Glycobiology vol. 17 no. 8 pp. 886–894, 2007
doi:10.1093/glycob/cwm051
Advance Access publication on May 19, 2007
Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): demonstration
of chondroitin in the developing nematocyst, the sting organelle, and structural
characterization of glycosaminoglycans
Shuhei Yamada2,3*, Hideto Morimoto2*,
Toshitaka Fujisawa4, and Kazuyuki Sugahara1 –3
Key words: chondroitin sulfate/cnidarian/glycosaminoglycan/
heparan sulfate/Hydra magnipapillata
2
Department of Biochemistry, Kobe Pharmaceutical University, Higashinadaku, Kobe 658-8558, Japan; 3Laboratory of Proteoglycan Signaling and
Therapeutics, Graduate School of Life Science, Frontier Research Center for
Post-Genomic Science and Technology, Hokkaido University, Nishi 11choume, Kita 21-jo, Kita-ku, Sapporo, Hokkaido 001-0021, Japan; and
4
Department of Developmental Genetics, National Institute of Genetics,
Mishima 411-0801, Japan
Introduction
Received on April 12, 2007; revised on May 1, 2007; accepted on May 5, 2007
The hydrozoan is the simplest organism whose movements
are governed by the neuromuscular system, and its de novo
morphogenesis can be easily induced by the removal of
body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in
vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been
implicated in the signaling functions of various growth
factors and play critical roles in the development of the
central nervous system, the isolation and characterization
of GAGs from hydrozoans have never been reported.
Here, we characterized GAGs of Hydra magnipapillata.
Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The
CS – PGs might furnish an environment for assembling
nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated
from Hydra and their structural features were examined.
A considerable amount of CS, three orders of magnitude
less heparan sulfate (HS), but no hyaluronan were found,
as in Caenorhabditis elegans. Analysis of the disaccharide
composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated Nacetylgalactosamine (GalNAc) but also 6-O-sulfated
GalNAc. The average molecular size of CS and HS was
110 and 10 kDa, respectively. It has also been established
here that CS chains are synthesized on the core protein
through the ubiquitous linkage region tetrasaccharide,
suggesting that indispensable functions of the linkage
region in the synthesis of GAGs have been conserved
during evolution.
*These authors contributed equally to this work.
1
To whom correspondence should be addressed; Tel: þ81 11 7069054;
Fax: þ81 11 7069056; e-mail: [email protected]
Glycosaminoglycans (GAGs) have been implicated in the
regulation and maintenance of cell adhesion, motility, proliferation, differentiation, tissue morphogenesis, and embryogenesis
(Kjellén and Lindahl 1991; Brickman and Gerhart 1994; Itoh
and Sokol, 1994). GAGs include chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS)/heparin. The
backbones of HS and CS consist of repeating disaccharide
units: D-glucuronic acid (GlcA)b1-4GlcNAca1-4 for HS and
GlcAb1-3N-acetylgalactosamine (GalNAc)b1-4 for CS.
These simple structures acquire a considerable degree of variability through extensive modifications involving sulfation and
uronate epimerization (Fransson 1985). This structural variability of GAGs is the basis for a wide variety of domain structures
with different biological activities (Kjellén and Lindahl 1991),
and is generated by the elaborate, concerted actions of biosynthetic enzymes.
Investigation of the occurrence of GAGs shows their wide
distribution in tissues of vertebrates and invertebrates (Nader
and Dietrich 1989), suggesting that GAGs have been phylogenetically conserved through evolution, and play fundamental, biological roles in animal development. The nematode
Caenorhabditis elegans and the fruit fly Drosophila melanogaster are ideal experimental organisms for studying a wide
range of fundamental biological disciplines in development
using modern techniques of molecular and cell biology
(Bellaiche et al. 1998; Sen et al. 1998; Seppo and Tiemeyer
2000). Biochemical studies of GAGs from Drosophila and
C. elegans have been reported in Yamada et al. (1999) and
Toyoda et al. (2000). Although HS chains are sulfated at
various positions in both organisms, the structure of the CS
of these organisms is unique in that the CS from Drosophila
is composed of nonsulfated and 4-O-sulfated disaccharides,
whereas only nonsulfated chondroitin is detected in C.
elegans. These results might suggest that GalNAc 4O-sulfation was acquired during evolution from nematodes
to arthropods and plays an important role in some special
function characteristic of higher animals. CS containing a
GalNAc 6-O-sulfated structure has never been detected in C.
elegans and in adult Drosophila. Although a small quantity of
6-O-sulfated GalNAc was detected in the third instar stage in
Drosophila larvae (Pinto et al. 2004), the specific CS 6-O-sulfotransferase in Drosophila has not been identified. Mollusks
such as squids are the lowest animals reported to contain
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Glycosaminoglycans in Hydra magnipapillata
GalNAc 6-O-sulfated or multi-sulfated structures in their CS
chains (Mathews and Person 1962; Suzuki et al. 1968).
