Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (52)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Vilela-Silva, A. C.
Right arrow Articles by Mourao, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vilela-Silva, A. C.
Right arrow Articles by Mourao, P. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Glycobiology Pages 927-933  

Structure of the sulfated [alpha]-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition
Introduction
Results and discussion
Materials and methods
Acknowledgments
References

Structure of the sulfated [alpha]-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition

Structure of the sulfated [alpha]-L-fucan from the egg jelly coat of the sea urchin Strongylocentrotus franciscanus: patterns of preferential 2-O- and 4-O-sulfation determine sperm cell recognition

Ana-Cristina E.S. Vilela-Silva, Ana-Paula Alves, Ana-Paula Valente1, Victor D. Vacquier2 and Paulo A.S. Mourão3

Laboratório de Tecido Conjuntivo, Hospital Universitário and Departamento de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil, 1Centro Nacional de Ressonância Nuclear Magnética, Departamento de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil and 2Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093-0202, USA

Received on January 11, 1999; revised on February 22, 1999; accepted on March 1, 1999

The egg jelly coats of sea urchins contains sulfated polysaccharides responsible for inducing the sperm acrosome reaction which is an obligatory event for sperm binding to, and fusion with, the egg. Here, we extend our study to the sea urchin Strongylocentrotus franciscanus. The egg jelly of this species contains a homofucan composed of 2-O-sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. This polysaccharide was compared with other sulfated [alpha]-l-fucans as inducers of acrosome reaction in conspecific and heterospecific sperm. Although all these fucans are linear polymers composed of 3-linked [alpha]-l-fucopyranosyl units, they differ in the proportions of 2-O- and 4-O-sulfation. The reactivity of the sperm of each species is more sensitive to the egg jelly sulfated fucan found in their own species. The reactivity of the sperm does not correlate with the charge density of the fucan, but with the proportion of 2-O- and 4-O- sulfation. The pattern of sulfation may be an important feature for recognition of fucans by the sperm receptor contributing to the species-specificity of fertilization.

Key words: sea urchin/fucan/acrosome reaction/polysaccharide/sulfation

Introduction

Sea urchin eggs are surrounded by a transparent gelatinous layer that induces the exocytotic sperm acrosome reaction. The acrosome reaction is an obligatory event for sperm binding to, and fusion with, the egg. It is a signal transduction event linked to ion fluxes, membrane depolarization and internal pH changes (Vacquier, 1986a; Darszon et al., 1996).

A major macromolecule of egg jelly coat, the one responsible for inducing the sperm acrosome reaction, is a sulfated polysaccharide (Mulloy et al., 1994; Alves et al., 1997, 1998; Vacquier and Moy, 1997). These compounds have simple, well-defined repeating structures and each species represents a particular pattern of saccharide chain and/or sulfate substitution. For example, the sea urchin Echinometra lucunter contains a homopolymer of 2-O-sulfated, 3-linked [alpha]-l-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated [alpha]-l-fucans with regular tetrasaccharide repeating units; the specific pattern of sulfation varies in the two species (Mulloy et al., 1994; Alves et al., 1997). These sulfated polysaccharides are extremely species-specific as inducers of the sperm acrosome reaction (Alves et al., 1997) and represent an unusually simple example of ligand-induced signal transduction leading to exocytosis (Alves et al., 1997; Vacquier and Moy, 1997).

More recently we reported two structurally distinct sulfated fucans in the egg jelly of the sea urchin Strongylocentrotus purpuratus (Alves et al., 1998). Approximately 90% of individual females of this species spawn eggs having only one of two possible fucans. Both purified fucans have equal potency in inducing the sperm acrosome reaction. The reason that eggs from this species possess two sulfated fucans isotypes remains unknown.

We now extend our study to another species, Strongylocentrotus franciscanus. The egg jelly layer of this sea urchin contains a homofucan composed of 2-O-sulfated, 3-linked units which is the simplest structure ever reported for a sulfated fucan. It contrasts with the very heterogeneous sulfated fucans from brown algae (Nishino et al., 1991; Patankar et al., 1993; Mulloy et al., 1994) and with similar compounds from other echinoderms in which the oligosaccharide repeating units differ in specific patterns of sulfation (Mulloy et al., 1994; Alves et al., 1997; Alves et al., 1998).

