Glycobiology vol. 13 no. 8 pp. 567±580, 2003
DOI: 10.1093/glycob/cwg070
Biochemical characterization of inner sugar chains of acrosome reaction±inducing
substance in jelly coat of starfish eggs
H.M.M. Jayantha Gunaratne2, Tohru Yamagaki3,
Midori Matsumoto4, and Motonori Hoshi1,4
2
Department of Biological Science, Graduate School of Bioscience and
Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta,
Midori-ku, Yokohama, Japan; 3Department of Chemistry, School of
Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan;
and 4Laboratory of Developmental and Reproductive Biology,
Department of Biosciences and Informatics, Keio University, 3-14-1,
Hiyoshi, Kohoku-ku, Yokohama, Japan
Received on January 18, 2003; revised on March 12, 2003; accepted on
March 13, 2003
The inception of the acrosome reaction (AR) in the starfish
Asterias amurensis is perceived to be strongly associated with
sulfated polysaccharide chains derived from an extremely
large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named
fragment 1 (Fr. 1), was composed of the repeating units of [ ! 4]b-D-Xylp-(1 ! 3)-a-D-Galp-(1 ! 3)-a-L-Fucp-4 (SO3±)-(1 ! 3)a-L-Fucp-4(SO3±)-(1 ! 4)-a-L-Fucp-(1 ! )n. In the current
study, this sugar chain is inferred to link to the peptide part
by O-glycosidic linkage through a sugar chain with different
structural features from Fr. 1. This inner sugar portion of
ARIS was isolated as Fr. 2 from the sonicated products of
pronase digest of ARIS. Fr. 2, which retains AR-inducing
activity to an admirable extent and has an apparent molecular
size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and
GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated),
Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the
major portion of the inner sugar chain of ARIS is composed of
the heptasaccharide units of ! 3)-Galp-(1 ! 3)-Fucp-(1 ! 3)G a l p -(1 ! 4)- G a lN A c p- (1 ! 4 )-G lcN A cp -6 (S O 3 ± )(1 ! 6)-Galp-4(SO3±)-(1 ! 4)-GalNAcp-(1 ! . This new
structure of inner sugar chains of ARIS is elucidated by
using electrospray ionization MS along with tandem mass
analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resinbased partial hydrolysis of Fr. 2. Furthermore, this study
corroborates that the sulfate groups are solely liable to the
anionic character of ARIS, which ought to be present in the
sugar chains of ARIS for its biological activity.
Key words: acrosome reaction/ARIS/CID MS-MS/egg jelly
coat/sulfated glycans
1
To whom correspondence should be addressed; e-mail:
[email protected]
Introduction
The acrosome reaction (AR) is an obligatory event for
fertilization in most metazoans. In starfish, the initiation
of AR is brought about by three major components in the
jelly coat of eggs: a highly sulfated proteoglycan-like molecule of an extremely large molecular size, referred to as
AR-inducing substance (ARIS); steroid saponins, named
Co-ARIS; and sperm-activating peptides known as asterosaps (Hoshi et al., 1994; Nishiyama et al., 1987; Nishigaki
et al., 1996). Among these components, ARIS itself is able
to trigger the AR in high calcium or high pH sea water,
whereas neither Co-ARIS nor asterosap has the ability to
induce the AR without the aid of ARIS (Nishigaki et al.,
1996; Ikadai and Hoshi, 1981a, b). Hence ARIS is considered the key component of the AR in the starfish. The
structural information of this unique molecule is therefore
essential in understanding the mechanism of AR in the
starfish.
Early studies performed in our laboratory indicate that
the AR-inducing activity of ARIS significantly remains
even after removing about 50% of proteins by pronase
digestion. Nevertheless, this activity is susceptible to periodate oxidation as well as desulfation (Koyota et al., 1997).
These results suggest that the intact sulfated sugar chains
are mostly responsible for the biological function of ARIS.
One of the sugar chains of ARIS with biological activity is
composed of a long linear polysaccharide chain consisting
of the pentasaccharide repeating units as [ ! 4]-b-D-Xylp(1 ! 3)-a-D-Galp-(1 ! 3)-a-L-Fucp-4(SO3 ÿ )-(1 ! 3)-a-LFucp-4(SO3ÿ )-(1 ! 4)-a-L-Fucp-(1 ! )n (Koyota et al.,
1997). This protein-free sugar fragment was isolated as
fragment 1 (Fr. 1) from the sonicated products of pronase
digest of ARIS (P-ARIS) on anion exchange chromatography. In this chromaotography, another fragment, which
was named Fr. 2 and also shown to have AR-inducing
activity to a certain extent, was obtained. This sugar portion
has been demonstated to retain the peptide part so that the
sugar chains of Fr. 2 are thought to derive from the inner
core region of ARIS. In this article, we describe the biochemical characterization of this biologically active core
region sugar portion of ARIS called Fr. 2 en route to the
complete structural elucidation of ARIS.
Results
The study of sugar chains of ARIS was carried out using
P-ARIS as the starting material because the elimination of
protein portion made it easier to handle the samples in
comparison with ARIS. The sonication of P-ARIS, by
which sample viscosity was considerably reduced, resulted
Glycobiology vol. 13 no. 8 # Oxford University Press 2003; all rights reserved.
567
H.M.M.J. Gunaratne et al.
in two major sugar fragments, Fr. 1 and Fr. 2. These sugar
fragments were successfully separated by anion-exchange
chromatography on DEAE Toyopearl 650 M under the
linear gradient of 0±1 M NaCl. In contrast to Fr. 1, the
second sugar fragment (Fr. 2) was shown to retain about
10% (w/w) of the peptide part, suggesting that sugar chains
of Fr. 2 are located in the region of carbohydrate-protein
linkage (core region). The estimated average molecular
mass of Fr. 2 by gel filtration on a calibrated column of
Sephacryl S-400 was demonstrated to be extremely large
(> 400 kDa), compared with Fr. 1 (10 kDa). Sulfate content
of Fr. 2 was estimated to be ~20% (w/w). Tests of Fr. 2 for
sialic acids, uronic acids, and phosphate groups, which
widely carry the anionic character of many glycans in addition to sulfate groups, were all negative. Altogether, with
the support of nuclear magnetic resonance (NMR) data, the
results conclude that sulfate groups are solely responsible
for the anionic character of ARIS.
Reductive elimination
Almost all the sugar portion of Fr. 2 was released by reductive b-elimination, indicating that the oligosaccharides of
Fr. 2 were O-linked. The gel filtration of the mixture of
b-eliminated products of Fr. 2 on a Sepharose CL-6B
resulted in one major sugar peak, which consisted of the
released O-glycans of Fr. 2. Then the major fractions were
collected and chromatographed on a DEAE-Toyopearl
column to find whether there were further separations.
However, further fractionation was not observed in this
chromatography, suggesting that released glycans would
be homogeneous as far as ionic character concerns. Thus
the fractions of this peak were collected, dialyzed against
water, and subjected to biochemical analysis. The results of
b-elimination of native ARIS also showed that the major
glycans derived from ARIS were O-linked.
Sugar composition analysis
Table I shows the results of sugar composition analysis.
Sugar compositions of native Fr. 2 and its released major
sugar chain were shown to be identical, indicating that all
the sugar chains of Fr. 2 were released by b-elimination.
Though Fr. 1 does not contain amino sugars, Fr. 2 was
shown to possess GalNAc and GlcNAc in addition to Fuc,
Gal, and Xyl. The data show that Xyl amount of Fr. 2 is one
third of Fr. 1. The result suggested the presence of novel
saccharide structures of ARIS. The most abundant sugar in
Table I. Sugar composition (approximate mol%) analysis of Fr. 1 and Fr. 2
Sugar
residue
Fr. 1
Native
Fr. 2
Released
sugar chains
of Fr. 2
Fr. 2-H1
Fuc
60
30
27
12
Xyl
17
06
06
Ð
Gal
23
29
28
40
GalNAc
Ð
22
24
34
GlcNAc
Ð
12
14
14
568
both Fr. 1 and Fr. 2 was observed to be Fuc, indicating that
Fuc is the major sugar residue in the carbohydrate portion
of ARIS. The data of Fr. 2-H1 will be discussed in the
following sections.