The hydrozoan has also been utilized as a model to study
tissue regeneration in vivo (Fujisawa 2003; Holstein et al.
2003). It is one of the lowest multicellular animals and
assumed to be a lower form of life than C. elegans. It has
an extracellular matrix (ECM) boundary zone between two
functionally distinct tissue layers, ectodermal and endodermal
epithelial layers, and the ECM components of Hydra
vulgaris have been characterized in an immunocytochemical
study (Sarras et al. 1991). Although the presence of the core
protein of HS – proteoglycan (PG) was indicated, it is not
clear whether it is glycanated and the structural characterization of hydra GAG chains has not been attempted. On the
basis of previous findings concerning the structure of CS/DS
chains from Drosophila and C. elegans (Yamada et al. 1999;
Toyoda et al. 2000), hydrozoans may be assumed to produce
only nonsulfated chondroitin or no CS.
Accumulating evidence suggests that GAGs play important
roles in neural development by regulating neuronal adhesion
and migration, the formation of neurites, and axonal guidance
(Sugahara et al. 2003). Immunological studies using monoclonal antibodies have revealed that CS isoforms, differing
in the position and degree of sulfation, perform distinct functions during development in the mouse and rat fetus (Mark
et al. 1990). Oversulfated disaccharides and L-iduronic acid
(IdoUA) residues from CS/DS chains play critical roles in
growth factor-binding and neuritogenic activities during development of the brain (Faissner et al. 1994; Clement et al. 1998;
Nadanaka et al. 1998; Deepa et al. 2002; Bao et al. 2004; Bao,
Mikami et al. 2005; Bao, Muramatsu et al. 2005; Li et al.
2007). Since the hydrozoan is the simplest organism, in
which movements are governed by the neuromuscular
system, it provides the most appropriate model system to investigate the basic mechanism of neuritogenesis. GAGs may be
present in hydrozoans and perform critical functions during
neurogeneration. To understand the development of nervous
system in terms of the evolution of GAG chains and their functions in the regeneration of the nervous system of hydrozoans,
structural information about GAGs will be required, which
can be used together with genetic and molecular data.
Hence, GAGs from Hydra magnipapillata were isolated and
their structural features were characterized in this study.
Results
Immunohistochemistry of GAGs in Hydra
Immunostaining of Hydra using five different antibodies,
including 473A12, 2H6, CS-56, HepSS-1, and F58-10E4,
against chondroitin, CS, and HS was carried out. Only the
anti-chondroitin antibody 473A12 detected the corresponding
epitope (Figures 1 and 2). The anti-HS antibodies (HepSS-1
and F58-10E4) and anti-CS antibodies (2H6 and CS-56)
showed no staining. In a whole-mount sample, clusters of
cells in the body column were stained (Figure 1). They are
nests of nematoblasts (Figure 2, lower panel). When stem
cells enter the nematocyte differentiation pathway, they
undergo a series of synchronous cell divisions up to five
rounds (David and Gierer 1974). However, the cytokinesis is
incomplete and the daughter cells are connected to each
other by cytoplasmic bridges, thus forming clusters or nests
of cells. Cells at this stage are called dividing nematoblasts.
Upon the cessation of division, nests of usually 8 or 16 cells
start producing nematocysts, the stinging organelle. First, a
hole appears in the cytoplasm and becomes a larger hollow
space. Finally, the nematocyst structures develop. Cells at
this stage are called differentiating nematoblasts. In a single
nest, nematocysts of the same type are synchronously produced. There are four types of nematocytes. To examine the
precise stage of the stained cells, polyps were dissociated
into single cells by maceration and then immunostained with
473A12 for chondroitin (Figure 2). Stained cells had a large
hollow space in the cytoplasm (Figure 2B), where the staining
occurred as can be seen in a merged image (Figure 2C). These
cells are actively producing nematocysts, but the nematocysts
have not yet fully developed.
Characterization of GAGs in Hydra
To characterize GAGs in Hydra chemically, a GAG – peptide
fraction was prepared from the acetone powder of the crude
homogenate of Hydra by digestion with actinase E, followed
by trichloroacetic acid treatment and ethanol precipitation.