The purified sulfated [alpha]-l-fucan from S.franciscanus was tested as an inducer of acrosome reaction in homospecific and heterospecific sperm. Our results suggest that the 2-O- and 4-O-sulfate esters may constitute a structural feature for recognition of fucans by the receptor on the sperm surface.

Results and discussion

Purification of a sulfated [alpha]-l-fucan from the egg jelly coat of the sea urchin S.franciscanus

Sulfated polysaccharides extracted from the egg jelly coat of S.franciscanus were purified by anion exchange chromatography on DEAE-cellulose (Figure 1). A peak rich in sialic acid was eluted completely by 0.5 M NaCl and denominated as a 'sialic acid-rich glycoconjugate" in analogy with similar compounds described in other species of sea urchin (SeGall and Lennarz, 1979). A second peak, eluted at higher salt concentration, corresponds to the sulfated fucan.


Figure 1. Purification of the sulfated [alpha]-l-fucan from the egg jelly coat of S.franciscanus. The crude polysaccharides (50 mg) were purified on a DEAE-cellulose column as described under Materials and methods. Fractions containing the sulfated [alpha]-l-fucan, as indicated by the Dubois and methachromatic positive tests (horizontal bar) were pooled, dialyzed against distilled water, and lyophilized. SG indicates sialic acid-glycoconjugate.

The sulfated fucan and the sialic acid-rich glycoconjugate migrate on agarose gel electrophoresis and stain with toluidine blue (Figure 2), indicating both are highly anionic charged polymers. Interestingly, the unfractionated samples have a slightly retarded electrophoretic mobility when compared with the isolated compounds. This observation suggests an interaction between the sulfated fucan and the sialic acid-glycoconjugate.


Figure 2. Agarose gel electrophoresis of the sulfated polysaccharides purified from S.franciscanus. The crude polysaccharides and the purified sulfated [alpha]-l-fucan and the sialic acid-glycoconjugate (~15 µg of each) were applied to a 0.5% agarose gel, and the electrophoresis was run for 1 h at 110 V in 0.05 M 1,3-diaminopropane:acetate buffer (pH 9.0). The gel was fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5,v/v).

Table I. Chemical composition and specific optical rotation of the sulfated fucan from the egg jelly coat of S.franciscanus
Chemical composition (molar ratio) [[alpha]]D20°C
Fucose Sulfate
1.00 1.14 -57°

Chemical analysis of the purified sulfated fucan revealed fucose as the only sugar with a content of ~1.1 sulfate ester groups per residue (Table I). The strongly negative specific rotation is compatible with residues of [alpha]-l-fucopyranose.

Structure of the sulfated [alpha]-l-fucan

Methylation analysis. Three rounds of methylation of the native sulfated [alpha]-l-fucan from S.franciscanus yields mainly 4-O-methyfucose, whereas 2,4-di-O-methylfucose is the predominant methyl ether derivative from the desulfated [alpha]-l-fucan (Table II). These results are consistent with a linear polysaccharide composed mainly of 3-linked and 2-O-sulfated l-fucose residues. Possibly 2-O-methylfucose and non-methylated fucose obtained from the desulfated fucan are products of incomplete methylation, whereas the formation of 4-O-methylfucose indicates some 2-O-sulfated residues are still present after the solvolytic desulfation process. One round of methylation of the desulfated fucan yields almost the same proportions of methyl ether derivatives as in Table II, except for a higher proportion (45%) of 2-O-methylfucose and a lower proportion (35%) of 2,4-di-O-methylfucose. Thus, apparently position 4 is more resistant to methylation than position 2 in the case of this polysaccharide. This observation suggest that the small proportion (6%) of 4-O-methylfucose is a product of 2-O-sulfated residues that are still present after desulfation reaction rather than a result of incomplete methylation. The small proportion (5%) of unmethylated fucose obtained from the native fucan is not merely a product of an incomplete reaction since its proportion remains unchanged after an additional methylation (not shown). It may indicate small amounts of 2,4-di-O-sulfated residues in the native fucan.

Table II. Methylated fucose derivatives obtained from native and desulfated fucan from the egg jelly coat of S.franciscanus
Alditola tRb (min) % of total peak area
Native Desulfated
2,4-Met2-Fuc 26.9 7 64
2-Met-Fuc 29.1 <1 15
4-Met-Fuc 30.8 88 6
Fuc 32.4 5 15
aThe identity of each peak was established by mass spectrometry.
bRetention time on a DB-1 capillary column.

