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Glycobiology Pages 939-946  

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans
Introduction
Results and discussion
Materials and methods
Acknowledgments
References

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

Ana-Paula Alves, Barbara Mulloy1, Gary W.Moy2, Victor D.Vacquier2, Paulo A.S.Mourão3

Departamento de Bioquímica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Caixa Postal 68041, Rio de Janeiro, RJ, 21941-590, Brazil, 1National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, United Kingdom, and 2Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA, 92093-0202, USA

Received on February 6, 1998; revised on March 8, 1998; accepted on March 21, 1998

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1->3-linked [alpha]-l-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O-2 position but with a heterogeneous sulfation pattern at O-4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-[alpha]-l-Fucp-2,4(OSO3)-1->3-[alpha]-l-Fucp-4(OSO3)-1->3-[alpha]-l-Fucp-4(OSO3)-1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.

Key words: sulfated fucan/acrosome reaction/sea urchin egg jelly

Introduction

Most animal spermatozoa must undergo an exocytotic acrosome reaction as a prerequisite for attachment to and fusion with the egg. The acrosome reaction is an ion channel regulated event whose signal transduction pathway remains to be elucidated (Darszon et al., 1996). Sea urchin spermatozoa are terminally differentiated cells that can be obtained in vast quantities which undergo the acrosome reaction with a high degree of synchrony in a time span of seconds. These cells provide an important model for studying the molecular events of sperm-egg recognition and transmembrane signaling during fertilization.

The sea urchin egg is surrounded by a transparent jelly coat, which contains molecules inducing potent physiological changes in sperm (Garbers, 1989). 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; Vacquier and Moy, 1997). These compounds have simple, well-defined repeating structures and each species represents a particular pattern of sulfate substitution. For example, the sea urchin Echinometra lucunter contains a homopolymer of 2-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 example of ligand-induced signal transduction (Alves et al., 1997; Vacquier and Moy, 1997).

Electrophoresis of the sulfated fucans isolated from the egg jelly of individual females of the sea urchin S.purpuratus showed that approximately 90% of the females possessed eggs containing one of two possible sulfated fucans, a fast migrating or a slow migrating electrophoretic isotype. Ten percent of the females spawned eggs with both sulfated fucan isotypes. We have now determined the chemical structures of both sulfated fucan isotypes and tested the purified material for its ability to induce the acrosome reaction in sperm of this species. Both sulfated fucans are linear polysaccharides but with a different sulfation pattern and are of approximately equal potency in inducing the sperm acrosome reaction. These results show expanded possibilities for structural variations among the sulfated fucans of echinoderms and constitutes a valuable tool for future studies aiming to determine the relationship between structure and biological activity of these polysaccharides.

Results and discussion

Agarose gel electrophoresis and Mono Q-FPLC chromatography of the sulfated fucans from S.purpuratus egg jelly

Agarose gel electrophoresis in 1,3-diaminopropane:acetate buffer followed by toluidine blue staining showed that crude egg jelly, isolated from individual females, contained either a slow (sulfated fucan I) or fast (sulfated fucan II) migrating fucan isotype (Figure 1), confirming similar results obtained by SDS-PAGE (Vacquier and Moy, 1997). The electrophoretic migration of sulfated polysaccharides in 1,3-diaminopropane:acetate buffer depends on the structure of the polysaccharide, which forms a complex with the diamino groups (Dietrich et al., 1977). Thus, the different electrophoretic mobility of sulfated fucans I and II in this system is a preliminary indication of distinctive structures of the two polysaccharides.


Figure 1. Agarose gel electrophoresis of the sulfated fucans extracted from the egg jelly of different individual females.Sulfated fucans were extracted from the egg jelly of different females using papain digestion and partially purified by ethanol precipitation. The sulfated fucans (~15 µg) were then 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). Gels were fixed with 0.1% N-cetyl-N,N,N-trimethylammonium bromide solution. After 12 h, the gels were dried and stained with 0.1% toluidine blue in acetic acid:ethanol:water (0.1:1:5, v/v).