The GAG – peptide fraction was further fractionated by
anion-exchange chromatography on a Sep-Pak Accell Plus
QMA anion-exchange cartridge (Waters Corp., Milford, MA)
using stepwise elution with a 0.3 M phosphate buffer, pH
6.0, containing 0.15, 0.5, 1.0, or 1.5 M NaCl. Each fraction
was analyzed by anion-exchange high-performance liquid
chromatography (HPLC) to identify the disaccharides produced by digestion with specific GAG lyases.
Upon digestion with chondroitinase ABC (Table I), disaccharides were detected mainly in the fraction eluted with
1.0 M NaCl on the Accell Plus QMA cartridge. The major
disaccharide was the nonsulfated disaccharide 4-deoxya-L-threo-hex-4-enepyranosyluronic acid (DHexA) – GalNAc
(Figure 3 and Table I). Small proportions of monosulfated disaccharides were also detected. Each peak was co-eluted with
the corresponding standard disaccharide (results not shown),
and upon digestion with chondro-4- or 6-sulfatase, the peak
that eluted at the position of DHexA – GalNAc(4-O-sulfate)
or DHexA –GalNAc(6-O-sulfate) was shifted to the position,
respectively, of DHexA – GalNAc (data not shown), confirming the identity of these structures. To evaluate whether the
Hydra GAG fraction contains DS, enzymatic digestions with
chondroitinase AC-I and B were carried out. Chondroitinase
AC-I and B split GalNAc – GlcA and GalNAc– IdoUA linkages,
respectively (Yoshida et al. 1993). The Hydra GAG fraction was
resistant to the action of chondroitinase B but sensitive to that
of chondroitinase AC-I (data not shown), suggesting that Hydra
synthesizes sulfated CS but not DS chains. Under the anionexchage HPLC conditions used, DHexA–GalNAc can be
separated from the unsaturated disaccharides from hyaluronate.
However, no disaccharides from hyaluronate were detected
in the digest with chondroitinase ABC, although the enzyme
depolymerizes not only CS but also hyaluronate, indicating
the absence of hyaluronate in Hydra. Approximately 1 mmol of
the unsaturated CS disaccharide was recovered by digestion
with chondroitinase ABC from 120 g of the Hydra.
The Hydra GAG fraction was also subjected to a compositional analysis of HS disaccharides after digestion with a
mixture of heparinase and heparitinase. The total amount of
HS disaccharides produced by digestion with HS lyases was
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Fig. 1. Whole mount immunostaining with anti-chondroitin antibody 473A12. The upper body of a hydra is shown. A closeup of a nest is shown in the upper left
panel. The positive cells in nests are nematoblasts.
only 0.35 nmol per 120 g of the Hydra, three orders of
magnitude less than that of CS. The chromatogram obtained
after labeling with 2-aminobenzamide (2AB) is shown in
Figure 4. The yield of each disaccharide was calculated
based on the fluorescence intensity, and the data are summarized in Table I. Peaks of unidentified contaminants derived
from the 2AB reagent were often observed as a result of the
sensitivity of the analysis just before the elution of monosulfated disaccharides, as indicated by asterisks in Figure 4.
The identity of the disaccharide peaks was established based
on co-chromatography with standard disaccharides.
When the digest of the 1.0 M NaCl-eluted fraction with
chondroitinase ABC was analyzed by gel-filtration HPLC
after 2AB labeling, only disaccharides but no oligosaccharides
were detected (data not shown). The amount of uronic acid in
the 1.0 M NaCl-eluted fraction was determined by the carbazole reaction and its concentration in the starting material
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was calculated to be 1.56 mg/g wet tissue. Taking this value
as 100%, the recovery of the total disaccharides generated by
chondroitinase ABC, heparinase, and heparitinase was 99%,
suggesting that essentially all the GAG polysaccharides were
degraded to disaccharides by the GAG lyases.
The molecular size of the Hydra CS and HS was analyzed
by gel-filtration chromatography on a Superdex 200 column
(Figure 5). To monitor small amounts of Hydra GAGs, aliquots of individual fractions collected at 4.0 min intervals
were lyophilized and digested with chondroitinase ABC or a
mixture of heparinase and heparitinase, then the products
were derivatized using a fluorophore, 2AB, and analyzed by
anion-exchange HPLC. The amounts of the derivatized disaccharide products were calculated based on their fluorescence
intensity. From comparisons with the calibration plot generated using the data obtained with size-defined commercial
polysaccharides (Figure 5, inset), the average molecular size
Glycosaminoglycans in Hydra magnipapillata
Fig. 2. Immunostaining on macerated cells with anti-chondroitin antibody 473A12. (A) Immunostaining. (B) Part of a nematoblast nest under phase contrast optics.