NMR spectroscopic analysis. The one-dimensional 1H spectrum of the sulfated fucan has a single anomeric signal at 5.33 p.p.m. and agreeing with the presence of a homopolymer of sulfated [alpha]-l-fucan (Figure 3) as is the methyl signal at 1.25 p.p.m.. It was not possible to record NMR spectra of the desulfated fucan since it has low solubility in water.


Figure 3. Expansions of the 5.5-3.0 and 1.5-1.0 p.p.m. regions of the 1H spectrum at 600 MHz of the sulfated [alpha]-l-fucan from S.franciscanus. The spectrum was recorded at 60°C for sample in D2O solution. Chemical shifts are relative to internal trimethylsilylpropionic acid at 0 p.p.m.. The HOD signal was partially suppressed by presaturation. × are signals from noncarbohydrate contaminants.

The assignment of peaks was achieved by analysis of 1H COSY (Figure 4A), 1H TOCSY (Figure 4B), and 1H/13C HMQC (Figure 5) spectra. All spectra have the simplicity compatible with the presence of a homopolymer.

   A
   B

Figure 4. COSY (A) and TOCSY (B) spectra of the sulfated [alpha]-l-fucan from S.franciscanus, at 600 MHz, 60°C, in D2O. The spin system for [alpha]-l-fucopyranose is traced through both spectra.

All fucan protons, with the exception of H4, could be assigned in the COSY spectrum (Figure 4A). The cross peaks show unambiguously the correlation between the spin systems. The TOCSY shows the same result with strong correlation between H1 to H2, H2 to H3, and H5 to H6 (Figure 4B). The 1H chemical shifts are presented in Table III.

Table III. Proton chemical shifts (p.p.m.) for residues of [alpha]-l-fucose in the sulfated [alpha]-l-fucan from S.franciscanus and in standard compounds
Compound Chemical shiftsa
H1 H2 H3 H4 H5 H6
Present work 5.33 4.55 4.10 4.07 4.42 1.25
-3-[alpha]-l-Fucp-(2,4SO3-)-1-b 5.40 4.58 4.39 4.91 4.37 1.25
-4-[alpha]-l-Fucp-(2SO3-)-1-c 5.31 4.56 4.23 4.01 4.52 1.35
-3-[alpha]-l-Fucp-1-b 5.03 3.96 4.01 3.96 4.35 1.21
aThe 1H spectrum was recorded at 600 MHz in 99.9% D2O. Chemical shifts are referenced to internal trimethylsilylpropionic acid. Values in boldface indicate positions bearing sulfate ester.
bData from a sea cucumber sulfated [alpha]-l-fucan (Ribeiro et al., 1994).
cData from a sulfated [alpha]-l-fucan from the sea urchin Arbacia lixula (Alves et al., 1997).

Table IV. Carbon chemical shifts (p.p.m.) for residues of [alpha]-l-fucose in the sulfated [alpha]-l-fucan from S.franciscanus and in standard compounds
Compound Chemical shifts (p.p.m.)a
C1 C2 C3 C4 C5 C6
Present work 96.77 75.52 75.94 71.16 68.55 17.32
-3-[alpha]-l-Fucp-1-b 98.29 69.00 77.60 71.10 69.00 18.00
-4-[alpha]-l-Fucp-1-c 103.10 nr nr 82.50 nr 18.20
-[alpha]-l-Fucp-O-Med 100.50 69.00 70.60 72.90 67.50 16.50
a13C chemical shifts relative to trimethylsilylpropionic acid. These values were obtained from the 1H/13C HMQC spectrum (see Figure 5). Values in boldface indicate positions involved in the glycosidic linkage.
bData from Ribeiro et al., (1994).
cData from Alves et al., (1997).
dData from Gorin and Mazurek, (1975).
nr, Not reported.


The carbon chemical shifts were obtained using the HMQC spectrum. The 1H/13C HMQC spectrum shows six major peaks although some contaminants are also seen. The HMQC was interpreted using the information obtained in the COSY and TOCSY spectra. The H4 overlaps with H3 in the proton dimension but they have well resolved carbon chemical shifts (Figure 5). The other components were also assigned and their chemical shifts are presented in Table IV.


