Glycobiology vol. 7 no 8 pp. 1175-1180, 1997
Structural variations in the glycosaminoglycan-protein linkage region of recombinant
decorin expressed in Chinese hamster ovary cells
Hiroshi Kitagawa, Mika Oyama, Kimiko Masayama, Yu
Yamaguchi1 and Kazuyuki Sugahara2
Department of Biochemistry, Kobe Pharmaceutical University ^
Higashinada-ku, Kobe 658, Japan and 'The Bumham Institute, La Jolla, CA
92037, USA
^To whom correspondence should be addressed at: Department of
Biochemistry, Kobe Pharmaceutical University, 4-19-1
Motoyamakita-machi, Higashinada-ku, Kobe 658, Japan
Decorin is a small fibroblast proteoglycan consisting of a
core protein and a single chondroitin/dennatan sulfate
chain. The structure of the carbohydrate-protein linkage
region of the recombinant decorin expressed in Chinese
hamster ovary cells was investigated. The decorin was secreted in the culture medium and isolated by anionexchange chromatography. The glycosaminoglycan chain
was released from the decorin by P-elimination using alkaline NaBH4, and then digested with chondroitinase ABC.
These treatments resulted in a major and a few minor
hexasaccharide alditols derived from the carbohydrateprotein linkage region. Their structures were analyzed by
enzymatic digestion in conjunction with high-performance
liquid chromatography. Two of these compounds have the
conventional hexasaccharide core, AHexAotl-3GalNAcpi4GlcApi-3Galpl-3Gaipi-4Xyl-ol. One is nonsulfated,
and the other is monosulfated on C4 of the GalNAc residue.
They represent 12% and 60% of the total linkage region,
respectively. The other compound has the hexasaccharide
alditol with an internal iduronic acid residue AHexAal3GalNAc(4-sulfate)pi-4IdoAal-3Gaipi-3Gaipi^4Xyl-ol,
which was previously demonstrated in one of the five linkage hexasaccharide alditols isolated from dennatan sulfate
proteoglycans of bovine aorta (Sugahara et aL, J. BioL
Chenu, 270, 7204-7212,1995). The compound accounts for
11% of the total linkage region. These structural variations
in the linkage hexasaccharide region of the decorin strikingly contrast to the uniformity demonstrated in the linkage hexasaccharide structure of human inter-a-trypsin inhibitor (Yamada et aL, Glycobiology, 5, 335-341,1995) and
urinary trypsin inhibitor (Yamada et aL, Eur. J. Biochem.,
233, 687-693, 1995), both of which have a single chondroitin sulfate chain with a uniform linkage hexasaccharide
structure, AHexAal-3GalNAc(4-sulfate)pl-4GlcAp 13Gal(4-sulfate)pl-3Gaipi-4Xyl, containing a 4-0-sulfated
Gal residue.
Key words: chondroitin sulfate/decorin/dermatan sulfate/
glycosaminoglycan/proteoglycan
Introduction
Decorin, a small chondroitin/dermatan sulfate proteoglycan, is
a ubiquitous component of the extracellular matrix of many
C Oxford University Press
tissues, including the adventitia of blood vessel walls, the dermis of the skin, tendon, sclera, articular cartilage, and other
connective tissues (for reviews, see Ruoslahti, 1988; Kresse et
al., 1993; and Iozzo and Murdoch, 1996). The decorin consists
of a core protein of about 40 kDa with a single covalently
attached glycosaminoglycan (GAG) chain (Ruoslahti, 1988).
Most of the biological functions ascribed to decorin are believed to form complex with other molecules via the GAG or
core protein moieties. For example, decorin has been demonstrated to bind to fibrillar collagens including type L, type n,
and type VI and inhibit fibrillogenesis (Iozzo and Murdoch,
1996). Decorin has also been shown to bind to a variety of
other proteins, including fibronectin, transforming growth factor- p, as well as a high affinity cell surface receptor found on
human osteosarcoma cells and fibroblasts, which mediates endocytosis of the proteoglycan (Iozzo and Murdoch, 1996).
The GAG chain of decorin is either chondroitin sulfate or
dermatan sulfate, depending on the tissue source. Chondroitin/
dermatan sulfate has a linear polymer structure that possesses
repetitive, sulfated disaccharide units containing hexuronic
acid (HexA) and A^-acetlygalactosamine (GalNAc) (Rode"n,
1980; Hascall and Hascall, 1981). Sulfated GAGs including
chondroitin/dermatan sulfate and heparin/heparan sulfate are
covalently bound to Ser residues in the core proteins through
the common carbohydrate-protein linkage structure, GlcApi3Gal31-3Gaipi^Xyipi-O-Ser (for reviews, see Rode"n,
1980, and Hascall and Hascall, 1981). Chondroitin/dermatan
sulfate is synthesized once GalNAc is transferred to the common linkage region, while heparin/heparan sulfate is formed if
GlcNAc is first added. The two distinct transferases, which
catalyze the transfer of GalNAc or GlcNAc, respectively, to the
common linkage region, are the key enzymes in determining
the type of GAGs to be synthesized. However, molecular
mechanisms are unknown for the synthesis of different GAGs
on the common linkage region.
