC-II-resistant fraction were obtained
from a single experiment due to the limited amount. Note that the chondroitinase AC-II-
resistant subfraction of F6-b contained most of the materials of F6-b eluted with the high
concentrations of salt (0.3– 0.5 MNaCl).
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(36), FGF2 (36, 37), FGF4 (38), FGF7 (37), and hepatocyte growth factor
(39).
Comparison of the oligosaccharides isolated from the PTN-bound
(F5-b) and -unbound (F5-ub) octasaccharide subfractions revealed a
pivotal role for the oversulfated disaccharides, particularly the D or iD
unit, in the binding of E-CS/DS oligosaccharides to PTN with a low yet
significant affinity (Fig. 2Band TABLES TWO and THREE). This find-
ing is consistent with our previous results that a low affinity fraction of
E-CS/DS, eluted with 0.4 MNaCl from the PTN column, was rich in D
and/or iD units among the unbound (0.15 MNaCl-eluted), low affinity
(0.4 MNaCl-eluted), and high affinity fractions (1.0 MNaCl-eluted) of
E-CS/DS (25). This is also in agreement with a recent report (24) that
CS-D from shark cartilage binds PTN, and a small proportion of D
(1.3%) in the CS chains of phosphacan is required for effective binding to
PTN. Our results demonstrated for the first time that certain octasac-
charide sequences with at least one D or iD unit, derived from E-CS/DS,
are required for the binding to PTN at a physiological salt concentra-
tion. The failure to bind PTN of a hexasaccharide fraction (F6-b-II),
which contained the ⌬D-A-D and/or ⌬D-iA-D structure as a major
component (70%), suggested that not onlyaDoriDunit but size is
critical for E-CS/DS oligosaccharides to interact with PTN (TABLE
FOUR).
Previous findings (24, 25, 40) suggested that E- or iE-containing
structures have higher affinity for PTN than D- or iD-containing struc-
tures. In this study, the chondroitinase AC-II digest of PTN-bound
decasaccharides contained the high affinity components and was
enriched with E and/or iE and B and/or iB units, but not D or iD units,
from the PTN-bound decasaccharide subfraction (F6-b) (Fig. 5Band
TABLES ONE and FOUR). These findings support previous results and
suggest the involvement of these particular disulfated disaccharide units
in the high affinity interaction between E-CS/DS and PTN.
Chondroitinase AC-II cleaves exolytically almost all galactosaminidic
linkages bound to GlcUA from the non-reducing end but not those
bound to IdoUA in DS or CS/DS hybrid chains. Digestion of CS/DS
FIGURE 6. Characterization of F6-b-I and F6-b-II
by MALDI-TOF-MS and enzymatic digestion. A
and B, MALDI-TOF-MS spectra. Approximately 4
pmol of F6-b-I (A) or F6-b-II (B) was individually
mixed with (Arg-Gly)
15
and gentisic acid, analyzed
by MALDI-TOF-MS in a positive mode. The signals
at m/z 3213.5, 3529.1, 3843.5, 3213.8, and 3529.3
were from the (Arg-Gly)
15
preparation. Note that
the signals in Aat m/z 5805.1 and 5884.3 corre-
sponded to a decasaccharide with seven and eight
sulfate groups, respectively, whereas the signal in
Bat m/z 4885.7 corresponded to a hexasaccharide
with five sulfate groups. Cand D, anion-exchange
chromatograms. F6-b-I (C) and F6-b-II (D) were
individually digested with a mixture of chondroiti-
nases ABC and AC-II, labeled with 2AB, and ana-
lyzed by HPLC with an amine-bound silica PA-03
column. The elution positions of 2AB-derivatized
authentic unsaturated di- and oligosaccharides
are indicated by arrows.1,⌬O; 2,⌬C; 3,⌬A; 4,⌬D; 5,
⌬B; 6,⌬E; 7,⌬A-D; 8,⌬T; and 9,⌬E-A (for the abbre-
viations, see the legend to Fig. 4). The peaks
marked by asterisks were derived from the buffers
used for the enzymatic treatments, and those
marked by # symbols were from the column.
TABLE FOUR
Properties of the chondroitinase AC-II-resistant subfractions of PTN-bound decasaccharides
The PTN-bound decasaccharide fraction isolated from E-CS/DS was digested with chondroitinase AC-II and separated by gel filtration on a Superdex peptide
column into F6-b-I to -IV (Fig. 5A). The resolved peaks were subjected to analyses for molecular size, disaccharide composition and activity to bind a PTN-coupled
affinity column as described below.