(C) A merged image of (A) and (B). The lower panel illustrates the differentiation of multipotent interstitial stem cells in Hydra. Cells in the red square correspond
to those shown in the upper panels. They are at a later stage of nematocyst differentiation.
Table I. Disaccharide composition of Hydra CS and HSa
CS disaccharides
Proportion
(%)
Total amount in
wet tissue (nmol/g)
DHexA– GalNAc
71
6.01
DHexA– GalNAc(4-O-sulfate)
15
1.29
DHexA– GalNAc(6-O-sulfate)
14
1.18
b
Total
100 (0.29)
HS disaccharides
Proportion
(%)
DHexA– GlcNAc
24
0.70
DHexA– GlcNAc(6-O-sulfate)
18
0.52
DHexA– GlcN(2-N-sulfate)
12
0.36
DHexA– GlcN(2-N-,6-O-disulfate)
25
0.73
8.48
Total amount in
wet tissue (pmol/g)
DHexA(2-O-sulfate) –GlcN(2-N-sulfate)
10
0.30
DHexA(2-O-sulfate) –GlcN(2-N-,6-Odisulfate)
11
0.31
Total
b
100 (1.33)
2.93
a
The purified GAG fraction was digested with chondroitinase ABC or a
mixture of heparinase and heparitinase, and the digests were analyzed by
HPLC as described in the “Materials and methods” section.
b
Total sulfate groups/100 disaccharides are given in parentheses.
Fig. 3. HPLC analysis of the chondroitinase ABC digest of the Hydra GAG
fraction. The chondroitinase ABC digest of the Hydra GAG fraction was
analyzed by reversed-phase ion-pair chromatography with post-column
detection. The positions of authentic unsaturated disaccharides are indicated
by arrows. 1, DHexAa1-3GalNAc; 2, DHexAa1-3GalNAc(4-O-sulfate);
3, DHexAa1-3GalNAc(6-O-sulfate); 4, DHexAa1-3GalNAc(4,6-O-disulfate);
5, DHexA(2-O-sulfate)a1-3GalNAc (6-O-sulfate); 6, DHexA (2-O-sulfate)a13GalNAc(4,6-O-disulfate).
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Fig. 4. HPLC analysis of the Hydra GAG fraction digested with a mixture of
heparinase and heparitinase. The 2AB derivative of the digest of the Hydra
GAG fraction obtained using a mixture of heparinase and heparitinase was
analyzed by HPLC on an amine-bound silica PA-03 column. The positions
of authentic 2AB-derivatized unsaturated disaccharides are indicated by
arrows. 1, DHexAa1-4GlcNAc; 2, DHexAa1-4GlcNAc(6-O-sulfate);
3, DHexAa1-4GlcN(2-N-sulfate); 4, DHexAa1-4GlcN(2-N,6-O-disulfate);
5, DHexA(2-O-sulfate)a1-4GlcN(2-N-sulfate); 6,DHexA(2-O-sulfate)
a1-4GlcN(2-N, 6-O-disulfate). The peaks marked by asterisks are due to
unknown substances derived from the GAG preparation or the column resin.
of CS and HS was estimated to be 110 and 10 kDa,
respectively.
The structure of the GAG – protein linkage region was also
analyzed. The GAG – peptide fraction was treated with LiOH
to liberate O-glycan chains including GAGs from the core
Fig. 5. Molecular size of the Hydra CS and HS determined by gel-filtration
chromatography. The Hydra GAG fraction (corresponding to 250 and
0.088 nmol of CS and HS, respectively) was subjected to gel-filtration
chromatography on a Superdex 200 column. The GAG lyase digests of
individual fractions were derivatized with 2AB, then analyzed by anionexchange HPLC on an amine-bound silica column as described in the legend
to Figures 3 and 4. The amount of the 2AB derivative of unsaturated CS
(circles) or HS disaccharides (squares) was calculated based on fluorescence
intensity. V0 and Vt were determined using human umbilical cord hyaluronic
acid and NaCl, respectively. The inset shows the calibration curve giving a
linear relationship between log Mr and elution volume, which was prepared
using size-defined commercial polysaccharides; dextran (average Mr: 65,500,
37,500, and 18,100) and HS (average Mr: 7500).