Mono Q-FPLC chromatography of a mixed sample of sulfated fucans I and II showed that sulfated fucan I eluted before sulfated fucan II and also that both fucans carried high net negative charge density. Both sulfated fucans, purified as in Figure 2A, migrated on agarose gels (Figure 2B) identically as shown in Figure 1, indicating that the toluidine blue staining material in crude egg jelly is the sulfated fucan component. Chemical analysis of the purified sulfated fucans (Table I) revealed a high content of sulfate ester with fucose as the only sugar, while the strongly negative specific rotations are compatible with residues of [alpha]-l-fucopyranose.


Figure 2. Purification of the sulfated fucans by Mono Q-FPLC chromatography (A) and analysis of the purified sulfated fucans by agarose gel electrophoresis (B). (A) a mixed sample of sulfated fucans from several S.purpuratus females (10 mg) was applied to a Mono Q-FPLC column (HR 5/5), equilibrated with 20 mM Tris/HCl buffer (pH 8.0). The column was developed by a linear gradient of 0.15-2.0 M NaCl in the same buffer. Fractions were assayed by metachromasia using 1,9-dimethylmethylene blue and those containing sulfated fucans I and II were pooled, dialyzed against distilled water and lyophilized. (B) A mixed sample of sulfated fucans and purified sulfated fucan I or sulfated fucan II (~15 µg of each) were applied to a 0.5% agarose gel and the electrophoresis was run and stained as described in the Figure 1 caption.

Table I. Chemical composition and specific optical rotation of the sulfated fucans from the egg jelly coat of S.purpuratus
Polysaccharidea Chemical composition (molar ratios) [[alpha]]D20°C
l-Fucose Sulfate
Mixed sulfated fucans 1.00 1.10  
Sulfated fucan I 1.00 1.13 -100°
Sulfated fucan II 1.00 1.08 -120°
aPurified sulfated fucans I and II were obtained by Mono Q-FPLC chromatography (see Figure 2).

Apparently, the slight difference in the sulfate content of sulfated fucans I and II is in contrast with the elution of these two polysaccharides on a Mono Q-FPLC column (Figure 2A). However, these results must be interpreted carefully. It is possible that the assay for sulfate is not accurate enough to distinguish small differences in sulfate content. In addition, it is not possible to exclude the influence of the molecular weight or structure of the polysaccharides on the elution from the Mono Q-FPLC column.


Methylation analysis of the sulfated fucan indicates a 1->3 linkage

The methylation analysis used a sample of sulfated fucans prepared from a pool of egg jelly from different females since these analyses require a large amount of polysaccharide. The sulfated fucan pool was purified by DEAE-cellulose which does not separate sulfated fucan I from sulfated fucan II. This preparation is hereby designated as 'mixed sample of sulfated fucans."

Table II. Methylated derivatives obtained from a mixed sample of native and desulfated fucans representing sulfated fucans I and II
Alditolsa Native Desulfatedb
2,3-Me2-Fuc <1 5
2,4-Me2-Fuc <1 60
2-Me-Fuc 41 30
4-Me-Fuc 18 3
Fuc 41 2
Data are % of total peak area.
aThe identity of each peak was established by mass spectrometry.
bDesulfation of the sulfated L-fucan was performed by solvolysis in dimethylsulfoxide for 6 h, as described previously (Mourão and Perlin, 1987; Vieira et al., 1991).

Methylation analysis of the mixed sample showed the two principal products to be non-methylated fucose and 2-methylfucose, indicating that the sample consisted largely of 2,3,4- and 3,4-substituted fucose (Table II). A smaller proportion of 4-methylfucose indicates the presence of 2,3-substituted fucose in the native sample. After desulfation, methylation products were principally 2,4-di-O-methylfucose and 2-methylfucose. These results are consistent with a completely 1->3 linked fucan, partially sulfated at the 2- and 4-positions, with some 4-sulfated material still present after the solvolytic desulfation process. The fucose-linked sulfate esters are known to be more resistant than other sulfated polysaccharides to solvolysis in dimethyl sulfoxide (Vieira et al., 1991).