Subfractions Size
a
CSases ABC/AC-II followed by 2AB labeling
b
Deduced major
component Binding activity
c
mol%
F6-b-I 10-mer 7S and 8S
d
⌬C⫹⌬A⫹⌬D⫹⌬B⫹⌬E⫹⌬A-D ⫹⌬T⫹
⌬E-A (22.8:26.2:14.1:4.6:11.3:6.5:3.4:11.1)
ND
e
⫹
F6-b-II 6-mer 5S ⌬C⫹⌬A⫹⌬D⫹⌬E⫹⌬A-D ⫹⌬T⫹⌬E-A
(4.7:9.9:31.4:8.3:27.5:10.6:7.6)
⌬D-A-D or ⌬D-iA-D (⬃70)
f
⫺
F6-b-III 4-mer 3S ⌬A-D ⫹⌬E-A (58.2:41.8) ⌬A-D (60) ⫹⌬E-A (40) ⫺
F6-b-IV 2-mer 1S ⌬C⫹⌬A (46.4:53.6) ⌬C (46) ⫹⌬A (54) ⫺
a
The sizes of F6-b-I and -II were assessed by MALDI-TOF-MS (see the legend to Fig. 6) and those of F6-b-III and -IV were determined by gel filtration by comparison with the
elution positions of structurally defined oligosaccharides. S, sulfate group.
b
F6-b-I and F6-b-II were digested with CSase ABC and CSase AC-II, followed by derivatization with 2AB, and then analyzed by anion-exchange HPLC to identify and quantify
unsaturated di- and tetrasaccharides. F6-b-III and -IV were directly analyzed by anion-exchange HPLC without enzymatic digestion. For the abbreviations C, A, D, B, E, T, and
⌬, see the legend to Table III.
c
Plus (⫹) and negative (⫺) indicate positive and negative binding to a PTN affinity column, respectively.
d
Minor components of 10 mer-6S or 9S were also detected.
e
ND, not determined.
f
The major digestion products ⌬D and ⌬A-D were most likely linked together to form these hexasaccharide sequences in F6-b-II.
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hybrid chains with this enzyme generates structures with a backbone of
⌬HexUA-GalNAc-IdoUA-GalNAc-(GlcUA/IdoUA-GalNAc)
n
(nⱖ0)
(30, 41). In this study, the chondroitinase AC-II-resistant components
(F6-b-I) were large (decasaccharides) and had a moderate proportion
(18%) of D and/or iD units. The results suggest that these compounds
might contain IdoUA-containing disaccharides at the penultimate posi-
tion from their non-reducing ends, although this enzyme has limited
ability to digest small CS oligosaccharides (hexa- or tetrasaccharides)
that contain a cluster of D units.
6
This assumption was supported by the
observation that a large portion (60%) of this chondroitinase AC-II-
resistant PTN high affinity subfraction (F6-b-I) was cleavable by chon-
droitinase AC-I (Fig. 5A). This observation suggested that the resistance
to chondroitinase AC-II was not due to the clustering of D units,
because chondroitinase AC-I has no activity to cleave D unit-containing
structures (41). Hence, it is reasonable to assume that the chondroiti-
nase AC-I-sensitive compounds of the chondroitinase AC-II-resistant
decasaccharides shown in Fig. 5Acontained unique IdoUA-containing
disaccharides, which were resistant to chondroitinase B as well.