We have been analyzing die structure of the carbohydrateprotein linkage region of various sulfated GAGs to investigate
the structure—function relationship and the biosynthetic mechanisms of these GAGs. These structural studies revealed that
sulfation of C6 on both Gal residues and C4 of Gal adjacent to
GlcA were characteristic of chondroitin sulfate (Sugahara et
al., 1988, 1991, 1992a; de Waard et al, 1992). In particular,
uniform sulfation of C4 of one of the Gal residues was observed in the linkage region of chondroitin sulfate isolated
from human inter-a-trypsin inhibitor (Yamada et aL, 1995a)
and urinary trypsin inhibitor (Yamada et al., 1995b). Sulfation
of C4 of the Gal residue was also demonstrated in the linkage
region of bovine aorta dermatan sulfate (Sugahara et al.,
1995a). Sulfated Gal residues have not been found in the linkage region of heparin or heparan sulfate (Sugahara et al.,
1992b, 1994, 1995b). In this study, we isolated and characterized the carbohydrate-protein linkage region of recombinant
decorin consisting of a single GAG chain to examine possible
1175
H.KJtagawa et al
structural similarity and/or variability as compared with this
region especially from human inter-ot-trypsin inhibitor and urinary trypsin inhibitor, both of which bear a single chondroitin
sulfate chain with the uniform linkage region structure.
Results
3
Isolation of recombinant decorin containing the H-labeled
GAG chain
Decorin-expressing CHO cells were metabolically labeled with
[3H]galactose, and the culture medium was fractionated on
DEAE-Sepharose (Figure 1) since nearly all of the decorin
produced by the cells was shown to be secreted in the culture
medium (Yamaguchi and Ruoslahti, 1988). The radiolabeled
fraction eluted with 0.45-0.55 M NaCl (indicated by the bar in
Figure 1) contained recombinant decorin as described previously (Yamaguchi et aL, 1990) and was pooled. The fraction
was depolymerized in part by chondroitinase B and completely
by subsequent chondroitinase AC-II digestion as examined by
gel permeation chromatography (data not shown), indicating
that the GAG chain carried at least some IdoA residues and
hence can be classified as dermatan sulfate. Disaccharide
analysis of the chondroitinase ABC digest of the fraction by
HPLC showed predominantly the ADi-4S component as expected for the presence of dermatan sulfate (Table I). A significant amount of ADi-diSB, which may be associated with an
anticoagulant activity (Maimone and Tollefsen, 1990), was
also identified.
Isolation of the linkage hexasaccharides
The decorin-containing fraction was then treated with alkaline
NaBH 4 to release the GAG chain from the core protein. The
3
H-labeled reduced fractions were isolated by gel filtration on
Sephadex G-50, pooled, and digested exhaustively with chon-
0
10
20
30
40
50
60
Table L Disaccharide composition of the GAG chain of
recombinant decorin
Component
Distribution (%)
ADi-OS
8.1
88.3
ND*
ND
3.6
ND
ADi-6S
ADi-diSD
ADi-diSB
ADi-triS
Disaccharide composition was determined by chondroitinase ABC digestion
followed by HPLC analysis as described (Sugahara et al., 1989). The
distribution of 'H-radioactivity is expressed as a percentage of the total
radioactivity co-eluting with disaccharide standards.
"ND, not detected.
droitinase ABC to obtain the linkage hexasaccharide fraction
by degrading the repeating disaccharide region. The oligosaccharide fraction expected to contain the linkage hexasaccharides (indicated by the bar in Figure 2) was separated from
disaccharides by gel filtration on a Superdex 30 column. No
absorbance at 232 nm in the putative hexasaccharide fraction
was due to the small quantity.
Characterization of the linkage hexasaccharides
The 3H-labeled linkage hexasaccharide fraction was analyzed
by HPLC on an amine-bound silica column before and after
chondro-4-sulfatase digestion as described (Yamada et al.,
1995a). Before digestion, a single major peak (Fraction II)
and two minor peaks (Fractions I and HI) were observed.
The major peak was detected at the elution position of the authentic monosulfated hexasaccharide alditol, AHexAal3GalNAc(4S)Bl-*GlcApl-3GalBl-3Ga^l^Xyl-ol (Fraction C derived from whale cartilage chondroitin 4-sulfate
(Sugahara et al., 1991; Figure 3A)). This peak accounted for
71% of the applied radioactivity. Fraction I was detected at the
elution position of the authentic nonsulfated hexasaccharide
alditol, AHexAal-3GalNAc0 l-4GlcAB l-3Galpl-3GalB 1-
I
FRACTION NUMBER
Fig. 1. Purification of recombinant decorin expressed in CHO cells by
FPLC on a DEAE-Sepharose column. The decorin-expressing CHO cells
were labeled metabolically with [l- 3 H]galactose, and the labeled
conditioned medium was fractionated by DEAE-Sepharose chromatography
as described in Materials and methods. The fraction size was 2 ml, and 1
(jj aliquots were used for determination of 3 H radioactivity. The
radiolabeled fraction eluted with 0.45-0.55 M NaCl, indicated by the bar,
contained recombinant decorin as described previously (Yamaguchi et al..
1990). This fraction was pooled and used for preparation of
carbohydrate-protein linkage region oligosaccharides. The flow-through
fractions with radioactivity primarily contained [3rT|galactose (data not
shown).