Linkage analysis
Glycosidic linkage analysis of released glycans of Fr. 2 and
the small fragments of Fr. 2 generated by partial acid hydrolysis (Fr. 2-H1) were carried out using methylation analysis
with some modifications (the results of the analysis of
Fr. 2-H1 will be discussed later in this article). Modifications
involved conversion of Fr. 2 to triethylammonium salt prior
to the methylation, which enhanced the solubility of the
sulfated sugar chains (Stevenson and Furneaux, 1991). Consequent methylations for three cycles were carried out to
ensure complete methylation. The desulfated Fr. 2 was
methylated without the modifications of original procedure.
Identification of partially methylated alditol acetates
(PMAAs) was made by gas chromatography mass spectrometry (GC-MS). The results are summarized in the Table II.
The quantitative data shown in the table were approximately calculated from the area of each peak corresponding
to each sugar alditol of the GC profile together with sugar
composition data of Fr. 2. The results clearly revealed
abundant occurrences of 4- and 2-O-sulfated Gal residues,
4-O-sulfated Fuc residue, and 6-O-sulfated GlcNAc residue. Glycosidic linkages of GlcNAc were shown to be
only 4-linked, whereas those of GalNAc are either 4- or
6-linked. A relatively low amount of Gal terminal residues
was detected as 2,4-O- and 2,3-O-disulated and 4-Omonosulfated. Terminal GalNAc residues appeared only
in the desulfated product, indicating that desulfation
might cause some partial degradation of sugar chains of
Fr. 2. Furthermore, considerable amounts of terminal Gal
and Fuc residues were also observed.
Although quite significant amounts of terminal sugar
were detected, no comparable amount of branching for
such terminals was reported in the methylation analysis of
Fr. 2. Detection of 3,6-Gal in both sulfated and desulfated
samples indicated either the presence of possible branching
points at C-6 or incomplete desulfation of the sample in
spite of the thorough desulfation. However the detected
amount of such linkage was too small to draw a conclusion.
Because Fr. 2 was obtained from the sonication of P-ARIS,
the sample may contain a mixture of sugar fragments generated from the breaking of different positions of the intact
sugar chains of P-ARIS, and such fragments are indistinguishable in the chromatographic separations due to the
small weight differences between such fragments. This
could probably account for the detection of many terminal
products in the methylation analysis of Fr. 2, which are not
necessarily representing the nonreducing sugar terminals of
the original sugar chains of ARIS. The linkages, 4-Osulfated -3-Fuc, 4-Fuc, 3-Gal, and 4-Xyl, which represent
the Fr. 1 sugar linkages, were all present in Fr. 2, too,
suggesting that Fr. 2 sugar chains are partially composed
of Fr. 1±like sugar chains.
Besides the information about linkage positions of individual sugar residues, the methylation analysis of Fr. 2
could be used to determine the ring size of some of the
Inner sugar chains of acrosome reaction±inducing substance
Table II. Linkage analysis of released glycans of Fr. 2
Mole ratio of glycosidic linkage
Sugar residue
Before
desulfation
After
desulfation
Location
of sulfate
substitution
3-linked Fuc
2.27
4.10
Ð
4-linked Fuc
0.55
0.58
Ð
3,4-linked Fuc
1.64
Ð
4 -O
1.10
Terminal Fuc
0.90
4-linked Xyl
1.00
1.00
3-linked Gal
2.90
3.36
6-linked Gal
0.36
1.10
Ð
4-linked Gal
0.36
Ð
4-O (terminal)
3,6-linked Gal
0.18
0.10
Ð
4,6-linked Gal
0.80
Ð
4 -O
2,4-linked Gal
0.18
Ð
2,4-O (terminal)
Ð
2,3-linked Gal
0.36
Ð
2,3-O (terminal)
Terminal Gal
0.27
1.00
Ð
4-linked GalNAc
3.28
2.70
Ð
6-linked GalNAc
0.64
0.50
Ð
Terminal-GalNAca Ð
0.80
Ð
4-linked GlcNAc
1.20
2.20
Ð
4,6-linked GlcNAc
0.90
Ð
6-O
Linkage positions were deduced by correlating the retention times on gas
chromatogram and fragmentation patterns of PMAAs of Fr. 2. Molar ratio
was calculated by setting the area under the 4-linked Xyl peak as 1.00 and
assuming equal molar response for the derivatives. Values for HexNAc
were calculated from their relative percentage distribution and from the
amounts of HexNAc determined by sugar composition analysis.
a
Because terminal GalNAc was detected only in the desulfated sugar
chains, it should arise from artifacts resulted by degradation during
desulfation. The detection of a quite significant amount of sugar terminal
products may account for the presence of sugar fragments with different
non-reducing terminals generated from the breaking of different positions
of the intact sugar chains of P-ARIS by sonication.
sugar residues. The data of tandem mass spectrometry
(MS/MS) analysis of PMAAs of Fr. 2 showed the presence
of 1,3,5-tri-O-acetyl-(1-deuterio)-2,4-di-O-methyl fucitol
(m/z 234, 131, 118) and 1,3,5-tri-O-acetyl-(1-deuterio)2,4,6-tri-O-methyl galactitol (m/z 234, 161, 118, 45), which
can only originate from the corresponding alditols of pyranose form of 3-Fuc and 3-Gal, respectively (the m/z values
are only the values of primary fragments). Detection of
1,5,6-tri-O-acetyl-(1-deuterio)-2,3,4-tri-O-methyl galctitol
(m/z 233, 189, 162, 118), 1,5-di-O-acetyl-(1-deuterio)2,3,4,6-tetra-O-methyl galctitol (m/z 249, 206, 205,162,161,
118), and 1,5-di-O-acetyl-(1-deuterio)-2,3,4-tri-O-methyl
fucitol (m/z 175, 162, 131, 118) indicate the presence of 6Gal, terminal-Gal, and terminal-Fuc in their pyranose ring
form. Therefore, all the sugar residues, which are major
neutral hexoses present in Fr. 2, are in the pyranose form.
In the case of HexNAc, detection of 1,5,6-tri-O-acetyl(1-deuterio)-(2-N-methylacetamide)-3-,4-di-O-methyl galctitol (m/z 247, 233, 203, 189, 159) and 1,5-di-O-acetyl-(1deuterio)-(2-N-methylacetamide)-3,4,6-tri-O-methyl galctitol
Fig. 1. 1D proton NMR spectrum at 400 MHz of released sugar chains of
Fr. 2 (a) and its expanded spectrum of anomeric region (b). The spectrum
shows that amino sugars are acetylated and fucose residues are in more
than three different environments. The NMR integrals from A to G in the
anomeric region mainly represent the presence of a anomeric resonance.
The signals from I to M may be attributable to the anomeric resonance of
b-linked Gal and HexNAc residues. The spectrum was recorded at 343 K,
and the HDO peak was at d 4.35 ppm.
(m/z 247, 205, 203, 161, 159, 45) indicate the presence of 6GalNAc and terminal GalNAc, respectively, in their pyranose form in Fr. 2. However, the alditols of GlcNAc and
other linked GalNAc could not be identified whether they
derived from pyranose ring form by using MS/MS analysis
of PMAAs of Fr. 2 because both C-4 and C-5 are acetylated
in suggested 4-linked HexNAcs. According to the facts that
the naturally occurring HexNAc generally exists in pyranose form and 5-linked HexNAc is unusual, it may not be
irrational to assume that such HexNAcs exist as pyranose
form of 4-GalNAc and 4-GlcNAc. Further, electrospray
ionization (ESI) MS/MS analysis of Fr. 2-H1, as will be
discussed later in this article, showed the presence of
4-HexNAcs, indicating that they should exist in pyranose
form. As will be discussed later in this article, Xyl is suggested to come from remaining Fr. 1 sugar units, thus it
would exist as pyranose ring form (Koyota et al., 1997).