Figure 5. 1H/13C HMQC spectrum of the sulfated [alpha]-l-fucan from S.franciscanus at 60°C, in D2O. Starting from the proton chemical shifts, it was possible to obtain the values of carbon chemical shifts of the [alpha]-l-fucopyranosyl residues.

Comparing the 1H and 13C chemical shifts with the values in the literature show they are near to those reported for 2-O-sulfated and 3-linked [alpha]-fucose residues. The position of sulfation was deduced from the strong (0.6-0.7 p.p.m.) downfield shift in the position of H2 (4.55 p.p.m.). The position of the glycosidic linkage is not easily deduced from the 1H chemical shifts, but from the 13C chemical shifts, based on the interpretation of the HMQC spectrum (Figure 5), as the carbon 3 shows the characteristic shift of a 3-linked [alpha]-fucan (Table IV).


NMR analysis shows no sign of 4-O-sulfation or of 2,4-di-O-sulfation, although one cannot rule out a small proportion of these residues. Overall, the combination of methylation and NMR spectroscopic analysis confirm the structure of the sulfated [alpha]-l-fucan from the egg jelly of S.franciscanus, as shown in Figure 6.


Figure 6. Deduced structure of the sulfated [alpha]-l-fucan from the egg jelly of S.franciscanus. This fucan is a 1->3 linked linear polysaccharide with a regular sulfation at O-2 position. The structure was deduced from the methylation (Table II) and NMR spectroscopic (Figures 3-5, Tables III and IV) analysis. It is not possible to rule out a small proportions of 4-O- or 2,4-O-sulfated units, but NMR analysis show no sign of these types of residues.

Sulfated [alpha]-l-fucan induces the acrosome reaction

After we had isolated, purified and characterized the structure of the sulfated [alpha]-l-fucan, we tested its ability to induce the acrosome reaction in conspecific sperm. As shown in Figure 7A (open circles), the purified, homologous sulfated [alpha]-l-fucan from S.franciscanus induces the acrosome reaction in a dose-dependent fashion at concentrations above 0.1 µg hexose/ml. Purified, sulfated [alpha]-l-fucans from two other sea urchin species, having a similar backbone structure, were also compared using sperm of S.franciscanus. The sulfated [alpha]-l-fucan from L.variegatus, enriched in the proportion of 2-O-sulfated fucose units (Figure 7C), induces the acrosome reaction with approximately the same potency as the homologous fucan (Figure 7A, solid circles). In contrast, the mixture of sulfated [alpha]-l-fucans I and II from S.purpuratus, which contains a preponderance of 4-O-sulfated fucose, induces the acrosome reaction only at concentrations above 10 µg hexose/ml (Figure 7A, solid squares).

   A, B
   C

Figure 7. Induction of acrosome reaction in the sperm of S.franciscanus (A) and S.purpuratus (B) by purified sulfated fucans from the egg jelly of three species of sea urchins (C). (A) and (B) percent of acrosome reaction is plotted against log ng hexose/ml. After lyophilization, the sulfated [alpha]-l-fucans from S.franciscanus (open circles), L.variegatus (solid circles), and S.purpuratus (solid squares) were dialyzed into sea water and assayed for acrosome reaction at 18°C (Vacquier, 1986b; Vacquier and Moy, 1997). Between 200-300 spermatozoa were scored per data point. (C) Structures of the sulfated [alpha]-l-fucans used as inducers of the acrosome reaction. The structure of the sulfated fucan from L.variegatus was determined in previous work (Alves et al., 1997). Sulfated fucans prepared from a pool of egg jelly from different females of S.purpuratus contains a mixture of two different polysaccharides as shown in the panel (see Alves et al., 1998). These two sulfated fucans are approximately equal in acrososome reaction inducing potency in sperm of S.purpuratus.

In order to evaluate further the species-specificity of sulfated fucans to induce the acrosome reaction, sperm of S.purpuratus were also investigated. In this sea urchin, the potency of sulfated fucans to induce the acrosome reaction decreased in the order: S.purpuratus > L.variegatus > S.franciscanus (Figure 7B). This is in agreement with a decrease in the proportion of 4-O-sulfated units in the polysaccharide chain (Figure 7C). We have previously demonstrated that purified sulfated fucans I and II are approximately equal in potency in induction of acrosome reaction in sperm of S.purpuratus (Alves et al., 1998).