1176
50
60
70
80
FRACTION NUMBER
Fig. 2. Gel filtration of die chondroitinase ABC digest of the 3H-labeled
GAG chain from recombinant decorin. The chondroitinase ABC digest of
the 3H-labeled GAG chain was chromatographed on a column of Superdex
30 (1.6 x 60 cm) with 0.25 M NH 4 HCO/7% 1-propanol as eluent.
Fractions of 2 ml were collected arid monitored by absorbance at 232 nm
(dashed line) and 3H-radioactivity (solid line). The radiolabeled fraction
containing the linkage hexasaccharides, indicated by the bar, was pooled
and subjected to enzymatic analysis as described in Materials and methods.
Vo was at around fraction 28 (not shown).
Glycosamlnoglycan structure of decorin
A
BC
10
0.6
0.4
0.2
-4+
0.0
B
0.6
lli
m 10
0.4
0-2
X
I»
0.0
<£
c
h 0.6 _
0.4
o
5
|
g
5
CO
0.0
10
0.6
0.4
1
c c ...--"
" 0.2
HI
0.0
10
20
30
40
TIME (min)
50
60
Fig. 3. HPLC analysis of the chondroitinase ABC digest of the 3H-labeled
GAG chain from recombinant decorin. The oligosaccharide alditols (2.2 x
104 d.p.m ) were obtained from the linkage region by chondroitinase ABC
digestion, and analyzed by HPLC on an amine-bound silica column before
(A) and after chondro-4-sulfatase (B), alkaline phosphatase ( O , and
chondroitinase AC-II (D) digestions as described in Materials and methods.
The eluates were collected at 1 min intervals for radioactivity measurement
by liquid scintillation counting. Arrows indicate the elution positions of the
authentic hexa- and tetrasaccharide alditols and unsaturated
chondro-disaccharides: A, AHexAal-3GalNAcpi^H31cA31-3Gal313Gaipi-4Xyl-ol; B, AHexAal-3GalNAc(6S)pi-4GlcApi-3Gaipil; C, AHexAal-3GalNAc(4S)pi^*GlcApl-3Gaipil; C , AHexAal-3GalNAc(4S)31^HdoAal-3GaI31l; D, AHexAal-3GalNAc(4S)pi^K31cA31-3Gal(4S)pi3GalBl^Xyl-ol; and Tetra, AHexAal-3Galf$l-3Gaipi^tXyl-ol. An
asterisk (see D) indicates an unidentified substance due to the absence of a
co-eluting authentic standard, and the peak most likely resulted from the
chondroitinase AC-II digestion of the peak observed at around 35 min in
(A).
3GalNAc(4S)pi-4GlcA31-3Gal(4S)pi-3Gal31^Xyl-ol
(Fraction D derived from whale cartilage chondroitin 4-sulfate;
Sugahara et al., 1991; Figure 3A), suggesting that the fraction
contained a novel hexasaccharide alditol. The two minor peaks
(Fractions I and IE) accounted for 12% and 17% of the applied
radioactivity, respectively. After chondro-4-sulfatase digestion, Fractions II and HI were shifted to the position of the
authentic A H e x A a l - 3 G a l N A c p i - 4 G l c A p i - 3 G a i p i 3Gaipi^lXyl-ol (Figure 3B and Table II). These results suggest that Fractions II and HI contained at least one 4-O-sulfated
residue. However, since considerable phosphatase activity was
detected in this chondro-4-sulfatase preparation (data not
shown), Fractions II and in might have contained phosphorylated residue(s). Indeed, the linkage hexasaccharide
AHexAal-3GalNAcpi-4GlcApi-3Gaipi-3Gaipi^Xylol(2-O-phosphate) (Sugahara et al., 1992a) was dephosphorylated by this enzyme preparation under the standard incubation
conditions as described in Materials and methods. Hence, alkaline phosphatase digest of the 3H-labeled linkage hexasaccharide fraction was analyzed in the same HPLC system, revealing that only Fraction El was shifted on HPLC by 6 min
upon the alkaline phosphatase digestion (Figure 3C), which
corresponds to a loss of one phosphate group. These findings
altogether indicate that Fraction III contained 1 mol each of a
phosphate and a 4-O-sulfated residue, while Fraction II had
only one 4-O-sulfated residue.