Proton NMR spectroscopy
The proton NMR spectrum of the released sugar chains of
Fr. 2 is presented in Figure 1. Much structural information
was not available in this NMR analysis due to complexity of
569
H.M.M.J. Gunaratne et al.
the spectrum (because of high molecular weight) with many
unresolved signals. However, the spectrum revealed some of
the preliminary structural information of Fr. 2. The unresolved strong signals between d 1.18 ppm and 1.30 ppm
represented methyl hydrogen atoms of fucosyl residues
(total relative area of the signals is 14.63), which should
exist in more than three different environments in the
sugar chains of Fr. 2. Unresolved but prominent signals
corresponding to the methyl protons of acetyl groups of
amino sugars with the total area of 16.85 confirmed the
presence of N-acetylated amino sugars. These NMR integrals are in good agreement with the sugar composition
data. Though not exact, the signals in anomeric region
revealed preliminary information of anomeric resonance
of sugar chains of Fr. 2.
Anomeric signals of A, B, C, D, E, and F were clearly
shown to originate from a-linked monosaccharide residues.
The relatively small coupling constants (below J ˆ 3) of
signals G (d 4.72 ppm) and H (d 4.65) implied that they
were not originated from H-1 of b-linked residues even
though they located as down field as d 4.70 ppm. They
may be attributable to either Hs attached to the sulfatesubstituted carbon atoms or anomeric Hs of a-linked
monosaccharide residues. Typical doublet signals between
d 4.63 ppm and d 4.40 ppm with significant coupling constants (from I to M) may be originated from anomeric
resonance of b-linked sugar residues, particularly from
Gal and HexNAc. Some of the anomeric signals of this
spectrum could be corresponding somewhat to the anomeric signals of repeating units of Fr. 1 sugar chains. Furthermore, the signals around d 2.8±2.5 ppm and 1.9±1.5 ppm,
which are characteristic for H-3eq and H-3ax of sialic acid,
respectively, were observed to be absent in the spectrum,
confirming the absence of sailic acid in Fr. 2.
ELISA
Linkage analysis and NMR analysis provided some evidence for the presence of the sugar chains similar to Fr. 1 in
Fr. 2. Enzyme-linked immunosorbent assay (ELISA), using
specific antibody for Fr. 1, was performed for Fr. 2 to get
further evidence for this observation. Our laboratory has
developed a specific ELISA for Fr. 1 (unpublished data).
This antibody was previously tested to be positive for ARIS,
P-ARIS, and Fr. 1, whereas these became negative after
periodate oxidation or desulfation of the sugar chains, suggesting that the antibody is specific for sulfated intact sugar
chains of Fr. 1. In the present study, this assay was applied
to native Fr. 2, released glycans of Fr. 2 by b-elimination,
and oxidized (by periodate) and desulfated Fr. 2.
We observed positive results for both the native and
released glycans of Fr. 2 and negative results for the oxidized and the desulfated products. These results provided
further evidence of the presence of some Fr. 1 repeating
units
as
[ ! 4]-b-D-Xylp-(1 ! 3)-a-D-Galp-(1 ! 3)a-L-Fucp-4(SO3 ÿ )-(1 ! 3)-a-L-Fucp-4(SO3 ÿ )-(1 ! 4)-a-LFucp-(1 ! ) in Fr. 2, which is likely the source for the
positive result of the ELISA for Fr. 1. As will be discussed
later in this article, Fr. 1 is assumed to be located in the
outermost region of ARIS sugar chains, which is linked to
the protein part through the inner core region, namely, Fr. 2.
570
Fr. 1 and Fr. 2 are generated by the sonication of P-ARIS.
Thus there is a good possibility that some sugar-repeating
units similar to Fr. 1 remain in the protein-free end of Fr. 2.
Partial acid hydrolysis of Fr. 2
Further structural studies of Fr. 2, especially sugar sequencing studies, were restricted by high molecular mass of the
sugar chains of Fr. 2. Therefore, either specific or nonspecific degradation procedures were required to obtain easily
manageable fragments from the intact chain. For this purpose, we attempted specific degradation methods, such as
Smith degradation, deamination, and so on with Fr. 2 (data
not shown). All of the methods, except hydrolysis with a
strong acidic-ion exchanger (as will be described later in this
article), failed, probably because of a number of sulfate
groups associated with Fr. 2 and its largeness. The sample
of released sugar chains of Fr. 2 was subjected to partial
acid hydrolysis using Dowex 50W (H ‡ form) to obtain
easily manageable fragments. In contrast to the common
procedures of partial hydrolysis, this method allows cleaving of HexNAc glycosidic bounds successfully in a single
step with the prevention of cleaving acetyl groups (Mark
et al., 2000). Hence, the method is fit for Fr. 2 because it is
composed of a significant amount of HexANc.
On the resin hydrolysis of Fr. 2, the hydrolysate fractions
were isolated by spin filtrations. Because the total fractions
were not amenable to mass analysis, smaller fractions were
isolated as Fr. 2-H1 (containing sugar fragments 5 5 kDa)
as described in Materials and methods. Although this hydrolysis procedure was successful in providing the fragments
amenable for the mass analysis, the yield obtained per cycle
of hydrolysis was very low, indicating that the reaction was
still restricted by its molecular size and/or sulfates. Therefore, the hydrolysis was repeated for the remaining bigger
part of sugar chains (45 kDa) to obtain adequate amounts
of the sample for further analysis. Finally, ~5% of the yield
was obtained with the repeating of four cycles of hydrolysis.
However, the fractions obtained after the fourth cycle of
repeated hydrolysis were accompanied with high noises in
MS experiments. This may be due to the presence of degradative products of the resin molecules in hydrolysates.
Sugar composition
The result of sugar composition analysis of Fr. 2-H1 is
tabulated in Table I. The results showed the absence of
Xyl and the presence of considerable amount of HexNAc
and Gal in Fr. 2-H1.
MS analysis of hydrolysates of Fr. 2
A sample of the major sugar fragments generated by the
partial hydrolysis of Fr. 2 (Fr. 2-H1) was subjected to MS
analysis to obtain the sequencing information pertaining to
the major sugar portion of Fr. 2. This ESI-MS analysis was
carried out in positive ion mode, and ions were detected as
mainly sodium-adduct ions. According to the sugar composition data, Fr. 2-H1 is composed of high amounts of
HexNAc and Gal and less Fuc. Though not precise, we
have observed that more than 10% (w/w) of sulfate is
present in Fr. 2-H1 by sulfate content analysis of Fr. 2-H1.