NMR spectroscopic analysis shows the patterns of sulfation of the fucans

NMR spectroscopic studies were undertaken on both samples of the purified sulfated fucans I and II, and on a larger sample in which both sulfated fucans were present. One-dimensional 1H NMR spectra of sulfated fucans I and II are shown in Figure 3; a single broad anomeric signal can be seen in the spectrum of sulfated fucan I (Figure 3A) and integration of the spectrum of sulfated fucan II indicated the presence of three anomeric signals (Figure 3B and inset). Two-dimensional correlated spectroscopy gave poor results for the samples of both purified sulfated fucans, but TOCSY spectrum of the mixed sample (Figure 4) allowed correlations to be traced for four spin systems, three from the anomeric signals present in the spectrum of sulfated fucan II (designated II A, II B, and II C) and one from the anomeric signal of sulfated fucan I (designated I). All the spin systems were attributable to [alpha]-l-fucose residues and were continuous from H1 to H4 of each fucose residue; for sulfated fucan I, two H4 signals could be identified. The TOCSY spectrum did not give H4-H5 cross-peaks, due to the small (<2 Hz) H4-H5 coupling constant. Completion of the assignments of the 1H spectra was achieved using a NOESY spectrum of the mixed sample (Figure 5), which gave H4-H5, H4-H6 and H5-H6 cross-peaks. NOESY spectra (not shown) of purified samples of sulfated fucans I and II confirmed the assignments. 1H NMR data are summarized in Table III.

The one-dimensional 1H spectrum of the mixed sample is shown in Figure 3C, with the spectrum of its desulfated derivative in Figure 3D. The spectrum of the desulfated sample has a single anomeric signal and is compatible with the presence of a homopolymer of [alpha]-l-fucose. Chemical shifts were similar to those previously recorded for 1->3 linked [alpha]-l-fucans (Table III), indicating that both sulfated fucans I and II have the same 1->3 linked backbone structure, varying in their sulfation patterns. Positions of sulfation were deduced from strong (0.6 - 0.7 ppm) downfield sulfation shifts in the 1H spectra. Fucan I is 2-O-sulfated at all its residues, and partially 4-O-sulfated. Fucan II has three distinct residues, all 4-O-sulfated, residue A also being 2-O-sulfated.

Table III. Proton chemical shifts (ppm) for residues of [alpha]-l-fucose in native and chemical desulfated fucans from S.purpuratus
Polysaccharide Residueb Chemical shifts (ppm)a
H1 H2 H3 H4 H5 H6
Sulfated fucan I Ia 5.38 4.59 4.28 4.90 4.55 1.32
Ib       4.09 4.48 1.26
Sulfated fucan II A 5.53 4.57 4.50 4.92 4.50 1.29
Sulfated fucan II B 5.14 3.90 4.07 4.76 4.55 1.32
Sulfated fucan II C 5.13 3.92 4.11 4.82 4.50 1.29
Desulfated mixed fucan   5.12 3.96 4.02 4.04 4.33c 1.25
[alpha]-Fuc-O-Med   4.47 3.80 3.80 3.80 4.04 1.23
-3-[alpha]-l-Fuc-1-e   5.08 3.92 4.00   4.27 1.21
[alpha]-l-Fuc-(1->2)-[alpha]-l-FucOMef   5.02 3.79 3.91 3.82 4.25 1.24
[alpha]-l-Fuc-(1->2)-[alpha]-l-FucOMef   4.95 3.85 3.92 3.84 4.04 1.24
aThe 1H spectrum was recorded at 500 MHz, 60°C in 99.8% D2O. Chemical shifts are relative to internal trimethylsilylpropionic acid. Values in bold indicate positions bearing sulfate.
bSee Figure 3.
cMeasured at 40°C.
dRao et al., 1985.
eRibeiro et al., 1994.
fBaumann et al., 1991.