The chondroitinase AC-II digest of the 3H-labeled linkage
hexasaccharide fraction was also analyzed in the same HPLC
system. Chondroitinase AC-EI degrades a linkage hexasaccharide into a disaccharide unit and a tetrasaccharide core (Sugahara et al., 1991) except for the iduronic acid—containing linkage hexasaccharides like AHexAal-3GalNAc(4S)pi4 I d o A a l - 3 G a i p i - 3 G a i p i - 4 X y l - o l and A H e x A a l 3GalNAc(4S)P l-4IdoAa l-3Gal(4S)P l-3Gal£ l-4Xyl-ol
(Sugahara et al., 1995a). HPLC analysis of the chondroitinase
AC-II digest gave rise to a single major peak emerging at the
position of authentic AHexAal-3Gal(31-3Galpl^lXyl-ol or
AHexAal-3GalNAc (ADi-OS) and three minor peaks at the
elution positions of AHexAal-3GalNAc(4S) (ADi-4S), authentic AHexActl-3GalNAc(4S)31-4IdoAal-3Gaipi3Gaipi-4Xyl-ol and intact Fraction m (Figure 3D). In addi-
Table II. Enzymatic analysis in conjunction with HPLC of the 3H-labeled
linkage hexasaccharide fraction from recombinant decorin
Fraction
I
II
Enzymes
Reaction products*
Chondroitinase AC-II
Cbondroitinase B
Chondro-4-sulfatase
Chondroitinase AC-II
AHA-G-G-X and ADi-OS
AHA-GN-UA-G-G-X
AHA-GN-UA-G-G-X
AHA-GN(4S)-IA-G-G-X, AHA-G-G-X
and ADi-4S
AHA-GN(4S)-IA-G-G-X and
AHA-GN(4S)-UA-G-G-X
AHA-GN-IA-G-G-X and
AHA-GN-UA-G-G-X
Unidentified hexasaccharide
alditol (Fraction QT)
Unidentified hexasaccharide
alditol (Fraction m)
Chondroitinase B
Chondro-4-sulfatase
ID
Chondroitinase AC-H
Cbondroitinase B
4Xyl-ol (Fraction A derived from whale cartilage chondroitin
4-sulfate; Sugahara et al., 1991; Figure 3A). Fraction HI was
detected at the elution position between AHexAotl3GalNAc(4S)pi^U31cApi-3Ga]pi-3Gaipi^lXyl-ol and the
disulfated hexasaccharide alditol, AHexAotl-
The 3H-labeled linkage hexasaccharide fraction was digested with
chondroitinase AC-H or B or chondro-4-sulfatase and the digest was
analyzed by HPLC as described in Materials and methods.
'HA, UA, IA, GN, G, X and 4S stand for HexA, GlcA, IdoA, GalNAc, Gal,
Xyl-ol and 4-O-suIfate, respectively.
1177
H.Kitagawa et al
tion, all the hexasaccharide fractions (Fractions I-HI) were
resistant to chondroitinase B digestion (data not shown), suggesting that these fractions do not contain the linkage hexasaccharide AHexAa l-3GalNAc(4S)pi-4IdoAal-3Gal(4S)pi-3Gaipi-4Xyl-ol demonstrated previously in dermatan
sulfate from bovine aorta (Sugahara et al, 1995a). This hexasaccharide is cleaved by the enzyme whereas another iduronic
acid-containing monosulfated hexasaccharide AHexAa 13GalNAc(4S)pi-4IdoAal-3Galpl-3Gaipi^Xyl-ol is not
(Sugahara et al, 1995a). The data are summarized in Table n.
Taken together, the compound in Fraction I was concluded to
be AHexAal-3GalNAcpi^GlcApi-3Gaipi-3Gal(31^Xylol, and Fraction El was found to consist of two oligosaccharides, AHexAal-3GalNAc(4S)pi^K}lcA31-3Gaipi-3Ga^l4Xyl-ol and AHexAal-3GalNAc(4S)|31-4IdoAal-3Ga^l3Galf$l—4Xyl-ol. Fraction III seems to be a novel, sulfated and
phosphorylated linkage hexasaccharide alditol with an internal
iduronic acid residue based on the findings that it was resistant
to chondroitinase AC-EI but sensitive to chondro-4-sulfatase
and alkaline phosphatase. However, since there were no authentic standards coeluted with these digests, we could not
determine the detailed structure. All these findings indicate that
the linkage hexasaccharide alditols from recombinant decorin
is primarily composed of the three structures as summarized in
Table m .
Discussion
In this study, we determined the structure of three major hexasaccharide alditols from the carbohydrate-protein linkage region of recombinant decorin with a single chondroitin/
dermatan sulfate chain. Although all of them were previously
isolated from bovine aorta dermatan sulfate where two additional hexasaccharide structures were also demonstrated (Sugahara et al, 1995a), the present findings unexpectedly revealed
that there are structural variations in the carbohydrate-protein
linkage region of decorin with a single chondroitin/dermatan
sulfate. The heterogeneity was in marked contrast to the uniformity demonstrated in the linkage hexasaccharide structure
of human inter-a-trypsin inhibitor (Yamada et al, 1995a) and
urinary trypsin inhibitor (Yamada et al, 1995b), both of which
also bear a single chondroitin sulfate chain and whose linkage
regions are always 4-sulfated on the Gal residue preceding the
GlcA residue.