Chondroitinase B is the only enzyme currently available to cleave the
galactosaminidic linkages bound to IdoUA in DS or CS/DS hybrid
chains, although its substrate specificity is not yet fully understood. This
enzyme is known to efficiently generate ⌬A units from various DS prep-
arations but does not work on non-sulfated dermatan (41). Although
commercial porcine skin CS-B (or DS) results in ⌬C, ⌬A, and ⌬Bina
molar ratio of 5.6:88:6.4 upon chondroitinase ABC digestion, sequential
digestions with chondroitinases AC-I and AC-II, and then chondroiti-
nase B produced tetrasaccharides (8%) and disaccharides (92%) (data
not shown), suggesting that chondroitinase B cleaves most, but not all,
the galactosaminidic bonds linked to IdoUA in DS or CS/DS hybrid
chains. In addition, it has been reported that chondroitinase B generates
⌬E unit from hagfish notochord CS-H, which is rich in E/iE and T/iT
units (18, 42), and ⌬B unit from DS of bovine aorta (41) and ascidian
Halocynthia roretzi, which is rich in iB units,
7
respectively. These find-
ings indicate that the galactosaminidic bonds linked to iE and iB units
were sensitive to this enzyme. In contrast, DS from ascidian Ascidia
nigra, which contains iD units and iC units as a major (⬃80%) and a
minor (⬃20%) component, respectively, was totally resistant to chon-
droitinase B (20). Nevertheless, the sequences obtained in this study
clearly indicate that chondroitinase B cleaved E-CS/DS chains at the
non-reducing sides of some, if not all, iC and iD units as well as iA and iE
units (TABLES TWO and THREE). These results together indicate that
chondroitinase B does not cleave all galactosaminidic linkages bound to
IdoUA in DS or CS/DS hybrid chains, but the size and sulfate pattern of
the substrates could be important for the recognition and digestibility
by this enzyme. This may explain why the chondroitinase B-resistant
oligosaccharides obtained in this study still appeared to contain some
IdoUA-containing structures, which were involved in the high affinity
interaction between E-CS/DS oligosaccharide and PTN. The sequences
of such unique oligosaccharide structures remain to be determined.
Oversulfated disaccharides, such as D/iD, B/iB, and E/iE, have been
implicated in the development of the brain (7, 17, 28). However, the
analytical discrimination of D, B, and E units from their iD, iB, and iE
counterparts in CS/DS chains remains difficult but necessary for a full
understanding of the structure-function relationship of the CS/DS
chains. Our recent study (20) demonstrated the presence of functional
iD-containing domains in the developing mouse brain using a mono-
clonal antibody, 2A12, against the DS from ascidian. This has been
confirmed in this study by the structural determination of F5-b-III,
whose predominant component has a ⌬D unit at the non-reducing end.
The ⌬D unit should be derived from an iD-containing structure in the
parent E-CS/DS polymers in view of the specificity of chondroitinase B
used for fragmentation of E-CS/DS. Similarly, the isolation of the struc-
ture ⌬E-D-A-D or ⌬E-D-iA-D (F5-b-V) from E-CS/DS indicated the
presence of iE unit in the mammalian brain. Interestingly, among the
five identified oligosaccharides isolated from the PTN-bound octasac-
charides, three contained two or more D/iD and/or E/iE units. This
finding indicates that these rare oversulfated disaccharides (⬃3%) in
E-CS/DS are not distributed randomly but tend to form clusters, which
appear to be involved in the binding of E-CS/DS to PTN with high
affinity and may be involved also in the binding of other growth factors.
In addition, the high affinity subfraction (F6-b-I) contained a mixture of
at least four decasaccharide sequences with six to nine sulfate groups
(TABLE FOUR and Fig. 6A), suggesting that these decasaccharides con-
tained one to four disulfated disaccharides in addition to monosulfated
disaccharides.
The results altogether demonstrate that the E-CS/DS chains possess
multiple minimal sequences capable of binding PTN, as reported also
for the binding of HS chains to FGF1, FGF2, FGF4, FGF7, and FGF8b
(36, 38). The presence of multiple structurally distinct sequences with
different affinity for a given growth factor or heparin-binding protein
may be a general tendency, which is applicable not only to HS but also to
CS/DS chains. The multiple PTN-binding sequences may be relevant in
the sliding of PTN along a long CS/DS chain from a lower affinity to a
higher affinity binding site as has been hypothesized for the binding of
heparin-binding growth factors to multiple binding sequences on an HS
chain (43). In addition, jumping of PTN from a lower affinity binding
site of a CS/DS or HS chain to a higher affinity binding site of another
CS/DS or HS chain may also be possible as has been suggested for
HS-binding growth factors and HS chains (43).
Acknowledgment—We thank Dr. Shuhei Yamada for the kind help in prepar-
ing the brain glycosaminoglycans.
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Pleiotrophin-binding Sulfated Oligosaccharides
35328 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280•NUMBER 42•OCTOBER 21, 2005
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Kazuyuki Sugahara
Xingfeng Bao, Takashi Muramatsu and
Hybrid Chains of Embryonic Pig Brains
Chondroitin Sulfate/Dermatan Sulfate
Oligosaccharide Sequences Isolated from
Demonstration of the Pleiotrophin-binding
Glycobiology and Extracellular Matrices:
doi: 10.1074/jbc.M507304200 originally published online August 23, 2005
2005, 280:35318-35328.J. Biol. Chem.
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