Table IV. Carbon chemical shifts (ppm) for unsulfated fucose and desulfated fucans
Compound Chemical shifts (ppm)a
C1 C2 C3 C4 C5 C6
[alpha]-l-Fuc-O-Meb 100.50 69.00 70.60 72.90 67.50 16.50
-3-[alpha]-l-Fuc-1-c 98.29 69.00 77.60 71.10 69.00 18.00
Present work 98.23 69.05 77.66 71.11 69.05 17.90
a13C spectra were recorded at 125 MHz, 60°C in D2O. Chemical shifts are relative to internal trimethylsilylpropionic acid.
bGorin and Mazurek, 1975.
cRibeiro et al., 1994.

Figure 3. Expansions of the 6.0-3.0 and 1.5-1.1 ppm regions of the 1H spectra at 500 MHz of the purified sulfated fucans I (A) and II (B) and of a mixed sample of both sulfated fucansbefore (C) and after chemical desulfation (D). Expansions of the 6.0-5.0 ppm region of the 1H spectra are shown in the insets in (B) and (C). The integrals listed under the proton spectra are normalized to a total of 100 protons. Signals corresponding to residues of sulfated fucan I in (C) are shaded. All chemical shifts are relative to internal trimethylsilylpropionic acid.


Figure 4. Expansion of the TOCSY spectrum of the mixed sample of sulfated fucans.Cross-peaks used in the assignment of H1-H4 of three sulfated fucose residues A, B, and C in sulfated fucan II, and H1-H4a and H1-H4b of sulfated fucan I are marked.

Interresidue cross-peaks in the NOESY spectrum of the mixed sample (Figure 5) indicate that the three residues in sulfated fucan II form a regular repeating unit. In this way cross-peaks from H1 of residue A to H3 of residue C, from H1 of residue B to H3 of residue A, and from H1 of residue C to H3 of residue B, give the order of the repeating unit of this fucan as -A-C-B-.

The 13C NMR spectra of the fucans are shown in Figure 6. The 13C spectrum of sulfated fucan I (Figure 6A) has a complex group of anomeric carbon resonances, and overlapping ring carbon signals. This indicates that sulfated fucan I does not have a simple repeating structure; the 1H spectrum shows full 2-O-sulfation, but only partial 4-O-sulfation, and the complex 13C spectrum may be a consequence of random or near-random distribution of the two types of residues. The 13C spectrum of sulfated fucan II (Figure 6B) contains 15 lines from ring carbons and three anomeric signals, as expected for a fucan with a three-residue repeating unit. Three signals near 82 ppm can be assigned to sulfated C4, four signals between 75 ppm and 79 ppm to glycosylated C3 and the sulfated C2 of residue A, and the signals between 69 ppm and 72 ppm to the three C5 and unsulfated C2s. The 13C spectrum of the mixed sample (Figure 6C) is the superposition of the spectra of the two purified sulfated fucans, and the desulfated fucan gives a 13C NMR spectrum (Figure 6D, Table IV) which, like the 1H spectrum, is consistent with a 1->3 linked [alpha]-l-fucan.

Overall, the combination of methylation and NMR spectroscopic analysis confirm the structures of sulfated fucans I and II as shown in Figure 7.

S.purpuratus contains unique variants of sulfated [alpha]-l-fucans

Sulfated fucans have been isolated from the cell walls of marine brown algae (Percival and McDowell, 1967; Painter, 1983; Nishino et al., 1991a), the sea cucumber body wall (Vieira and Mourão, 1988; Ribeiro et al., 1994), and the egg jelly coat of sea urchin eggs (SeGall and Lennarz, 1979; Mulloy et al., 1994; Alves et al., 1997; Vacquier and Moy, 1997). These polysaccharides are among the most widely studied of all the sulfated polysaccharides from nonmammalian origin that exhibit biological activities in mammalian systems (for a review, see Mulloy et al., 1994). Sulfated fucans are potent anticoagulant and antithrombotic polysaccharides (Church et al., 1989; Nishino et al., 1991b; Mauray et al., 1995) and thus their structures are of potential biomedical importance.