Structural variations were so far observed in the linkage
hexasaccharides isolated from all cartilaginous chondroitin sulfate examined, whose linkage regions are sometimes but not
ajways phosphorylated on C2 of the Xyl residue (Shibata et al.,
1992; Sugahara et al, 1992a,c) or sulfated on C4 and/or C6 of
the Gal residue(s) (Sugahara et al., 1988, 1991; de Waard et
al, 1992; Shibata et al., 1992; Cheng et al, 1996). It should be
noted that these heterogenous linkage regions may represent a
Table ED. Yields of the carbohydrate-protein linkage hexasaccharide
alditols isolated from recombinant decorin
Structure
Total (%)
AHexAa 1 -3GalNAc£ 1 -4GlcA01 -3Galp 1 -3Gaip 1 -4Xyl-ol
AHexAal-3GalNAc(4S)pi-4GlcApi-3Gaipi-3Gaipi^Xyl-ol
AHexAa 1 -3GalNAc(4S)p 1 -41doAa l-3Gal|3 l-3Gaip 1 -4Xyl-ol
Unidentified component
12
60
11
17
1178
number (up to 100) of different chondroitin sulfate attachment
sites of a cartilage proteoglycan aggrecan. In addition, similar
variations were also observed in the linkage hexasaccharides
isolated from bovine aorta dermatan sulfate chains most likely
derived from small proteoglycans, such as decorin and biglycan, as well as those from large proteoglycans, whose linkage
regions sometimes but not always contain an iduronic acid
residue as the innermost uronic acid and/or are 4-sulfated on
the Gal residue preceding the GlcA/IdoA residue (Sugahara et
al, 1995a). Therefore, we anticipated that the structural heterogeneity of the linkage hexasaccharide region in the cartilaginous chondroitin sulfate and the aorta dermatan sulfate
chains may reflect heterogeneous populations of different cell
types in those tissues which are responsible for synthesis of
distinct subclasses of chondroitin/dermatan sulfate chains with
different structure. However, the present study demonstrated
structural variations in the carbohydrate-protein linkage region
of the recombinant decorin with a single chondroitin/dermatan
sulfate which was synthesized in the cloned CHO cells, suggesting that even a single cell or a single population of a certain
cell type can synthesize a chondroitin/dermatan sulfate chain
with heterogeneous structures.
The present findings demonstrate that the linkage hexasaccharide alditols of the recombinant decorin contain the structure with an internal iduronic acid residue AHexAotl3GalNAc(4S)pi-4IdoAol-3Gaipi-3Gaipi^Xyl-ol, which
was first demonstrated in one of the five linkage hexasaccharide alditols isolated from dermatan sulfate proteoglycans of
bovine aorta (Sugahara et al, 1995a), indicating that the GAG
chain of the decorin carried at least some dermatan sulfate and
the structure containing an internal iduronic acid in the linkage
region does not seem to be rare in dermatan sulfates. In contrast to the previous findings (Sugahara et al, 1995a), the other
sulfated linkage hexasaccharide alditol with an internal iduronic acid residue, AHexAal-3GaLNAc(4S)31-4IdoAal3Gal(4S)pi-3Galpl-4Xyl-ol, was not detected in this study.
Furthermore, this disulfated linkage hexasaccharide alditol was
recently isolated from dermatan sulfate proteoglycans of human post-burn scar tissues (Sugahara et al, 1996). Nonetheless, linkage regions 4-sulfated on the Gal residue preceding
the GlcA/IdoA residue were notably absent in the decorin.
Taken together, the present findings indicate that
the 4-sulfotransferase responsible for the sulfation on the Gal
residue is probably distinct from that for the sulfation on the
GalNAc residue and that the 4-sulfation on both of the neighboring Gal/GalNAc residues is not a prerequisite for the epimerization reaction of GlcA to IdoA by C5 uronosyl epimerase
for dermatan sulfate (Malmstrom et al, 1975; Maimstrom, 1984).
Previously, we proposed that Xyl phosphorylation and Gal
sulfation in the carbohydrate-protein Linkage region of chondroitin suLfate do not coexist since they had never been demonstrated in a single chondroitin sulfate chain (Sugahara et al,
1992a,c). This appears to be true for dermatan sulfate as well.
As described in Results, no linkage regions with the sulfated
Gal residue were detected despite the finding that one of the
linkage hexasaccharide alditol fractions (Fraction HI) seemed
to contain at least one phosphate group based on its susceptibility to alkaline phosphatase. Although the biological significance, if any, is unknown, individual chondroitin/dermatan sulfate chains with distinct structural modifications in the linkage
region may reflect discrete subclass chains with different roles.
Moreover, this region could contain signals that influence the
pattern of epimerization and sulfation of the repeating disac-
Glycosaminogiycan structure of decorin
charide region. To address these questions, future studies attempting to separate chondroitin and dermatan sulfate-type
GAG chains derived from the recombinant decorin will be
required although there is a possibility that all the GAG chains
are the hybrid type.
Materials and methods
Materials
D-[l-3H]Galactose (20 Ci/mmol) was purchased from American Radiolabeled
Chemicals, Inc., St. Louis, MO. Chondroitinases ABC (EC 4.2.2.4), B (EC
4.2.2), AC-II (EC 4.2.2.5), and chondn>4-0-sulfatase (EC 3.1.6.9) were purchased from Seikagaku Corp., Japan. Special quality calf intestine alkaline
phosphatase for molecular biology was from Boehringer Mannheim GmbH,
Germany. Sephadex G-50, HiLoad 16760 Superdex 30 (prep grade) and
HTTRAP DEAE-Sepharose Fast Row were obtained from Pharmacia Biotech
Inc., Sweden. Glucose-free RPMI 1640 medium and a-minimum essential
medium were from Life Technologies Inc., USA. All other reagents and
chemicals were of the highest quality available.