Altogether, we could propose possible components
Inner sugar chains of acrosome reaction±inducing substance
Table III. Possible components generated from H ‡ resin hydrolysis of
Fr. 2 sugar chains
m/z
Assumed ion composition
1486
Hex3FucHexNAc3(SO3Na)2 ‡ Na ‡
1464
Hex3FucHexNAc3(SO3Na)2 ‡ H ‡
1384
Hex3FucHexNAc3(SO3Na) ‡ Na ‡
1324
Hex2FucHexNAc3(SO3Na)2 ‡ Na ‡
1302
Hex2FucHexNAc3(SO3Na)2 ‡ H ‡
1222
Hex2FucHexNAc3(SO3Na) ‡ Na ‡
1178
Hex2HexNAc3(SO3Na) 2 ‡ Na ‡
1156
Hex2HexNAc3(SO3Na) 2 ‡ H ‡
1076
Hex2HexNAc3(SO3Na) ‡ Na ‡
1016
HexHexNAc3(SO3Na) 2 ‡ Na ‡
994
HexHexNAc3(SO3Na) 2 ‡ H ‡
914
HexHexNAc3(SO3Na) ‡ Na ‡
813
HexHexNAc2(SO3Na) 2 ‡ Na ‡
791
HexHexNAc2(SO3Na) 2 ‡ H ‡
711
HexHexNAc2(SO3Na) ‡ Na ‡
610
HexHexNAc(SO3Na) 2 ‡ Na ‡
609
HexHexNAc2 ‡ Na ‡
549
HexNAc2(SO3Na) ‡ Na ‡
508
HexHexNAc(SO3Na) ‡ Na ‡
406
HexHexNAc ‡ Na ‡
349
HexFuc ‡ Na ‡
(as tabulated in Table III) in which these components correspond to the major signals appeared in ESI spectrum of
Fr. 2-H1 shown in Figure 2. In addition to the sodium
adduct forms, protonated forms, though not abundant,
were also observed in this ESI spectrum. The results showed
that both mono- and disulfated components were predominant. However the completely desulfated components were
less abundant. The highest molecular-related ion was
observed at m/z 1486 as sodium adduct or 1464 as protonated form, which could be derived from the chemical
species of Hex3FucHexNAc3(SO3Na)2. Because Gal was
detected as the only Hex in this sample, all Hex in the
table can be replaced by Gal. The molecular-related ions
at m/z 1324, 1178, 1016, and 813 were shown to possess two
sulfate groups. Except for the molecular-related ion at
m/z 1324, all of these ions were consist of only Gal and
HexNAc. Thus, the sulfate groups should attach to either
Gal or HexNAc, not Fuc.
The methylation analysis of Fr. 2-H1, discussed later in
this article, showed that there was no branching in the
hydrolysates. Therefore all the sugar fragments in Fr. 2-H1
should be linear. The molecular ions at 1324 and 1178 are
consistent with the ions resulted by the removal of Gal
and the removal of GalFuc from the ion at m/z 1486,
respectively. Thus the nonreducing terminal of the sequence
corresponding to the molecular ion at m/z 1486 presumed to
be composed of a Gal-Fuc-sequence. Observation of an ion
at m/z 349 supports the presence of GalFuc in Fr. 2-H1,
which may have originated from the nonreducing end terminal of the sequence corresponding to the molecular-related
ion at m/z 1486. The molecular ions at m/z 1016 and 813 can
be the partial fragments generated by the loss of Gal2Fuc
and Gal2FucHexNAc, respectively, from sugar chin, corresponding to the molecular-related ion at m/z 1486, implying
Fig. 2. ESI mass spectrum of the hydrolysate fragments of Fr. 2, Fr. 2-H1 (a) and its expanded high mass region (b). The spectrum (a) displays the
signals attributable to the components in the small sugar fragments, Fr. 2-H1 (5 kDa) generated by resin hydrolysis. Many of the molecular ions were
detected as Na ‡ adduct ions, but corresponding H ‡ adduct ions were also present. Both di- and monosulfated Na ‡ adduct precursor ions are predominant,
whereas completely desulfated ions are less abundant, indicating that one of the sulfate group is much more stable for this hydrolysis. The chemical
species suggested for major molecular ions are tabulated in Table III. See MS analysis of hydrolysates of Fr. 2 for a more detailed explanation.
571
H.M.M.J. Gunaratne et al.
that the nonreducing terminal may be composed of a GalFuc-Gal-HexNAc sequence. The molecular-related ion at
m/z 549 represents exclusively HexNac2(SO3Na)‡Na ‡ ion,
indicating that two HexNAc units must be directly linked
each other and one of them must be sulfated. On the other
hand, the very prominent ions at m/z at 813 and 711 and at
609 can be originated from a chemical species of GalHexNAc2, reflecting the extent of the sulfate substitution(s).
The results of collision-induced dissociation (CID) MS/
MS of all these three ions, to be discussed, suggested that
the sequence corresponding to all three would be HexNAcGal-HexNAc, which may represent the reducing-end
terminal sequence, the ion at m/z 813 being disulfated.
According to the data of methylation analysis of Fr. 2-H1,
discussed later in this article, the sample contained monosulfated Gal and monosulfated GlcNAc residues. We have
already stated that HexNAc2(SO3Na) must be present in
the sugar sequence. Taking all these observations into
account, the reducing-end terminal sequence would
be HexNAc-HexNAc(SO3Na)-Gal(SO3Na)-HexNAc. The
molecular-related ion at 610 can only be derived from the
formula of HexNAcGal(SO3Na)2, indicating that sulfated
HexNAc and sulfated Gal must be linked to each other.
This observation is a positive clue for the presence of the
reducing terminal sequence. Taking all the facts discussed
into consideration, we could assume that the sugar sequence
corresponding to the ion at m/z 1486 is Gal-Fuc-GalHexNAc-HexNAc(SO3Na)-Gal(SO3Na)-HexNAc. Except
for the Gal-Fuc sequence at nonreducing terminal of this
sequence, the rest was confirmed by the CID MS/MS
analysis of corresponding molecular ions as follows.
CID MS/MS analysis of major precursor ions
The CID MS/MS spectra of the major molecular ions were
dominated mainly by abundant, mainly Y- and B-type ions
(nomenclature of Domon and Costello, 1988). Under general conditions, particularly at low collision energy, Y- and
B-type fragmentations are predominant in CID MS/MS
analysis of oligosaccharides (Harvey, 2000a,b). The CID
MS/MS spectrum of the precursor ion at m/z 508 and the
schematic explanation of the fragmentation of the ion are
shown in Figures 3a and 3b, respectively. The ions at m/z
244 and 287 correspond to Y- and B-type glycosidic cleavages, respectively. The product ion at m/z 407 is attributable to the removal of 101 units from the reducing-end
HexNAc by 0 ,2 A cross-ring cleavage. It can be assumed
that this type of cleavage is observed in a sugar sequence,
which is terminated with -(1 ! 4)-HexNAc residue at the
reducing end. The results of methylation analysis of Fr.
2-H1 (discussed later in this article) have indicated that all
the HexNAcs of the suggested sugar chain for Fr. 2-H1 are
4-linked. Thus such type of cross-ring cleavage can be
observed from the sugar chains with -(1 ! 4)-HexNAc residue in ESI CID MS/MS mode, much like in matrix-assisted
laser desorption/ionization-post source decay as previously
reported by Yamagaki and Nakanishi (2000).
In addition to glycosidic cleavages, the product ions
obtained from the removal of sulfate group were also
observed. The removal of a sulfate group can be identified
by observing the loss of 120 units (NaHSO4) from the
572
Fig. 3. CID MS-MS spectrum of selected precursor ion at m/z 508 (a) and
the schematic illustration of the fragmentation patterns corresponding to
the major signals (b). The product ions were dominated by the Y- and
B-type fragment ions. A ring cleavage was observed at m/z 407 (0;2 A).
The sulfate removal was observed from the precursor ion and B-type ion
as indicated in the sketch (b).
precursor ion or product ions. This is also confirmed by
the fragment ions derived from the loss of 102 units from
sequence ions. The loss of 102 units is accounted for in
terms of exchange reaction of the sulfate group with hydrogen (Ii et al., 1995); therefore there is no possibility to
undergo this reaction from precursor ions themselves. It is
because CID MS/MS detects ions that resulted from the gas
phase unimolecular dissociation of the precursor ion only.
In other words, there is no extra hydrogen to replace the
(-SO3Na) group within the precursor ion. The removal of
the sulfate group through both routes from the precursor
ion at m/z 508 was observed at m/z 388 and 185 as indicated
in Figure 3b.