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proteins (Sakaguchi et al. 2001). The liberated saccharides
were labeled with 2AB, and the excess 2AB-derivatizing
reagents were removed by paper chromatography and gelfiltration chromatography as described in the Materials
and methods section. The flow-through fraction of the sample
was pooled and analyzed further. For the analysis of the
linkage region of CS, the sample was digested with chondroitinase ABC and/or AC-II, and the digests were subjected to
anion-exchange HPLC. Depolymerization of CS chains by
chondroitinase ABC results in sulfated disaccharide units
and core hexasaccharides derived from the linkage region
(Sugahara et al. 1988). Chondroitinase AC-II degrades a
linkage region hexasaccharide into a disaccharide unit and a
core tetrasaccharide (Sugahara et al. 1991).
When a chondroitinase ABC digest of the 2AB derivative
prepared after the LiOH treatment was analyzed by anionexchange HPLC, two major peaks were observed at the position of the authentic 2AB-labeled nonsulfated hexasaccharide,
DHexAa1-3GalNAcb1-4GlcAb1-3Galb1-3Galb1-4Xyl-2AB,
and monophosphorylated hexasaccharide, DHexAa13GalNAcb1-4GlcAb1-3Galb1-3Galb1-4Xyl(2-O-phosphate)2AB (Figure 6A). Upon digestion with chondroitinase ABC
and AC-II, two major peaks were observed at the positions
of the authentic 2AB-labeled tetrasaccharides, DHexAa13Galb1-3Galb1-4Xyl-2AB and DHexAa1-3Galb1-3Galb14Xyl(2-O-phosphate)-2AB, in a molar ratio of 55:45
(Figure 6B). These samples were co-eluted when co-chromatographed (data not shown) with the corresponding standard
linkage tetrasaccharides (Sakaguchi et al. 2001), confirming
Fig. 6. HPLC analysis of the linkage oligosaccharides prepared by enzymatic
digestion of Hydra CS. The 2AB derivative of Hydra GAG was digested with
chondroitinase ABC (A) and then AC-II (B). Each digest was analyzed by
HPLC on an amine-bound silica PA-03 column. The positions of authentic
2AB-derivatized linkage hexasaccharides and tetrasaccharides from whale or
shark cartilage (Sakaguchi et al. 2001) are indicated in (A) and (B),
respectively: 1, DHexAa1-3GalNAcb1-4GlcAb1-3Galb1-3Galb1-4Xyl-2AB;
2, DHexAa1-3GalNAc(6-O-sulfate)b1-4GlcAb1-3Galb1-3Galb1-4Xyl-2AB;
3, DHexAa1-3GalNAc(4-O-sulfate)b1-4GlcAb1-3Galb1-3Galb1-4Xyl-2AB;
4, DHexAa1-3GalNAcb1-4GlcAb1-3Galb1-3Galb1-4Xyl(2-O-phosphate)2AB; 5, DHexAa1-3Galb1-3Galb1-4Xyl-2AB; 6, DHexAa1-3Gal
(4-O-sulfate)b1-3Galb1-4Xyl-2AB; 7, DHexAa1-3Galb1-3Galb1-4Xyl
(2-O-phosphate)-2AB. The peaks marked by asterisks are due to unknown
substances derived from GAG, the enzyme preparation, or the column resin.
Glycosaminoglycans in Hydra magnipapillata
Fig. 7. Diagrammatic representation of the structure of the Hydra CS and HS.
The diagrams of the Hydra HS and CS chains are based on the findings
obtained from analyses of disaccharide composition (Table I), the GAG–
protein linkage region (Figure 6), and molecular size (Figure 5). 2P in the
linkage region represents 2-O-phosphate. The width of each box corresponds to
the proportion of each disaccharide unit based on Table I. The variously
shaded boxes for the repeating disaccharide region of CS indicate, DHexA
a1-3GalNAc, DHexAa1-3GalNAc(4-O-sulfate), and DHexAa1-3GalNAc
(6-O-sulfate), respectively, from the right. Boxes for the repeating disaccharide
region of HS indicate DHexAa1-4GlcNAc, DHexAa1-4GlcNAc(6-O-sulfate),
DHexAa1-4GlcN(2-N-sulfate), DHexAa1-4GlcN(2-N-,6-O-disulfate),
DHexA(2-O-sulfate)a1-4GlcN(2-N-sulfate), and DHexA(2-O-sulfate)
a1-4GlcN(2-N-,6-O-disulfate), respectively, from the right. Note that boxes for
the repeating disaccharide region represent compositions, but do not reflect the
location or clustering of the disaccharide units along the polysaccharide chains.
the identity of the peak. Upon subsequent digestion with alkaline phosphatase, the latter peak was shifted by approximately
15 min to the position of the nonsulfated or nonphosphorylated
tetrasaccharide (data not shown), suggesting that the compound in the peak contained a phosphate group most probably
at position C-2 of the Xyl residue in the linkage region tetrasaccharide (Oegema et al. 1984; Fransson et al. 1985;
Sugahara, Mizuno et al. 1992; Sugahara, Ohi et al. 1992).