The presumable small proportion (~5%) of 2,4-di-O-sulfated fucose residues is unlike to be an important acrosome reaction inducer in sperms of S.franciscanus. This unit accounts for 25% of the total residues in the fucan from L.variegatus (Figure 7C), but the polysaccharide has a weak potency to induce acrosome reaction in sperms of S.franciscanus (Figure 7A).

Sulfated polysaccharides as species-specific inducers of the acrosome reaction in sea urchin spermatozoa

We previously demonstrated the species-specificity of sulfated polysaccharides as inducers of the acrosome reaction in sea urchin sperm (Alves et al., 1997). However, in this previous study, sulfated polysaccharides expressing marked interspecific structural variations were used.

In order to evaluate the finer specificity of recognition in the acrosome reaction, we have now compared egg jelly sulfated fucans containing the same backbone of 3-linked [alpha]-l-fucopyranosyl units, but with different proportions of 2-O- and 4-O-sulfation. Although we observed a less strict species-specificity in sperm recognition of sulfated polysaccharides, the potency of acrosome reaction induction clearly depended on the extent of 2-O- and 4-O-sulfation (Figure 7). Thus, sperm from S.franciscanus are sensitive to either homologous or heterologous sulfated fucans enriched in 2-O-sulfated units, similar to their own egg jelly, but not to 4-O-sulfated fucans (Figure 7A). Alternatively, sperm of S.purpuratus have a more strict specificity to the polysaccharide with the closest structural similarity found in their own (Figure 7B). Notably, the reactivity of the sperm from the two species to the various sulfated [alpha]-l-fucans has no correlation with the charge density. However, there is a correlation with the specific positions of the sulfate esters on the polysaccharide chain (Figure 7).

Studies of membrane proteins of sperm of S.purpuratus indicate that glycoprotein binds to the egg jelly sulfated fucan; this is sufficient to induce the sperm acrosome reaction (Moy et al., 1996; Vacquier and Moy, 1997). This unique membrane protein was termed 'REJ" (receptor for egg jelly). REJ has the structure of a carbohydrate binding protein. If similar receptors are also found in the sperm of other species of sea urchins, we expect that the sulfated polysaccharides are the ligands for the receptor. Possibly, the structure of the polysaccharide and its sulfation pattern are important structural features for recognition of these molecules by the receptor in the sperm membrane. Variations in the structure of the egg jelly sulfated polysaccharide may represent one of the barriers which limit interspecific cross fertilization. In addition, our results with sperm of S.franciscanus strongly suggest that, even though recognition and binding to sulfated polysaccharides provide an important interspecific discrimination step in fertilization, additional receptor structures must be able to discriminate between homologous and heterologous polysaccharides with similar content of 2-O-sulfated units, as happens with egg jelly fucans from S.franciscanus and L.variegatus. Further studies with isolated sperm membrane receptors for sulfated [alpha]-l-fucans should provide additional insights into the mechanism of interaction of these 'pattern recognition" receptors in fertilization.

Materials and methods

Sulfated fucan from sea urchin egg jelly

Extraction. Adults of S.franciscanus and S.purpuratus were collected at La Jolla, CA, and L.variegatus was collected at Rio de Janeiro, Brazil. Eggs were spawned into sea water by intracelomic injection of 0.5 M KCl. The crude egg jelly was isolated by the pH 5.0 method and prepared as 30,000 × g supernatant and stored at -20°C, or lyophilized after dialysis against distilled water (Vacquier and Moy, 1997). The acidic polysaccharides were extracted from the jelly coat by papain digestion and partially purified by ethanol precipitation, as described previously (Albano and Mourão, 1986).

Purification. The crude polysaccharides (50 mg) were applied to a DEAE-cellulose column (10 × 1 cm), equilibrated with 50 mM sodium acetate (pH 5.0), and washed in 50 ml of the same buffer. The column was then eluted by a linear gradient prepared by mixing 100 ml of 50 mM sodium acetate buffer (pH 5.0) with 100 ml of 3.0 M NaCl in the same buffer. The flow rate of the column was 10 ml/h, and fractions of 2.0 ml were collected. Fractions were checked for fucose and sialic acid by the Dubois et al. reaction (1956) and by the Ehrlich assay (Kabat and Mayer, 1971), respectively, and also by their metachromasia (Farndale et al., 1986). The NaCl concentration was estimated by conductivity. Fractions containing the sulfated [alpha]-l-fucan and the sialic acid-glycoconjugate were pooled, dialyzed against distilled water, and lyophilized.