According to all the product ions, the sequence corresponding to this precursor ion must be Gal(SO3Na)HexNAc. The CID MS/MS spectrum of the precursor ion
at m/z 609 and the schematic explanation for the cleavages
are shown in Figure 4. All the Y- and B-type glycosidic
cleavages, as well as cross-ring cleavages from the reducing
terminal, were observed from this precursor ion. Two types
of cross-ring cleavages at m/z 508 (0 ,2 A) and 448 (2 ,4 A)
resulted from the reducing end clearly indicated that
Inner sugar chains of acrosome reaction±inducing substance
Fig. 4. CID MS-MS spectrum of selected precursor ion at m/z 609
(a) and the schematic illustration of the fragmentation patterns
corresponding to the major signals (b). The products ions were dominated
by the Y- and B-type fragment ions. The major ring cleavages, which
could originate from 4-linked reducing terminal HexNAc, were observed
at m/z 448 (2 ,4 A) and 508 (0 ,2 A). This observation (together with the
identical results illustrated by Figures 3 and 5) suggests that a HexNAc
residue is located at reducing terminal and is 4-linked to adjacent sugar
residue. The sketch explains the routes of formation of all major
product ions.
reducing sugar of this sequence should be 4-linked
HexNAc. A presence of product ions at m/z 406 (Y2) and
226 (B1), both sodiated, indicated that the nonreducing
sugar would also be HexNAc; thus the sequence corresponding to this ion should be HexNAc-Gal-HexNAc. The
CID MS/MS spectrum of precursor ion at m/z 711 showed
(the MS/MS spectrum is not shown) that its sequence was in
accord with that of the ion at m/z 609 but in monosulfated
form. Other expected cleavages were observed in the
MS/MS results of this ion. Among them, the presence of
product ion at m/z 328 originated by B1 (sodiated) cleavage
implied that the sulfate group would be located in nonreducing end HexNAc residue. The CID MS/MS spectrum of
ion at m/z 813 and the schematic explanation for the
formation of the product ions are shown in Figure 5. In
addition to the removal of sulfate group as -NaHSO4 and
(-SO3Na‡H ‡ ), the removal of Na2S2O7 (222 units) was
observed in this spectrum, indicating that the ion consists
of two sulfate groups. These sulfate removals were observed
from the precursor ion itself and the product ion at m/z 712.
Fig. 5. CID MS/MS spectrum of precursor ion at m/z 813 (a) and the
schematic illustration of the fragmentation patterns corresponding to the
major signals (b). Product ions corresponding to Y- and B-type glycosidic
cleavages, sulfate removal, and ring cleavage at reducing terminal was also
observed. The results are illustrated in the sketch (b). See CID MS/MS
analysis of major precursor ions for further details.
Other sulfate removal routes are shown in the Figure 5b.
The product ion at m/z 712 indicated ring cleavage fragmentation of 0 ,2 A from reducing HexNAc residue.
Taking all the CID MS/MS data discussed so far into
consideration, we confirm the reducing terminal sequence
of sugar Fr. 2-H1 as HexNAc(SO3Na)-Gal(SO3Na)HexNAc. The CID MS/MS analysis of the precursor ion
at m/z 1016 (the spectrum is shown in Figure 6b), corresponding to the component of GalHexNAc3(SO3Na)2‡
Na ‡ , yielded some Y-type fragment ions at m/z 813 (loss
of HexNAc), 508 (loss of HexNAc2[SO3Na]), and their
desulfated product ions as indicated in Figure 6a. A prominent ion at m/z 915 (the loss of 101 units by 0;2 A cross-ring
cleavage of reducing terminal HexNAc) indicated that the
reducing terminal of the putative sugar sequence of the
precursor ion at m/z 1016 should be HexNAc residue.
Therefore this ion is proof of the presence of a HexNAc at
the both terminals.
The peak at m/z 204 is attributable to the anhydrous form
of the [HexNAc‡H] ‡ product ion. The results and explanation of the CID MS/MS analysis of the ion at m/z 1076,
corresponding to the ion of Gal2HexNAc3(SO3Na)‡Na ‡ ,
are shown in Figure 7. The product ion at m/z 914 clearly
indicated the removal of Gal by Y-cleavage and the ion at
m/z 975 indicated the loss of 101 units by 0 ,2 A cross-ring
cleavage of reducing terminal HexNAc. Thus the sequence
573
H.M.M.J. Gunaratne et al.
Fig. 6. CID MS/MS spectrum of precursor ion at m/z 1016 (a) and the
schematic illustration of the fragmentation patterns corresponding to the
major signals (b). Though B-type ions were not observed from this
precursor ion, prominent Y-type ions and their product ions generated
from sulfate removal/s were abundant. The product ion at 915 represents
cross-ring cleavage, suggesting the sequence is terminated with reducing
HexNAc residue.
corresponding to this precursor ion should be terminated
with Gal at nonreducing end and HexNAc at reducing end.
The CID MS/MS spectrum of the precursor ion at
m/z 1178 (the spectrum is not shown), corresponding to the
components of Gal2HexNAc3(SO3Na)2‡Na ‡ has shown
product ions, at m/z 1016, 813, and 508, which were corresponding to the Y-type fragments. Among these ions, the
ion at m/z 1016 indicated the loss of Gal from the nonreducing terminal. The ion at m/z 813, which is likely to correspond to the component GalHexNAc2(SO3Na)2‡Na ‡ , as
the result of loss of GalHexNAc from the precursor ion,
was not prominent. However, the strong signals at m/z 711
and 609 indicated mono- and desulftaed components of the
corresponding component of m/z 813, respectively. Though
weak, the ion at m/z 1077, which is attributable to the
removal of 101 units from the reducing HexNAc by 0;2 A
cross-ring cleavage, was observed.
The CID MS/MS analyses for weak ions of m/z 1486 and
1324 could not be carried out. Therefore the presence of
GalFuc sequence is yet to be confirmed. Though we have
attempted to carry out MS/MS analyses of weak ions at
m/z 1486 and 1324, satisfactory results have not been
obtained. However, according to the ESI spectrum of
574
Fig. 7. CID MS/MS spectrum of precursor ion at m/z 1076 (a) and the
schematic illustration of the fragmentation patterns corresponding to the
major signals (b). Some Y-type ions and their product ions generated from
sulfate removal/s were prominent. The product ion at m/z 914 represents Y4
cleavage, suggesting that nonreducing sugar in this precursor is Gal. The
product ion at m/z 975 represents cross-ring cleavage, indicating that the
sequence is terminated with a reducing HexNAc residue.
Fr. 2-H1, this GalFuc would be at a nonreducing terminal.
This is because no molecular ions corresponding to reducing terminal fragments with this sequence (such as
HexNAcFuc/Gal or Gal(SO3Na)-HexNAc Fuc/Gal) were
observed in the ESI spectrum, whereas other reducing
terminal species (such as HexNAc(SO3Na)-Gal(SO3Na)HexNAc or Gal(SO3Na)-HexNAc) were abundant. Therefore we can suggest a sugar sequence for the biggest
fragment in Fr. 2-H1 as depicted in Figure 8a.
Methylation analysis of Fr. 2-H1 and linkage assignment
For the linkage analysis, sample of PMAAs of the hydrolysate of Fr. 2 (Fr. 2-H1) and its desulfated sample were
prepared. When compared with the intact sugar chains of
Fr. 2, sugar fragments in the hydrolysate of Fr. 2 used for
this analysis were small by means of quantity as well as the
molecular weight. Therefore desulfation and preparation of
PMAAs of the sample were carried out without modifications of the original procedure. The result of this analysis is
summarized in Table IV. Some nonreducing terminals,
liberated from the partial acid hydrolysis, were observed
in the linkage analysis of Fr. 2-H1. However they were not
included in the table because they were not so significant for
the linkage assignment.
Inner sugar chains of acrosome reaction±inducing substance
Fig. 8. Suggested structure of the major sugar chain of Fr. 2. A sugar sequence for the major sugar chain of Fr. 2 was obtained by the mass analysis
of the major product resulted from resin hydrolysis. A detailed structure (a) could be drawn by the assignment of the results of methylation analysis
for sugar A to G in the suggested sugar sequence (b).