Previously we compared the sulfated fucans from echinoderms with those from brown algae. The sulfated fucans from echinoderms have a unique structure, composed of tetrasaccharide repeating units in which the four residues differ by specific patterns of sulfation at the O-2 and O-4 positions. In addition, the specific pattern of sulfation and the position of the glycosidic linkage varies among different species. Thus, the regular repeating sequences of residues in the sulfated fucan from the sea urchin L.variegatus are as follows: [3-[alpha]-l-Fucp-2(OSO3)-1->3-[alpha]-l-Fucp-4(OSO3)- 1->3-[alpha]-l-Fucp-2,4(OSO3)-1->3-[alpha]-l-Fucp-2(OSO3)-1]n (Mulloy et al., 1994), while the sequences of residues in the sulfated fucan from the sea urchin A.lixula are: [4-[alpha]-l-Fucp-2(OSO3)-1->4-[alpha]-l-Fucp-2(OSO3)-1->4-[alpha]-l-Fucp-1->4-[alpha]-l-Fucp-1]n (Alves et al., 1997). Finally, the sea cucumber L.grisea contains a sulfated fucan with the following sequences of residues: [3-[alpha]-l-Fucp-2,4(OSO3)-1->3-[alpha]-l-Fucp-1->3-[alpha]-l-Fucp-2(OSO3)1->3-[alpha]-l-Fucp-2(OSO3)-1]n (Ribeiro et al., 1994).

Surprisingly, S.purpuratus egg jelly contains sulfated fucans of two different structures. Sulfated fucan II is composed of a regular repeating sequence of three residues, as follows: [3-[alpha]-l-Fucp-2,4(OSO3)-1->3-[alpha]-l-Fucp-4(OSO3)-1->3-[alpha]-l-Fucp-4(OSO3)-1]n (Figure 7). Sulfated fucan I is entirely sulfated at the O-2 position, but has a heterogeneous sulfation pattern at the O-4 position. These results show expanded possibilities for structural variations among the sulfated fucans of echinoderms.


Figure 5. Two expansions from the NOESY spectrum of the mixed sample of sulfated fucans. (A) nOe cross-peaks between H6 methyl resonances and H4 and H5 of each residue. (B) nOe cross-peak from H1 of each residue to other ring protons, in particular the sequence-defining nOes A1-C3, B1-A3, C1-B3 for sulfated fucan II.

One way to determine the relationship between structure and biological activity of sulfated polysaccharides is to compare the activities in various assays where the nature of the polysaccharide backbone and the extent and position of sulfation have been fully characterized. These echinoderm sulfated fucans constitute a valuable tool for such studies, especially concerning their anticoagulant and antithrombotic activities.

Induction of the acrosome reaction

Both sulfated fucan isotypes, purified by DEAE and Mono Q-FPLC, were lyophilized to dryness, resuspended in sea water, and dialyzed against sea water (3500 mol. wt. cut off membrane); total neutral hexose determined, and the ability to induce acrosome reaction was assayed. A plot of percentage of acrosome reaction versus log ng hexose per ml (Figure 8) showed that both sulfated fucan isotypes are approximately equal in acrosome reaction inducting potency. These data are consistent with experiments in which both fucans were purified by other methods (Vacquier and Moy, 1997). Both this study and the previous studies (SeGall and Lennarz, 1979; Alves et al., 1997; Vacquier and Moy, 1997) indicate that these sulfated fucans are the acrosome reaction inducers of sea urchin egg jelly.