Cell culture and metabolic labeling
A Chinese hamster ovary (CHO) cell line (D61, clone 61 in Yamaguchi and
Ruoslahti, 1988) that overcxpresses human decorin as a result of DNA transfection and subsequent gene amplification was used in this study. The CHO
cells were propagated in metbotrexate-contairung a-minimum essential medium to maintain the decorin expression with the highest level as described
(Yamaguchi and Ruoslahti, 1988). The CHO cells were passed once in methotrexate-free medium to eliminate possible methotrexate effects before use. The
cells were then labeled metabobcally with D-[l-3H]galactose (20 (iCi/ml) in
98% glucose-free RPMI 1640 medium containing 10% dialyzed fetal calf
serum and 2% complete a-minimum essential medium supplemented with
10% dialyzed fetal calf serum at 37°C for 24 h. Since nearly all of the deconn
produced by the cells in culture was shown to be secreted in the culture
medium (Yamaguchi and Ruoslahti, 1988), the medium was harvested and
freed of cellular debris by centrifugation for 20 min at 2000 x g. The labeled
conditioned medium containing decorin was then concentrated and washed
twice with 25 mM Tris-HQ buffer (pH 6.5) containing 0.15 M NaCl and 7.8
M urea to remove most of the free [l-3H]galactose using a Centriplus 30
(Amicon).
ml of 50 mM Tris-HCl buffer (pH 8.0) containing 50 mM sodium acetate and
100 u.g/ml bovine serum albumin as an enzyme stabilizer at 37°C overnight.
The digest was subjected to gel filtration on a column of Superdex 30 (1.6 x
60 cm) with 0.25 M NH 4 HCO/7% 1-propanol. The radioactive peak corresponding to the putative linkage hexasaccharide alditols (indicated in Figure 2
by the bar) was pooled and evaporated to dryness. Half of this fraction was
incubated with 100 mlU of chondroitinase AC-II in a total volume of 100 \L\
of 50 mM sodium acetate buffer (pH 6.0) at 37°C for 18 h. Chondro-4sulfatase digestion of the chondroitinase ABC or AC-II digest was conducted
as follows. Each sample corresponding to about 2.2 x 10* d.p.m. was digested
with 20 mlU of the enzyme in a total volume of 40 uJ of 80 mM Tris-HCl
buffer (pH 7.5) containing 80 mM sodium acetate and 100 ng/rnl bovine serum
albumin at 37°C for 3 h. Chondro-4-sulfatase digestion of the phosphorylated
linkage hexasaccharide AHexAal-3GalNAcBl^*GlcABl-3GalBl-3GalBl4Xyl-ol(2-0-phosphate) (Sugahara et al, 1992a) was carried out using 20 mlU
of the enzyme in a total volume of 40 p.1 of 80 mM Tris-HCl buffer (pH 7.5)
containing 80 mM sodium acetate and 100 p.g/ml bovine serum albumin at
37°C for 3 h. Alkaline phosphatase digestion of the chondroitinase ABC digest
was performed using 1 U of the enzyme in a total volume of 60 u.1 of 0.07 M
glycine/NaOH buffer (pH 9.9) containing 0.5 M MgCl2 at 37°C for 10 min.
The digestions with these enzymes were terminated by boiling for 1 min. The
digested samples were analyzed by HPLC on an amine-bound silica column as
described previously (Sugahara et al, 1989).
Cochromatography HPLC analysis with the structurally defined hexa- and
tetrasaccharide alditols
To identify linkage sugar alditols, chondroitinase ABC digest of the reduced
3
H-labeled GAG or a chondro-4-sulfatase or an alkaline phosphatase digest of
the chondroitinase ABC digest was cochromatograpbed on an amine-bound
silica column HPLC as described previously (Yamada et al, 1995a) with four
structurally defined hexasaccharide alditols isolated from whale cartilage
(Sugahara et al, 1991): AHexAal-3GalNAcBl-4GlcABl-3GalBl-3GalBl4Xyl-ol, AHexAal-3GalNAc(6S^l^M31cABl-3GalBl-3GalBl-4Xyl-ol,
AHexAa l - 3 G a l N A c ( 4 S ) B l - 4 G l c A B l - 3 G a l B l - 3 G a ^ l - 4 X y l - o l and
AHexAal-3GalNAc(4S)Bl-4GlcABl-3Gal(4S)Bl-3GalBl-4Xyl-ol. A
chondroitinase AC-II digest of the reduced 3H-labeled hexasaccharides or a
chondro-4-sulfatase digest of the chondroitinase AC-II digest was also cochromatographed with four structurally defined tetrasaccharide alditols isolated from shark and whale cartilage (Sugahara et al, 1991, 1992a; de Waard
et al, 1992): AHexAal-3GalBl-3Gaipi^lXyl-ol, AHexAa l-3Gal(4S)Bl3GalBl^»Xyl-ol, AHexAal-3GalBl-3Gal(6S)Bl^»Xyl-ol and AHexAal3Gal(6S)Bl-3Gal(6S)Bl^»Xyl-ol.
Preparation ofJH-labeled GAGs from recombinant decorin expressed m
CHO cells
Recombinant deconn was isolated from the labeled conditioned medium prepared above by chromatography on Hl'l'KAP DEAE-Sepharose Fast Flow in a
Pharmacia FPLC system as described previously (Choi et al, 1989; Mann et
aL, 1990; Yamaguchi et al, 1990), except for a slight modification. To the
concentrated medium, chondroitin 4-sulfate (4 mg) derived from whale cartilage (Sugahara et at, 1991) was added as a carrier. The mixed solution was
applied to DEAE-Sepharose previously equilibrated with 25 mM Tris-HCl
buffer (pH 6.5) containing 0.15 M NaCl and 7.8 M urea. The column was
washed with 3-column volumes of the same buffer, then eluted with a 0.15—1
M NaCl linear gradient at a flow rate of 2 ml/min. Fractions of 2 ml were
collected and monitored by 3H radioactivity. The radiolabeled fraction eluted
with 0.45-0.55 M NaCl (indicated by the bar in Figure 1) contained recombinant deconn as described (Yamaguchi et al, 1990) and was pooled. Although control CHO cells were also metabolically labeled arid the resulting
culture medium was applied to the same DEAE-Sepharose column as described above, radiolabeled materials eluted with 0.45-0.55 M NaCl were
negligible.