Table IV. Linkage analysis of Fr. 2-H1
Mole ratio of glycosidic linkage
Sugar residue
Fr. 2-H1
Desulfated
Fr. 2-H1
3-linked Fuc
1.0
1.0
1.8
3-linked Gal
2.1
4,6-linked Gal
0.8
Ð
6-linked Gal
0.6
1.6
4-linked GalNAc
2.1
2.2
4,6-linked GlcNAc
0.8
Ð
4-linked GlcNAc
0.3
1.2
Location
of sulfate
substitution
4-O
6-O
Linkage positions were deduced by correlating the retention times on gas
chromatogram and fragmentation patterns of PMAAs of Fr. 2-H1. Molar
ratio was calculated by setting the area under the 4-linked Fuc peak as 1.0
and assuming equal molar response for the derivatives. Values for HexNAc
were calculated from their relative percentage distribution and from the
amounts of HexNAc determined by sugar composition analysis.
Furthermore, neither branching nor disulfated sugars
were detected by this methylation analysis of Fr. 2-H1.
The linkage information in Table IV shows that sulfate
substituted amino sugar is only GlcNAc, thus sugar C
(Figure 8b) should be GlcNAc. The study also showed the
absence of 4,6-linked GlcNAc and the presence of increased
amount of 4-linked in desulfated sample, indicating that
GlcNAc is 6-O sulfated. Because the total sugar composition of GalNAc:GlcNAc was shown to be ~2:1, the other
two HexNAc residues (A and D) should be GalNAc. The
results further showed that GalNAc and GlcNAc residues
were all 4-linked. Three linkage types were detected for Gal
as 3-, 6-, and 4,6-linked in the sulfated sample. However, the
detection of increased amount of PMAAs from 6-, the same
amount of 4-, and the absence of 4,6-Gal in desulfated
sample indicated that Gal should be 4-O-sulfated. Therefore, B should be 4,6-Gal, whereas the other two, E and G,
should be 3-Gal. The Fuc (F) should be 3-linked. The
detection of 6-Gal and 4-GlcNAc in the sample before
desulfation might be attributable for the presence of some
fragments with originally sulfate-free or desulfated sugars
in the hydrolysate.
Taking all the data of MS and the methylation analysis
into account, we propose a sugar structure for the major
fragment present in the hydrolysate as depicted in Figure 8a.
The total yield of Fr. 2-H1 obtained from four repeating
cycles of hydrolysis was nearly 55% (w/w) and the same
results in the mass analysis for the fragments generated
from every consequent hydrolysis were observed, evoking
conclusions that new sugar sequence seems to represent the
major part of Fr. 2. As discussed previously in this article,
some part of the sugar chains of Fr. 2 could be composed of
the similar sugar-repeating units of the Fr. 1. Providing that
all the Xyl residues of Fr. 2 are attributable to this part of
sugar chain, approximately a fourth of sugar residues of
Fr. 2 should be composed of Fr. 1 repeating units (nearly
100 units). If this assumption is the case, the other sugar
portion of Fr. 2 would be composed of Fuc:Gal:GalNAc:GlcNAc in a molar ratio of 1:2:2:1. Unlike Fr. 1, these
new sugar chains were shown to contain mono- and disulfated Gal, 6-linked Gal, sulfated GlcNAc, and GalNAc
besides Fuc. The suggested sugar structure (Figure 8a)
represents the major linkages shown, indicating that it consists of the major part of Fr. 2. The results shown in Table II
indicated that more than half of GlcNAcs were sulfated in
intact sugar chains of Fr. 2. Provided that all this amount of
sulfated GlcNAc is assigned to the proposed new sugar
chain of Fr. 2, sugar chains of Fr. 2 would be composed
of at least 120 units of suggested heptasaccharide domains.
As stated previously, Fr. 2 is assumed to consist of partly
the Fr. 1 repeating units. However, until recently it was not
clear whether they are derived from one single sugar chain
of ARIS or not. In the current study, we have obtained
a quite homogenous mixture of sugar chains after belimination by means of size and acidity, suggesting that
Fr. 1 repeating units are derived from the same sugar chain
of Fr. 2. Furthermore, Fr. 1 was observed to be protein free,
whereas Fr. 2 was shown to retain peptides. Based on all the
results discussed so far and with some assumptions, the
major glycans of ARIS can be suggested as follows:
(P)m4200 ± (Q) ± (R)n4100-(S) ± Ser/Thr (Protein), where
P is [ ! 4)-b-D-Xylp-(1 ! 3)-a-D-Galp-(1 ! 3)-a-L-Fucp4(SO 3 ÿ )-(1 ! 3)-a-L-Fucp-4(SO 3 ÿ )-(1 ! 4)-a-L-Fucp(1 ! and R is ! 3)-Galp-(1 ! 3)-Fucp-(1 ! 3)-Galp(1 ! 4)-GalNAcp-(1 ! 4)-GlcNAcp-6(SO 3 ÿ )-(1 ! 6)Galp-4(SO3 ÿ )-(1 ! 4)-GalNAcp-(1] ! . The structures of
parts Q and S have yet to be revealed. Furthermore, the
anomeric information is still not available for the suggested
new sugar sequence. In fact, we have attempted with glycosidase enzyme digestion to reveal this information. Because
of the limited sample availability, we have not yet obtained
fragments from these reactions amenable for mass analysis.
According to this proposed structure, the sugar chain of
Fr. 1 is located in the outermost region of ARIS, which is
linked to the peptide part through the sugar chain of Fr. 2,
575
H.M.M.J. Gunaratne et al.
called inner sugar chain. Therefore the sugar chains in outermost region of ARIS entirely differ from the inner sugar
chains in terms of sugar compositions and linkages as well
as sulfate substitutions. Although we have yet to reveal the
structural information of sugar chains of Q and S in Fr. 2,
the major sugar chains derived from ARIS (Fr. 1 and major
sugar part of Fr. 2) were shown to be linear and very long
as well. Thus ARIS is likely to be a proteoglycan-like
molecule.
Discussion
Despite the fact that drawing the structural features of core
region carbohydrate portion of ARIS (Fr. 2) is an extremely
difficult task because of its highly complex structure and
largeness, we have gained some important information pertaining to the primary chemical structure of core region
glycans of ARIS in current study. We have already shown
that ARIS is a proteoglycan-like molecule with very long
sulfated sugar chains. Even after removing of the protein by
b-elimination, the entire sulfated sugar chain of ARIS alone
was exhibited to possess much higher activity than the
fragments generated by sonication (Fr. 1 and Fr. 2), suggesting the necessity of the whole sugar chain for efficient
AR-inducing activity. Furthermore, the periodate oxidation and/or desulfation of these sugar chains abolished
the activity (data not shown). These results suggested
that initiation of AR in the starfish is associated with
more complex sugar chains. In contrast to the starfish, sea
urchins use the sugars with much less complex structures
for the same purpose. For instance, linear sulfated a-Lfucans in Arbacia lixula and Lytechinus variegatus (Alves
et al., 1997), [3a-L-Fucp-2(SO3 ÿ ),4( SO3 ÿ -)-1]n and
[3a-L-Fucp-2,4(SO3 ÿ )-1 ! 3a-L-Fucp-4(SO3 ÿ )-1 ! 3a-LFucp-4(SO3 ÿ )-1]n in Strongylocentrotus purpuratus (Alves
et al., 1998); a homofucan composed of 2-O-sulfated,
3-linked units in Strongylocentrotus franciscanus (AnaChristina et al., 1999); and [3a-L-Fucp-2(SO3 ÿ )-1 !
3a-L-Fucp-4(SO3ÿ )-1 ! 3a-L-Fucp-4(SO3ÿ )1 ! 3a-L-Fucp4(SO3ÿ )-1]n in Strongylocentrotus pallidus and Strongylocentrotus droebachiensis (Ana-Cristina et al., 2001) have been
shown to trigger the AR. Though sugars in the starfish
ARIS are also composed of sulfated fucose, it has been
shown that short chains of sulfated fucans in the ARIS,
obtained by chemical degradation, were not capable of
inducing the AR, suggesting that other sugar residues
(such as Gal and Xyl) should be involved in the sugar chains
of ARIS for its activity. Therefore the receptors for starfish
ARIS seem quite different from the receptors for sea urchin.