Chemical analyses

Total fucose was measured by the method of Dische and Shettles, (1948). After acid hydrolysis of the polysaccharide (5.0 trifluoroacetic acid for 5 h at 100°C), sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968). The presence of hexoses and 6-deoxyhexoses in the acid hydrolysates was estimated by paper chromatography in n-butanol:pyridine:water (3:2:1,v/v) for 48 h and by gas-liquid chromatography of derived alditols (Kircher, 1960). Optical rotation was measured using a digital polarimeter (Perkin-Elmer model 243-B).

Agarose gel electrophoresis

Sulfated polysaccharides were analyzed by agarose gel electrophoresis as described previously (Vieira et al., 1991; Alves et al., 1997). The sample (~15 µg) was applied to a 0.5% agarose gel and run for 1 h at 110 V in 0.05 M 1,3-diaminopropane:acetate buffer (pH 9.0). The sulfated polysaccharides in the gel were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gel was dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:5:5,v/v).

Desulfation and methylation of fucans

Desulfation of the sulfated [alpha]-l-fucan from S.franciscanus was performed by solvolysis in dimethylsulfoxide as described previously for desulfation of other types of polysaccharides (Mourão and Perlin, 1987; Vieira et al., 1991). The chemical analysis of the desulfated polysaccharide revealed <0.1 sulfate ester groups per fucose residue while the native fucan contains 1.14 sulfate groups per residue (Table I). The native and desulfated polysaccharides (5 mg) were subjected to three rounds of methylation as described previously (Ciucanu et al., 1984), with the modifications suggested by Patankar et al., (1993). The methylated polysaccharides were hydrolyzed in 6 M trifluoroacetic acid for 5 h at 100°C, reduced with borohydride and the alditols were acetylated with acetic anhydride:pyridine (1:1, v/v) (Kircher, 1960). The alditols acetates of the methylated sugars were dissolved in chloroform and analyzed in a gas chromatography/mass spectrometer.

NMR experiments

1H and 13C spectra were recorded using a Bruker DRX 600 triple resonance 5 mm probe. About 5 mg of the sulfated [alpha]-l-fucan was dissolved in 0.7 ml of 99.9% D2O (NMR grade from Cambridge Isotope Laboratories) and the spectra were recorded at 60°C, with HOD suppression by presaturation. TOCSY, COSY, and 1H/13C heteronuclear correlation (HMQC) spectra were recorded using states time proportional phase incrementation for quadrature detection in the indirect dimension. COSY spectrum was run with 2048 and 200 points. TOCSY spectrum was run with 4096 × 200 points with a spin-lock field of about 10 kHz and a mixing time of 80 ms, which was previously determined to give optimum results for these samples. HMQC spectrum was run with 1024 ×128 points, and globally optimized alternating phase rectangular pulses was used during acquisition for 13C decoupling. All chemical shifts are relative to internal or external trimethylsilylpropionic acid and methanol.

Sulfated fucans as inducers of the sperm acrosome reaction

Sperm of S.franciscanus and S.purpuratus were collected as undiluted semen and stored for up to 6 h on ice before dilution and testing for acrosome reaction induction. Assays for acrosome reaction by sulfated polysaccharides were done as described previously (Vacquier, 1986b; Vacquier and Moy, 1997).

Acknowledgments

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq: FNDCT, PADCT, and PRONEX), Financiadora de Estudos e Projetos (FINEP), Fundacão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and NIH Grant HD 12986 (to V.D.V.). We thank Adriana A.Eira and Fábio S.Araujo for technical assistance and Dr. George A.Reis for help in the preparation of the manuscript.