To release 3H-labeled GAG from the purified recombinant decorin, the
decorin-containing fraction was treated with 1 M NaBH«/0.05 M NaOH at
room temperature for 24 h. The reaction was stopped by adding 1 M glacial
acetic acid and neutralized with 1 M Na2CO3. Following repeated evaporation
of the sample with methanol, the residues were reconstituted in water. The
3
H-Iabeled sample was chromatographed on a column (1 x 47 cm) of Sephadex
G-50 (fine) using 0.25 M NH 4 HCO : /7% 1-propanol. The flow-through fractions containing H-labeled GAG were recovered.
Acknowledgments
We thank Maho Adachi for excellent technical assistance. This work was
supported in part by the Science Research Promotion Fund of the Japan Private
School Promotion Foundation (to K.S.), the Mizutani Foundation for Glycoscience (to K.S.), the Kanae Medical Research Promotion Fund (to H.K.), and
Grants-in-aid for Encouragement of Young Scientists 09772013 (to H.K.) and
for Scientific Research on Priority Areas 05274107 (to K.S.) from the Ministry
of Education, Science, Culture, and Sports of Japan.
Abbreviations
CHO cells, Chinese hamster ovary cells; FPLC, fast protein-liquid chromatography; GAG, glycosaminogiycan; GlcA, D-glucuronic acid; AHexA, 4,5unsaturated hexuronic acid or 4-deoxy-a-L-threo-hex-4-ene-pyranosyluronic
acid; HexA, hexuronic acid; HPLC, high-performance liquid chromatography;
IdoA, iduronic acid; ADi-OS, A°HexAa l-3GalNAc; ADi-6S, A 4 J HexAal3GalNAc(6-C»-sulfate); ADi-4S, A 4J HexAal-3GalNAc(4-0-sulfate); ADidiS B , A 4 - 5 HexA(2-0-sulfate)al-3GalNAc(4-0-sulfate); ADi-diS D ,
A"-3HexA(2-0-sulfate)al-3Ga]NAc(6-O-sulfate); ADi-diSE, A'-'HexAal3GalNAc(4,6-0-disulfate); ADi-triS, A 4 - 3 H e x A ( 2 - 0 - s u l f a t e ) a l 3GalNAc(4,6-O-disulfate); 2S, 4S and 6S represent 2-0-sulfate, 4-0-sulfale,
and 6-O-sulfate, respectively; Xyl-ol, xylitol.
References
Digestion with chondroitinases ABC, AC-ll, chondro-4-sulfatase, or
alkaline phosphatase
The 3H-labded GAG fraction corresponding to 4.3 x 10* d.p.rn. was exhaustively digested with 750 mlU of chondroitinase ABC in a total volume of 0.45
ChengJ3., Heinegard.D., Fransson,L.-A., Baylissjvt, BielickiJ., HopwoodJ.
and Yoshida,K. (1996) Variations in the chondroitin sulfate-protein linkage
region of aggrecans from bovine nasal and human articular cartilages. J.
Biol Chem., 271, 28572-28580.
1179
H.Kitagawa et al
Choi,H.U , Johnson,T.L., Pal.S., Tang,L.-H., Rosenberg.L. and Neame.PJ.
(1989) Characterization of the dermatan sulfate proteoglycans, DS-PGI and
DS-PGI1, from bovine articular cartilage and skin isolated by octylsepharose chromatography. / Biol. Chan., 264, 2876-2884.
de Waard,P., VliegenthartJ.F.G., HaradaJ". and Sugahara,K. (1992) Structural
studies on sulfated oligosaccharides derived from the carbohydrate-protein
linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. H
Seven compounds containing 2 or 3 sulfate residues. J. BioL Chem., 261,
6036-6043.
Hascall.V.C. and Hascall.G.K. (1981) Proteoglycans. In Hay,E.D. (ed.). Cell
Biology of Extracellular Matrix. Plenum, New York, pp. 39-63.
Iozzo,R.V. and Murdoch,A.D. (1996) Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in
molecular diversity and function. FASEB J., 10, 598-614.
Kresse.H., Hausser.H. and Schonherr.E. (1993) Small proteoglycans. Experientia, 49, 403-416.
Maimone^M.M. and TollefsenJ).M. (1990) Structure of a dermatan sulfate
hexasaccharide that binds to heparin cofactor II with high affinity. J. Biol.
Chem., 265, 18263-18271.
MalmstrOmA- (1984) Biosynthesis of dennatan sulfate. D. Substrate specificity of the C-5 uronosyl epimerase. J. BioL Chem., 259, 161-165.
MalmstrOm^A., Fransson,L.-A., HOOkJvl. and Lindahl.U. (1975) Biosynthesis
of dermatan sulfate. I. Formation of L-iduronic acid residues. J. Biol. Chem.,
250, 3419-3425.