In fact, our trials to detect the starfish homolog of the
putative receptor for egg jelly fucans in sea urchins (Moy
et al., 1996) have so far been unsuccessful.
The current study confirms that sulfate groups are solely
liable to the anionic character of ARIS, which are shown to
be essential in the induction of the AR. Our previous study
has shown that the removal of even one sulfate group from
a repeating unit of Fr. 1 entirely destroys its biological
activity. As discussed earlier, sea urchin AR-inducing molecules are also highly sulfated. The sulfation of sea urchin
egg jelly fucans was known to be essential for the sperm±egg
576
interactions (Hirohashi and Vacquier, 2002). In the ascidian
Halocynthia roretzi, sperm±egg binding was shown to be
mediated through sulfated glycans present in the vitelline
coat (Baginski et al., 1999). In not only echinoderms but
also mammals sulfate has been implicated in sperm±egg
interaction, including induction of the AR. The sulfated
glycans present in porcine zona pellucida (ZP), for example,
were shown to participate in sperm±egg interactions
(Yurewicz et al., 1991; Noguchi et al., 1992). It has been
shown that the murine ZP3 (mZP3) is also composed of
sulfated N- and O-glycans, although Wassarman and coworkers showed that sulfation is not directly involved in the
binding of sperm to mZP3 (Liu et al., 1997). In human, the
initial binding of the sperm to eggs has been shown to be
blocked by fucoidan, a sulfated fucan polymer (reviewed by
Dell et al., 1999). Thus in most animals it seems that the
sperm recognizes the spatial arrangement of sulfate moieties
in the egg jelly coat glycans to undergo sperm±egg
interactions, including AR.
One of the other important observations in this study
is that the major sugar chains of ARIS were found to be
O-linked, presumably via Ser or Thr residues. These
released O-glycans of ARIS, which have an admirable biological activity, would be used as good ligands to identify
ARIS receptors. Our group has made several attempts to
isolate the receptors for ARIS using Fr. 1 as a ligand, which
made it easier to handle in comparison with ARIS or
P-ARIS as a whole, although it is less active compared to
ARIS or P-ARIS. The efforts resulted in only partial isolation of some sperm proteins of 50±60 kDa, which were
likely to be some component related to ARIS receptor
(Kawamura et al., 2002).
In addition to the starfish, O-linked glycans in many
animals have also been documented to carry considerable
biological activity in fertilization events. The O-linked glycans isolated from the vitelline coats, for example, were
reported to carry a key function in sperm±egg interactions
of the mollusk Unio elongatulus (Focarelli and Rosati, 1995)
and the ascidian Halocynthia roretzi (Baginski et al., 1999).
In the amphibian Xenopus laevis, the sperm±egg binding
process was shown to be involved in O-linked glycans
(Tina et al., 1999). Though the analysis of porcine ZP
glycans yields conflicting results, some studies find that
O-linked glycans present in porcine ZP are responsible for
sperm±egg binding (Yurewicz et al., 1991, 1993). The presence of O-linked glycans in the glycoprotein mZP3, which
plays an important role in the induction of the AR in mice,
has been reported (Florman and Wassarman, 1985;
Wassarman, 1988; Easton et al., 2000). All the facts discussed suggest that sulfation and O-glycosylation would be
essential modifications of the glycosilated proteins involved
in sperm±egg interactions. Moreover, it seems that the
fucosylation could also be important for the activity of
such glycans. Our studies have already shown that starfish
ARIS possess all these three features. According to these
intrinsic features of ARIS and the other factorsÐsuch as
obtaining ample quantities of eggs and sperms on demand,
easily detectable morphological changes during AR and so
onÐthe starfish could be used as a very good model for
understanding the comprehensive mechanisms of sperm±
egg interactions, including the AR.
Inner sugar chains of acrosome reaction±inducing substance
Although they have very complex structures, sugar chains
of ARIS possess some unusual sugar structural features.
When Fuc is present in glycoprotein with other sugar residues, it is commonly located at the terminal or as a short
branch. However peripheral as well as core region sugar
chains of ARIS were shown to have in-chain Fuc residues,
suggesting that fucosylation is associated with elongation,
not termination, of the glycans in the starfish egg coats. The
other unusual feature of ARIS sugar chain is that a-Gal is
in in-chain, which is unexpected. Generally, a-Gal is present as a terminal sugar residue of many of glycans, for
example, O-glycans from the jelly coat of amphibian eggs
terminate with Gala1 ! 4(Fuca1 ! 2)Galb (Strecker et al.,
1992, 1995), blood group P1 antigen Gala1 ! 4Galb1!,
and blood group B antigen Gala1 ! 3(Fuca1 ! 2)Galb. It
has previously been shown that Gal in Fr. 1 is a-linked
(Koyota et al., 1997). Therefore, a-Gal residues can also
be present in Fr. 2, as Fr. 1 sugar chains were shown to
remain in Fr. 2. These a-Gal residues should be in-chain as
Fr. 2 represents inner core sugar structure of ARIS
(between Fr. 1 and peptide part). Other than the sugar
chains of starfish ARIS, very few cases having such an
unusual property of naturally occurring sugar chains were
previously reported in N-glycans isolated from the eggs of
flounder, Paralichthys olivaceus (Seko et al., 1989), and
O-glycans derived from cercarial glycocalyx of Schistosoma
mansoni (Khoo et al., 1995).
Although a significant activity was observed in the sugar
chains of ARIS, the best activity was always noted in the
intact ARIS, hinting that protein portion is also somewhat
important in triggering the AR. This may be attributable to
the clustering effect (Lee, 1992) for which we do not have
direct evidence so far. Recently we have isolated the protein
portion of ARIS using reductive-elimination reaction
described by Gerken et al. (1992). Its size seems to be over
100 kDa. The molecular cloning of this isolated protein is
currently under progress. The targets of our current study
are to figure out (1) the anomeric information of suggested
sugar chains of Fr. 2, (2) the rest of the sugar structures of
ARIS with reducing terminal information, and (3) the primary structure of protein part of ARIS.
Materials and methods
General methods
The starfish A. amurensis were collected in the breeding
season from Tokyo Bay and Otsuchi Bay on the Pacific
coast of Honshu, Japan, and from the coast of Tasmania
in Australia. Water was purified with a Milli-Q reagent
water system (Millipore, Bedford, MA). UV absorption
was monitored by using a Tosoh (Montgomeryville, PA)
UV-8000 detector in high-performance liquid chromatography (HPLC) separations. Proton NMR analysis was carried
out using a JEOL A400 NMR spectrometer. Gas-liquid
chromatography MS was performed on a Shimadzu GC14A instrument coupled with a Shimadzu GCMS-QP2000A
instrument (Kyoto, Japan.). Nano-ESI spectra were acquired with a quadrupole/time-of-flight 2 instrument
(Micromass, Manchester, UK). All chromatographic separations for neutral sugars were monitored by a microtiter
plate version of resorcinol-sulfuric acid method (Monsigny
et al., 1988) using a Tosoh MPR A4 microplate reader.
Sulfate contents were determined according to Terho and
Hartiala (1971). Tests for sialic acids, uronic acids, and
phosphate were performed by precolumn derivatization
procedure for sialic acid (Anumula, 1997), carbozol assay
(Bitter and Muir, 1962), and phosphorus assay described by
Bartlett (1959), respectively.