Together these results indicate that the Hydra CS is synthesized as PGs, being covalently bound probably to a Ser
residue of the core protein through the linkage region tetrasaccharide. The structure of the GAG – protein linkage region of
Hydra HS could not be detected because of the limited
amount of sample. The data obtained from the analyses of
the chain sizes, disaccharide composition, and linkage region
structure of Hydra GAGs are summarized in the diagrams
shown in Figure 7.
Discussion
In the present study, we characterized GAGs isolated from
Hydra and demonstrated the existence of CS as well as HS.
This is the first study to demonstrate the existence of GAGs
in Hydra, although the presence of HS – PG core protein in
Hydra vulgaris has previously been suggested by an immunocytochemical analysis (Sarras et al. 1991). It has been reported
that Drosophila and C. elegans synthesize GAGs (Yamada
et al. 1999; Toyoda et al. 2000). Although various positions
in HS chains of these model animals are sulfated, their CS
chains are structurally unique. Caenorhabditis elegans contains only nonsulfated chondroitin chains, and CS chains of
Drosophila are nonsulfated or sulfated predominantly at the
C4 position of GalNAc residues. On the basis of these findings
we had expected Hydra to have no CS chains or only
nonsulfated chondroitin chains, because Drosophila and C.
elegans are assumed to be higher forms of life than H. magnipapillata. Interestingly, however, Hydra CS turned out to be
more heterogeneous in terms of sulfation than C. elegans
chondroitin and Drosophila CS. Since the CS chains from
lower animals are more complex than those from higher
animals, there seems to be no direct correlation between CS
structure and evolution. It cannot be ruled out that Hydra
acquired the genes of various CS sulfotransferases through
horizontal transfer.
Hydra CS contains not only nonsulfated and 4-O-sulfated
GalNAc residues but also 6-O-sulfated GalNAc residues. In
contrast, CS chains from C. elegans and adult Drosophila
are not 6-O-sulfated on GalNAc residues (Yamada et al.
1999; Toyoda et al. 2000), and a very small quantity of 6-Osulfated GalNAc was detected only in the third instar stage
in Drosophila larvae (Pinto et al. 2004). The complete
genome sequences of C. elegans (1 108 bp) and
Drosophila (1.4 108 bp) have been determined, but no
ortholog of the human CS 6-O-sulfotransferase gene has
been reported for these genomes. Although the complete
genome sequence of Hydra has not been determined or
reported, some genes, such as those for CS 4-O-sulfotransferase, CS 6-O-sulfotransferase, HS 2-O-sulfotransferase, HS
6-O-sulfotransferase, HS–GlcA C5-epimerase, chondroitin
synthase, exostosin-like 3, and galactosyl-galactosyl-xylosylprotein 3-b-glucuronosyltransferase, presumably encoding
enzymes involved in the biosynthesis of GAGs, have been
found in the GenBank database. The findings of this study
suggest that the enzymes required for the 6-O-sulfation of
GalNAc residues of CS chains exist in Hydra. Indeed, a gene
(accession number CN554591) encoding a protein with significant homology to human CS 6-O-sulfotransferase is present in
the expressed sequence tags of Hydra.
We also isolated and characterized the Hydra CS – core
protein linkage region and demonstrated that Hydra CS
chains are attached to core proteins through the common
linkage region tetrasaccharide, 24GlcAb1-3Galb1-3Galb14Xylb1 – . This indicates that Hydra CS exists as a PG,
although the core proteins remain to be identified. Some bacterial strains synthesize GAG-like polysaccharides and are produced as extracellular polysaccharide capsules that serve as
virulence factors. These polysaccharides are not attached
to core proteins and are synthesized on the inner surface of
the cytoplasmic membrane (DeAngelis 2002). Studies on the
GAG – protein linkage region of invertebrate GAGs are
limited, and the linkage region tetrasaccharide GlcA-GalGal-Xyl was isolated as a discrete structure from chondroitin
of C. elegans as well as HS and CS of Drosophila (Yamada
et al. 2002). Although the hydrozoan is a lower form of life
than the nematode, it has now been clarified that at least the
conventional linkage region tetrasaccharide of PGs has been
conserved from Hydra through evolution. The tetrasaccharide
linkage structure was not detected in Hydra HS, because of
the limited amount of HS.
A chain length analysis of Hydra CS and HS was also
carried out in this study. The average molecular size of
Hydra CS was estimated to be approximately 110 kDa,
which is similar to that of mammalian CS, but relatively
large in size. In contrast, the average molecular size of HS
chains from Hydra was only approximately 10 kDa.