References

Albano ,R.M. and Mourão,P.A.S. (1986) Isolation, fractionation and preliminary characterization of a novel class of sulfated glycans from the tunic of Styela plicata (Chordata-Tunicata). J. Biol. Chem., 261, 758-765. MEDLINE Abstract

Alves ,A.P., Mulloy,B., Diniz,J.A. and Mourão,P.A.S. (1997) Sulfated polysaccharides from the egg jelly are species-specific inducers of acrosomal reaction in sperm of sea urchins. J. Biol. Chem., 272, 6965-6971. MEDLINE Abstract

Alves ,A.P., Mulloy,B., Moy,G.W., Vacquier,V.D. and Mourão,P.A.S. (1998) Females of the sea urchin Strongylocentrotus purpuratus differ in the structure of their egg jelly sulfated fucans. Glycobiology, 8, 939-946. MEDLINE Abstract

Ciucanu ,J. and Kerek,F. (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr. Res., 131, 209-217.

Darszon ,A., Lievano,A. and Beltran,C. (1996) Ion channels: key elements in gamete signaling. Curr. Top. Dev. Biol., 34, 117-167. MEDLINE Abstract

Dische ,Z. and Shettles,L.B. (1948) A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination. J. Biol. Chem., 175, 595-603.

Dubois ,M., Gilles,K.A., Hamilton,J.K., Rebers,P.A. and Smith,F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem., 28, 350-354.

Farndale ,R.W., Buttle,D.J. and Barret,A.J. (1986) Improved quantitation and discrimination of sulfated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta, 883, 173-177. MEDLINE Abstract

Gorin ,P.A.J. and Mazurek,M. (1975) Further studies on the assignment of signals in 13C magnetic spectra of aldoses and derived methyl glycosides. Can. J. Chem., 53, 1212-1220.

Kabat ,E.A. and Mayer,M.M. (1971) In: Experimental Immunochemistry, Charles C. Thomas, Springfield, IL, pp. 505-513.

Kircher ,H.W. (1960) Gas-liquid chromatography of methylated sugars. Anal. Chem., 32, 1103-1106.

Mourão ,P.A.S. and Perlin,A.S. (1987) Structural features of sulfated glycans from the tunic of Styela plicata (Chordata-Tunicata): a unique occurrence of L-galactose in sulfated polysaccharides. Eur. J. Biochem., 166, 431-436. MEDLINE Abstract

Moy ,G.W., Mendoza,L.M., Schulz,J.R., Swanson,W.J., Glabe,C.G. and Vacquier,V.D. (1996) The sea urchin sperm receptor for egg jelly is a modular protein with extensive homology to the human polycystic kidney disease protein PKD1. J. Cell. Biol., 133, 809-817. MEDLINE Abstract

Mulloy ,B., Ribeiro,A.C., Alves,A.P., Vieira,R.P. and Mourão,P.A.S. (1994) Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2 and O-4 positions. J. Biol. Chem., 269, 22113-22123. MEDLINE Abstract

Nishino ,T., Kiyohara,H., Yamada,H. and Nogumo,T. (1991) An anticoagulant fucoidan from the brown seaweed Ecklonia kurome. Phytochemistry, 30, 535-539. MEDLINE Abstract

Patankar ,M.S., Oehninger,S., Barnett,T., Williams,R.L. and Clark,G.F. (1993) A revised structure for fucoidan may explain some of its biological activities. J. Biol. Chem., 268, 21770-21776. MEDLINE Abstract

Ribeiro ,A.C., Vieira,R.P., Mourão,P.A.S. and Mulloy,B. (1994) A sulfated [alpha]-l-fucan from sea cucumber. Carbohydr. Res., 255, 225-240. MEDLINE Abstract

Saito ,H., Yamagata,T. and Suzuki,S. (1968) Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem., 245, 1536-1542.

SeGall ,G.K. and Lennarz,W.J. (1979) Chemical characterization of the components of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction. Dev. Biol., 71, 33-48. MEDLINE Abstract

Vacquier ,V.D. (1986a) Activation of sea urchin spermatozoa during fertilization. Trends Biochem. Sci., 11, 77-81.