Preparation of core region sugar fragments of
ARIS (Fr. 2)
Preparation of Fr. 2 was carried out essentially as described
by Koyota et al. (1997). Breifly, P-ARIS was prepared by
actinase E digestion of ethanol precipitation of the egg jelly
solution of the starfish A. amurensis followed by Sepharose
CL-4B gel filtration and DEAE-Toyopearl 650 M ion
exchange chromotography, respectively. ARIS was prepared in the same way using undigested egg jelly solution
instead of ethanol precipitation. The solution of the acidic
fraction containing sugar and protein (P-ARIS) was sonicated for 30 min using a Bransonic ultrasonic apparatus
(Danbury, CT), followed by DEAE Toyopearl 650 M anion
exchange chromatography. The column was eluted with a
linear gradient of 0±1 M NaCl. Fr. 1 was recovered in 0.7±
0.8 M NaCl solution, whereas Fr. 2 was recovered in ~0.9 M
NaCl concentration. The fractions were dialyzed and lyophilized to give purified Fr. 1 (~45%) and Fr. 2 (~14%).
Reductive b-elimination
b-Elimination was performed as described by Chaplin and
Kennedy (1994). Each sample (1 mg) was treated with
0.05 M NaOH-containing 1 M NaBH4 (1 ml) at 45 C,
terminating the reaction with 50% acetic acid after 48 h.
Released sugar chains were isolated by gel filtration on a
Sepharose CL-6B column (2.2 90 ml) using the eluent of
100 mM ammonium acetate buffer (pH 5.5) followed by
dialysis and lyophilization.
Sugar composition analysis
Sugar composition analysis was performed by 1-phenyl-3methyl-5-pyrazolone (PMP) derivertization method
described by Fu and O'Neill (1995). Each sample (50 mg)
was hydrolyzed using 4 M trifluoracetic acid at 120 C for 2
h. Hydrolyzed samples were directly labeled with PMP,
extracted, and separated with a C18 reverse-phase HPLC
column (4.6 250 mm, 5C18-AR, Waters, Nacalai
Tesque), essentially as described by the original procedure.
ELISA
Dried samples (50±500 ng) in a microtiter plate were incubated with 1% human serum albumin in phosphate buffered
saline (PBS) for 30 min. After removing the blocking buffer,
the samples were incubated with 50 ml mouse anti-Fr. 1
antibody (IgG) (prepared by our laboratory) in PBS for
1 h followed by washing each well with PBS. Each sample
was then treated with 200-fold diluted horseradish
peroxidase±conjugated goat anti-mouse IgG antibody
(CHEMI CON, Temecula, CA) in PBS (50 ml) for 30 min,
followed by washing with PBS to remove the excess antibody. Then the samples were labeled with freshly prepared
577
H.M.M.J. Gunaratne et al.
200 ml o-phenylenediamine (4 mg o-phenylenediamine in 10
ml 0.1 M citric acid buffer, pH 5.0) and 4 ml 30% hydrogen
peroxide for 20 min. The reaction was terminated by adding
20 ml 5 N sulfuric acid. The plate was read at 492 nm.
Desulfation
A solution of Fr. 2 was dialyzed against 0.1 M pyridinium
acetate buffer (pH 5.4) for 2 days. The resulting pyridinium salt (2 mg) was treated with 1 ml 10% methanol in
dimethyl sulfoxide or 5 h at 80 C, concentrated to dryness, and passed through a 10-ml column of Sephadex
G-25 (PD-10) (Nagasawa et al., 1979). Thoroughly desulfated sample was obtained by passing through a Dowex
1 8 column equilibrated with 1 M acetic acid. The desulfated sample (1.2 mg) was finally subjected to methylation
analysis. In the case of small fragments of Fr. 2 generated
by resin hydrolysis, the pyridinium salt was prepared by
passage through a microcolumn of Dowex 50 (8) in the
pyridinium form because of its limited sample availability.
Proton NMR spectroscopy
A sample of released sugar portion by b-elimination was
repeatedly exchanged in D2O (99.96%, Aldrich, St. Louis,
MO), with intermediate lyophilization. Lyophilized sample
was dissolved in 150 ml 99.996% D2O (Aldrich) and transferred to an NMR tube (3 mmf 18 cm). The spectra were
recorded at 70 C. Chemical shifts were reported in ppm
using HDO signal in D2O (d 4.35 ppm at 70 C) as the
internal reference.
Preparation of PMAAs
Prior to permethylation, samples were reduced with 1%
(w/v) NaBH4 for 2 h at room temperature. In the case of
sulfated sugars, reduced samples were converted into
triethylammonium salts by passage through a microcolumn
of Dowex 50 (8) in the triethylammonium form (Stevenson
and Furneaux, 1991). Then PMAAs were prepared as
described by Anamula and Taylor (1992) with consequent
methylations for three cycles.
GC-MS analysis
PMAAs were dissolved in chloroform prior to injection. An
aliquot of the sample was applied on a HP-5 column (5%
phenylmethylsilicone, 0.25 mm 30 mm, Hewlett-Packard,
Wilmington, DE) at 100 C. This temperature was held for 2
min and then increased to 250 C over 60 min at 2.5 C/min.
Partial acid hydrolysis of Fr. 2 and isolation of
hydrolysates
A sample of released sugar portion of Fr. 2 (300 mg) was
treated with H ‡ form of Dowex 50WX2-400 strong acidic
ion exchange resin at 75 C for 24 h as described by Mark
et al. (2000). The oligosacchraide fragments were isolated
by centrifuging the resin-containing solution through a
100,000 MWCO spin-filter at 3500 g. Because the
resulted hydrolysate was shown not to be amenable to
mass analysis, the mixture was further separated by centrifuging through a 5000 MWCO spin-filter at 3500 g
followed by a gel filtration on a Superdex Peptide
578
PC 3.2/30 column on a SMART system (Pharmacia,
Uppsala, Sweden) using water as the eluent to remove
the small molecules as the total volume. The other
major fractions, named Fr. 2-H1, were collected (~160 mg,
~55%) and subjected to sugar composition and mass and
methylation analysis. The procedure was repeated for the
remaining part obtained after the second filtration
(45 kDa) to give the adequate amount of sample for the
further analyses.
MS measurements
ESI quadrapole/time-of-flight MS was used for the sequencing analysis of the small fragments of Fr. 2 generated from
the resin hydrolysis. An aliquot of each sample was dissolved in methanol±water (1:1, v/v) and mixed with equal
amount of 1 mM NaCl in 1 mM acetic acid water solution.
An aliquot of this sample solution (8 ml) was deposited in a
metal-coated borosilicate nanoelectrospray tip (F type tips,
Micromass). The needle voltage was 1000 V, and the ion
source was maintained at 80 C.
Acknowledgments
We express our gratitude to Prof. Ann Dell of the Imperial
College of Science, Technology and Medicine, U.K., for
critical commentary on this manuscript and Prof. M. Ueda
of Keio University, Japan, and Prof. Tachibana of University
of Tokyo for making arrangements for the NMR analysis
and mass analysis. We also thank Dr. W.M.S. Wimalasiri of
University of Peradeniya, Sri Lanka; Drs. H. Nishida,
T. Baginzki, and M. Kawamura of Tokyo Institute of
Technology; and Prof. Y. Ohashi of University of Electrocommunications, Japan, for helpful advice and discussions.
We thank the directors and the staff of Otsuchi, Misaki,
Ushimado Marine Biological Centers and Stations in Japan
and Marine and Coastal Research, Tasmania, Australia,
for help in collecting starfish. H.M.M.J.G. is supported by
the Ministry of Education, Science and Culture, Japan, and
is currently on leave from the Faculty of Medicine, University of Ruhuna, Galle, Sri Lanka.
Abbreviations
AR, acrosome reaction; ARIS, acrosome reaction±inducing substance; CID MS/MS, collision-induced dissociation; ELISA, enzyme-linked immmunosorbent assay; ESI,
electrospray ionization; GC, gas chromatography; HPLC,
high-performance liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NMR,
nuclear magnetic resonance; P-ARIS, pronase digest of
ARIS; PBS, phosphate buffered saline; PMAA, partially
methylated alditol acetate; PMP, 1-phenyl-3-methyl-5pyrazolone; ZP, zona pellucida.
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