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Although the chains of Drosophila HS have been demonstrated
to be relatively short (20 kDa) compared with mammalian HS
chains (Yamada et al. 2002), the average molecular size of the
HS chains from Hydra was half that of the Drosophila HS
chains. The reason why the chains of Hydra HS are much
shorter than those of Hydra CS remains to be elucidated.
Immunostaining showed that the epitope of the antichondroitin antibody 473A12 was present in the developing
nematocysts. Although their precise role in Hydra is unknown,
chondroitin PGs might furnish an environment for assembling
the components of nematocysts, or themselves be components.
Since chondroitin in C. elegans is indispensable for cytokinesis early during embryonic development (Mizuguchi et al.
2003), Hydra chondroitin may also be essential in cell division
and morphogenesis. The lack of staining with anti-HS antibodies might be because of the small amount of HS present
in Hydra. Anti-CS antibodies 2H6 and CS-56, which presumably recognize CS chains containing GlcA – GalNAc
(6-O-sulfate)–GlcA–GalNAc(6-O-sulfate) and GlcA–GalNAc
(4-O-sulfate)–GlcA(2-O-sulfate)–GalNAc(6-O-sulfate) sequences, respectively (Deepa et al. 2007), also showed no staining,
indicating the lack of the epitope structures recognized by these
antibodies in Hydra.
Although chondroitinase ABC depolymerizes not only CS
but also hyaluronate, no disaccharides from hyaluronate were
detected in the digest, suggesting its absence in Hydra. This
observation is consistent with the fact that no homologous
genes of human hyaluronan synthase 1 (accession number,
NM_001523.1) are found in the genome database of H. magnipapillata constructed by the J. Craig Venter Institute
(Rockville, MD), which is not yet available to the public.
Hyaluronan has been demonstrated to be involved in cell division in higher organisms (Brecht et al. 1986). As reported for
C. elegans, in which hyaluronan is not synthesized (Yamada
et al. 1999) and chondroitin plays critical roles in cytokinesis
and cell division (Mizuguchi et al. 2003), CS – PGs may have
similar roles in Hydra. Thus, Hydra is a useful organism for
studying functions of CS chains especially in cell division,
cytokinesis, and regeneration in the absence of hyaluronan.
Immunohistochemistry
Fluorescence immunohistochemical staining for whole-mount
and macerated samples was performed as described previously
(Nishimiya-Fujisawa and Sugiyama 1993; Yum et al. 1998).
Single cell samples were prepared by a maceration procedure
(David 1973). The following monoclonal antibodies against
GAGs used in this study were obtained from Seikagaku
Corp.: the anti-HS antibodies HepSS-1 and F58-10E4, antiCS antibodies 2H6 and CS-56, and anti-chondroitin antibody
473A12.
Preparation of a GAG-peptide fraction from Hydra
Frozen tissue of H. magnipapillata (120 g wet weight) was
homogenized in ice-cold acetone and lyophilized as described
previously (Yamada et al. 1999). The acetone powder was
digested with actinase E, treated with 5% trichloroacetic
acid, and precipitated with 80% ethanol as described previously (Yamada et al. 2002). The resultant precipitate, a
GAG – peptide fraction, was subjected to anion-exchange
chromatography on an Accell QMA Plus cartridge using stepwise elution with a 0.3 M phosphate buffer, pH 6.0, containing
0.15, 0.5, 1.0, or 1.5 M NaCl. Each fraction was desalted by
gel filtration on a PD-10 column using 50 mM pyridineacetate, pH 5.9, as the eluent, lyophilized, and dissolved in
water. Uronic acid in each fraction was determined by the
carbazole method (Bitter and Muir 1962).
Materials and methods
Enzymatic digestion
The Hydra samples fractionated by chromatography with the
Accell QMA Plus cartridge (corresponding to 0.1– 6.0 g of
Hydra) were digested with chondroitinase ABC, AC-I, AC-II
or B, chondro-4- or 6-sulfatase, or a mixture of heparinase
and heparitinase as described previously (Sugahara et al.
1994, 1995; Yamada et al. 1994; Yamada and Sugahara
2003). Unsaturated disaccharides produced enzymatically
from HS and CS were derivatized with 2AB, and then analyzed by anion-exchange HPLC on an amino-bound silica
PA-03 column (YMC Co., Kyoto, Japan) and gel-filtration
HPLC on a Superdex Peptide column (Amersham Pharmacia
Biotech) as reported previously (Kinoshita and Sugahara
1999; Bao, Mikami et al. 2005), or directly analyzed by
reversed-phase ion-pair chromatography with post-column
detection (Toyoda et al. 1999).