Vacquier ,V.D. (1986b) Handling, labeling and fractionating sea urchin spermatozoa. Methods Cell Biol., 27, 15-40. MEDLINE Abstract

Vacquier ,V.D. and Moy,G.W. (1997) The fucose sulfate polymer of egg jelly binds to sperm REJ and is the inducer of the sea urchin sperm acrosome reaction. Dev. Biol., 192, 125-135. MEDLINE Abstract

Vieira ,R.P., Mulloy,B. and Mourão,P.A.S. (1991) Structure of a fucose-branched chondroitin sulfate from sea cucumber: evidence for the presence of 3-O-sulfo-[beta]-d-glucuronosyl residues. J. Biol. Chem., 266, 13530-13536. MEDLINE Abstract

3To whom correspondence should be addressed

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: jnl.info{at}oup.co.uk
Last modification: 25 Aug 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
V. H Pomin and P. A S Mourao
Structure, biology, evolution, and medical importance of sulfated fucans and galactans
Glycobiology, December 1, 2008; 18(12): 1016 - 1027.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
L. P Cinelli, M. O Castro, L. L Santos, C. R Garcia, A.-C. E. Vilela-Silva, and P. A. Mourao
Expression of two different sulfated fucans by females of Lytechinus variegatus may regulate the seasonal variation in the fertilization of the sea urchin
Glycobiology, August 1, 2007; 17(8): 877 - 885.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
V. H. Pomin, A. P. Valente, M. S. Pereira, and P. A.S. Mourao
Mild acid hydrolysis of sulfated fucans: a selective 2-desulfation reaction and an alternative approach for preparing tailored sulfated oligosaccharides
Glycobiology, December 1, 2005; 15(12): 1376 - 1385.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. R. Melo, M. S. Pereira, D. Foguel, and P. A. S. Mourao
Antithrombin-mediated Anticoagulant Activity of Sulfated Polysaccharides: DIFFERENT MECHANISMS FOR HEPARIN AND SULFATED GALACTANS
J. Biol. Chem., May 14, 2004; 279(20): 20824 - 20835.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
H.M.M. J. Gunaratne, T. Yamagaki, M. Matsumoto, and M. Hoshi
Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs
Glycobiology, August 1, 2003; 13(8): 567 - 580.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
O. Berteau and B. Mulloy
Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide
Glycobiology, June 1, 2003; 13(6): 29R - 40R.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
M. S. Pereira, F. R. Melo, and P. A.S. Mourao
Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?
Glycobiology, October 1, 2002; 12(10): 573 - 580.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Hirohashi and V. D. Vacquier
Egg Sialoglycans Increase Intracellular pH and Potentiate the Acrosome Reaction of Sea Urchin Sperm
J. Biol. Chem., March 1, 2002; 277(10): 8041 - 8047.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A.-C. E. S. Vilela-Silva, M. O. Castro, A.-P. Valente, C. H. Biermann, and P. A. S. Mourao
Sulfated Fucans from the Egg Jellies of the Closely Related Sea Urchins Strongylocentrotus droebachiensis and Strongylocentrotus pallidus Ensure Species-specific Fertilization
J. Biol. Chem., January 4, 2002; 277(1): 379 - 387.
[Abstract] [Full Text]

Home page
 GlycobiologyHome page
A.-C. E.S. Vilela-Silva, C. C. Werneck, A. P. Valente, V. D. Vacquier, and P. A.S. Mourao
Embryos of the sea urchin Strongylocentrotus purpuratus synthesize a dermatan sulfate enriched in 4-O- and 6-O-disulfated galactosamine units
Glycobiology, June 1, 2001; 11(6): 433 - 440.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
K. J. Mengerink and V. D. Vacquier
Glycobiology of sperm-egg interactions in deuterostomes
Glycobiology, April 1, 2001; 11(4): 37R - 43R.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
W. R. L. Farias, A.-P. Valente, M. S. Pereira, and P. A. S. Mourao
Structure and Anticoagulant Activity of Sulfated Galactans. ISOLATION OF A UNIQUE SULFATED GALACTAN FROM THE RED ALGAE BOTRYOCLADIA OCCIDENTALIS AND COMPARISON OF ITS ANTICOAGULANT ACTION WITH THAT OF SULFATED GALACTANS FROM INVERTEBRATES
J. Biol. Chem., September 15, 2000; 275(38): 29299 - 29307.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Hirohashi and V. D. Vacquier
High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction
J. Biol. Chem., January 4, 2002; 277(2): 1182 - 1189.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (52)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Vilela-Silva, A. C.
Right arrow Articles by Mourao, P. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vilela-Silva, A. C.
Right arrow Articles by Mourao, P. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?