Two sulfated fucan isotypes

The reason that the majority of individual females spawn eggs possessing only one of the two sulfated fucan isotypes remains unknown. The inheritance of such sulfation patterns is unknown. Whether the two sulfated fucan isotypes occur because of genetic or environmental factors, or age of the egg clutch stored in the ovary, remains unknown. The animals used in this study were collected from the same population at the same time and held under identical conditions. If the difference between sulfation pattern was temporal and related to the stage of ovogenesis, one would expect to see intermediate bands between sulfated fucan I and sulfated fucan II. However, SDS-PAGE (Vacquier and Moy, 1997) and agarose gel electrophoresis (Figure 1) never show intermediate bands between fast (II) and slow (I) migrating sulfated fucan isotypes.

Figure 6. 13C spectra at 125 MHz of the purified sulfated fucan I (A) and II (B) and of a mixed sample of sulfated fucansbefore (C) and after desulfation (D). Expansion of the 105-95 ppm of the spectrum is shown in the inset in (B). The integrals listed under the carbon region of the spectrum are normalized to a total of 100 carbons.



Figure 7. Deduced structures of the sulfated fucans I and II isolated from the egg jelly of the sea urchin S.purpuratus. Sulfated fucan II is a 1->3 linked linear polysaccharide with a regular trisaccharide repeat unit defined by the pattern of O-sulfation. Two residues are sulfated at the O-4 position and one residue is sulfated at both O-2 and O-4 positions. In contrast,sulfated fucan Ihas a more heterogeneous structure. It is also a 1->3 linked linear polysaccharides, entirely sulfated at the O-2 position, but with a heterogeneous sulfation pattern at O-4 position.

Figure 8. Induction of the S.purpuratus sperm acrosome reaction by purified sulfated fucan I and sulfated fucan II. Percent acrosome reaction is plotted against log ng hexose/ml. After lyophilization, both sulfated fucans were dialyzed into sea water and assayed for acrosome reaction at 18°C (Vacquier and Moy, 1997; Vacquier, 1986). Between 200-300 spermatozoa were scored per data.

The pattern of sulfation is produced by site specific sulfotransferases. Sulfated fucan I may require two transferases, one for position C2 and the other for position C4. However, sulfated fucan II could require a minimum of four sulfotransferases, two for C2 and C4 on the first fucose residue, a third to recognize the 2,4-disulfated fucose and then sulfate C4 on the second fucose residue, and a fourth transferase to recognize the sulfation pattern of fucose residues one and two and then to sulfate the third residue at the C4 position to obtain the repeating trisaccharide pattern.

An alternative hypothesis to explain the presence of either sulfated fucan I or sulfated fucan II in separate females is to postulate that in both types of females all fucose residues of sulfated fucan become sulfated on C2 and C4, but in the females containing sulfated fucan II a sulfatase removes the sulfate groups at C2 on only the second and third fucose residues. Of 71 individual females, 40 had eggs with sulfated fucan II, 22 had eggs with sulfated fucan I, and 9 had eggs with both sulfated fucans (Vacquier and Moy, 1997).

Studies of membrane proteins from the sperm of the sea urchin S.purpuratus indicate that one glycoprotein of Mr 210,000 binds the egg jelly sulfated fucan; this is sufficient to induce the sperm acrosome reaction (Moy et al., 1996; Vacquier and Moy, 1997). This unique sperm membrane protein is therefore termed 'REJ" (receptor for egg jelly). REJ possesses two carbohydrate recognition domain modules (CRDs) of the 'intron plus" C-type lectin variety. Of the 1450 amino acids in the open reading frame of REJ, 929 comprise the 'REJ module," a motif shared only with human polycystin. Polycystin is the protein mutated in autosomal dominant polycystic kidney disease, one of the most common genetic disease of humans (Moy et al., 1996). Because REJ regulates ion channel activity in sea urchin sperm cells, the hypothesis has been presented that polycystin may serve a similar function in human cells (Moy et al., 1996).