Mann.D.M., Yamaguchi.Y., Bourdon,M.A. and Ruoslahti.E. (1990) Analysis
of glycosaminoglycan substitution in decorin by site-directed mutagenesis.
J. BioL Chem., 265, 5317-5323.
Roderi.L. (1980) Structure and metabolism of connective tissue proteoglycans.
In Lennarz.WJ. (ed.), The Biochemistry of Glycoprvteins ami Proteoglycans. Plenum, New York, pp. 491-517.
Ruoslahri,E. (1988) Structure and biology of proteoglycans. Annu. Rev. Cell
Biol., 4, 229-255.
Shibata,S., MiduraJtJ. and Hascall.V.C. (1992) Structural analysis of the
linkage region oligosaccharides and unsaturated disaccharides from chondroitin sulfate using CarboPac PA1. J. Biol. Chem., 267, 6548-6555.
Sugahara.K., YamashinaJ-, de Waard.P., van Halbeek.H. and VliegenthartJ.F.G. (1988) Structural studies on sulfated glycopeptides from the
carbohydrate—protein linkage region of chondroitin 4-sulfate proteoglycans
of Swarm rat chondrosarcoma. Demonstration of the structure, GlcAfil3Gal(4-O-sulfate)pi-3Galpl^»Xyipi-O-Ser. J. Biol. Chem., 263, 1016810174.
Sugahara,K., Okumura.Y. and YarnashinaJ. (1989) The Engelbreth-HolmSwarm mouse tumor produces undersulfated heparan sulfate and oversulfated galactosaminoglycans. Biochem. Biophys. Res. Commun., 162, 189—
197.
Sugahara,K., Masuda^l., Harada.T., YamashinaJ., de WaardJ". and Vh'egenthartJ.F.G. (1991) Structural studies on sulfated oligosaccharides derived
from the carbohydrate-protein linkage region of chondroitin sulfate proteoglycans of whale cartilage. Eur. J. Biochem., 202, 805-811.
Sugahara.K., Ohi.Y., Harada,T., de WaarcM5. and VUegenthartJ.F.G. (1992a)
Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark
cartilage. I. Six compounds containing 0 or 1 sulfate arid/or phosphate
residue. J. Biol. Chem., 267, 6027-6035.
Sugahara.K., Yamada,S., Yoshida.K., de Waard,P. and Vu'egentharU.F.G.
(1992b) A novel sulfated structure in the carbohydrate-protein linkage region isolated from porcine intestinal heparin. J. BioL Chem., 267, 15281533.
Sugahara.K., Mizuno,N., Okumura,Y. and Kawasaki.T. (1992c) The phosphorylated and/or sulfated structure of the carbohydrate-protein linkage
region isolated from chondroitin sulfate in the hybrid proteoglycans of Engelbreth-Holm-Swarm mouse tumor. Eur. J. Biochem., 204, 401-406.
Sugahara,K., Tohno-okaJ*., Yamada,S., Khoo,K.-H., Morris.H.R. and Dell A
(1994) Structural studies on the oligosaccharides isolated from bovine kidney heparan sulfate and characterization of bacterial heparitinases used as
substrates. Glycobiology, 4, 535—544.
Sugahara, K., Ohkita, Y., Shibata, Y., Yoshida, K. and Ikegami, A. (1995a)
Structural studies on the hexasaccharide alditols isolated from tbe carbohydrate-protein linkage region of dermatan sulfate proteoglycans of bovine
aorta. /. BioL Chem., 270, 7204-7212.
Sugahara,K., Tsuda,H., Yoshida,K., Yamnda.S., de BeeT.T. aod Vliegenthart J F . G . (1995b) Structure determination of the octa- and decasaccharide
sequences isolated from the carbobydrate-protein linkage region of porcine
intestinal heparin. J. Biol. Chem., 270, 22914-22923.
Sugahara,K., Masayama,K_, SieberU.W. and Garg.H.G. (1996) Structural
analysis of the carbohydrate-protein linkage region of dermatan sulfate
1180
proteoglycans from post-burn scar tissues. XV1I1 International Carbohydrate Symposium (abstracts). Milano, Italy, p. 119.
Yamada,S., Oyama.M., Kinugasa,H., Nakagawa,T., Kawasaki.T., Nagasawa,S., Khoo,K.-H., Moriss.H.R., Dell,A- and Sugahara,K. (1995a) The sulfated carbohydrate-protein linkage region isolated from chondroitin 4sulfate chains of inter-a-trypsin inhibitor in human plasma. Glycobiology, 5,
335-341.
Yamada^., Oyamajd., Yuki.Y., Kato.K. and Sugahara,K. (1995b) The uniform galactose 4-sulfate structure in the carbohydrate-protein linkage region
of human urinary trypsin inhibitor. Eur. J. Biochem., 233, 687-693.
Yamaguchi.Y. and Ruoslahu'JE. (1988) Expression of human proteoglycan in
Chinese hamster ovary cells inhibits cell proliferation. Nature, 336, 244246.
Yamaguchi.Y., MannJXM. and Ruoslahti,E. (1990) Negative regulation of
transforming growth factor-^ by the proteoglycan decorin. Nature, 346,
281-284.
Received on March 4, 1997; revised on June 3, 1997; accepted on June 6, 1997