Materials
Chondroitinases ABC (EC 4.2.2.4), AC-I (EC 4.2.2.5), AC-II
(EC 4.2.2.5), and B (EC 4.2.2.), chondro-4-sulfatase (EC
3.1.6.9), chondro-6-sulfatase (EC 3.1.6.10), heparinase (EC
4.2.2.7), heparitinase (EC 4.2.2.8), and standard unsaturated
disaccharides derived from CS were obtained from
Seikagaku Corp. (Tokyo, Japan). Unsaturated standard HS disaccharides were prepared as described previously (Yamada
et al. 1992). Calf intestine alkaline phosphatase (EC 3.1.3.1)
of special quality for molecular biology was obtained from
Roche Molecular Biochemicals (Tokyo, Japan). Sephadex
gels, a prepacked Superdex 200 column (10 300 mm),
and prepacked disposable PD-10 columns were purchased
from Amersham Pharmacia Biotech (Tokyo, Japan). Sep-Pak
Plus AccellTM QMA anion-exchange cartridges were from
Waters Corp. Hydra was cultured as described previously
(Sugiyama and Fujisawa 1977).
Preparation of the GAG – protein linkage region from Hydra
The major Hydra GAG – peptide fraction, which was eluted
using 1.0 M NaCl on an Accell QMA Plus cartridge (corresponding to 30 g of Hydra), was treated with LiOH to liberate
O-linked saccharides including GAG chains from the core
proteins as described previously (Sakaguchi et al. 2001). The
purified sample was derivatized with 2AB. The excess 2ABderivatizing reagents were removed by paper chromatography
and gel-filtration chromatography (Ueno et al. 2001). The
2AB derivative of the Hydra GAG preparation (corresponding
to 6.0 g of hydra as the starting material) was digested with
10 mIU of chondroitinase ABC in a total volume of 25 mL
of 50 mM Tris –HCl buffer, pH 8.0, containing 60 mM
sodium acetate at 37 8C for 1 h. The digest was mixed with
15 mL of water and 10 mL of 250 mM sodium acetate buffer,
pH 6.0, containing 10 mIU of chondroitinase ACII at 37 8C
892
Glycosaminoglycans in Hydra magnipapillata
for 30 min. Alkaline phosphatase treatment of the chondroitinase digest was carried out using 4 IU of the enzyme in a
total volume of 100 mL of 0.08 M glycine – NaOH buffer,
pH 9.9, containing 0.5 mM MgCl2 at 37 8C for 30 min
(Sugahara, Mizuno et al. 1992). The enzymatic reactions
were terminated by heating at 100 8C for 1 min, and each
digest was analyzed by anion-exchange HPLC on an aminebound silica PA-03 column as described previously
(Kinoshita and Sugahara 1999).
Gel-filtration chromatography of the Hydra GAG preparation
The major Hydra GAG fraction, which was eluted with 1.0 M
NaCl on an Accell Plus QMA cartridge (corresponding to
30 g of hydra as the starting material), was analyzed by
gel-filtration chromatography on a column (10 300 mm)
of Superdex 200 eluted with 200 mM ammonium bicarbonate
at a flow rate of 0.3 mL/min. Fractions were collected at
4.0 min intervals, lyophilized, and digested with chondroitinase ABC or a mixture of heparinase and heparitinase. The
digests were derivatized with 2AB then analyzed by HPLC
on an amine-bound silica column (Kinoshita and Sugahara
1999).
Conflict of interest statement
None declared.
Acknowledgments
We thank Tomomi Nishimura and Eri Maegawa for technical
assistance. The work at Kobe Pharmaceutical University was
supported in part by HAITEKU (2004– 2008) from the
Japan Private School Promotion Foundation, Grant-in-aid for
Scientific Research C-17590078 (to S.Y.), Scientific
Research (B) 16370097 (to T.F.) and Scientific Research (B)
18390030 (to K.S.) from MEXT, The Human Frontier
Science Program, and CREST, JST.
Abbreviations
2AB, 2-aminobenzamide; CS, chondroitin sulfate; DS, dermatan
sulfate; ECM, extracellular matrix; GAG, glycosaminoglycan;
GalNAc, N-acetylgalactosamine; GlcA, D-glucuronic acid;
DHexA, 4-deoxy-a-L-threo-hex-4-enepyranosyluronic acid;
HPLC, high-performance liquid chromatography; HS, heparan
sulfate; IdoUA, L-iduronic acid; 2P, 2-O-phosphate; PG,
proteoglycan
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