Both sulfated fucan isotypes bind to isolated sperm REJ coupled to an Affigel column (Vacquier and Moy, 1997). The two CRDs of REJ, each 120 residues in length and separated by a 14 residue spacer, suggest that REJ is a carbohydrate binding protein. The two CRDs are 50% identical, indicating that considerable divergence has occurred following their duplication (Moy et al., 1996). The discovery of two CRDs on sperm REJ and two structurally different sulfated fucans in egg jelly suggests that each CRD may be specific for one of the two sulfated fucans, occupancy of only one CRD being necessary for the acrosome reaction induction.

The discovery of two structurally distinct acrosome reaction inducers in sea urchin egg jelly increases the level of complexity in considering the evolutionary forces acting on gamete recognition systems (Vacquier et al., 1997).

Materials and methods

Sulfated polysaccharides from the sea urchin egg jelly coat

Extraction. Eggs were spawned into sea water from individual adults of S.purpuratus (collected at La Jolla, CA) by injection of 0.5 M KCl. The crude egg jelly was isolated by the pH 5.0 method and prepared as a 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 cetylpyridinium chloride and ethanol precipitation as described previously (Vieira et al., 1991).

Purification. The crude polysaccharides (~50 mg) were applied to a DEAE-cellulose column (15 × 2 cm), equilibrated with 50 mM sodium acetate (pH 5.0), and washed with 250 ml of the same buffer. The column was eluted in three steps. First, the column was eluted with a linear gradient prepared by mixing 50 ml of 50 mM sodium acetate buffer (pH 5.0) with 50 ml of 1.0 M NaCl in the same buffer. Second, the column was washed with 100 ml of the sodium acetate buffer containing 1.0 M NaCl. Third, the column was eluted with a linear gradient prepared by mixing 100 ml of 1.0 M NaCl with 100 ml of 5.0 M NaCl, both in the same sodium acetate buffer. The flow rate was 15 ml/h; fractions of 3.5 ml were collected in the different elution steps. Fractions were assayed by the metachromasia produced by sulfated polysaccharides with 1,9-dimethylmethylene blue (Farndale et al., 1986) and for fucose content by the Dubois et al. reaction (Dubois et al., 1956). The NaCl concentration was estimated by conductivity. Fractions containing the sulfated [alpha]-l-fucan were pooled, dialyzed against distilled water and lyophilized.

The DEAE-cellulose-purified sulfated fucan (~10 mg) was applied to a Mono Q column-FPLC (HR 5/5) (Pharmacia Biotech Inc.) equilibrated in 20 mM Tris-HCl (pH 8.0). The column was developed with a linear gradient of 0-2.0 M NaCl in the same buffer. The flow rate of the column was 0.45 ml/min, and fractions of 0.5 ml were collected and assayed by metachromasia using 1,9-dimethylmethylene blue (Farndale et al., 1986).

Chemical analyses. Total fucose was measured by the method of Dische and Shettles (Dische and Shettles, 1948). After acid hydrolysis of the polysaccharides (5.0 M trifluoroacetic acid for 5 h at 100°C), sulfate was measured by the BaCl2/gelatin method (Saito et al., 1968). The percentages of hexoses and 6-deoxyhexoses in the acid hydrolysates were estimated by paper chromatography in n-butanol:pyridine:water (3:2:1, v/v) for 48 h and by gas-liquid chromatography of derived alditol acetates (Kircher, 1960). Optical rotations were 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). Sulfated [alpha]-l-fucans (~15 µg) were 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 L-fucans 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 native and desulfated polysaccharides (~5 mg) were subjected to three rounds of methylation as described previously (Ciucanu and Kerek, 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) (Kircher, 1960). The alditol acetates of the methylated sugars were dissolved in chloroform and analyzed in a gas chromatography/mass spectrometer.

NMR spectroscopy. 1H spectra were recorded at 500 MHz and 13C spectra at 125 MHz using a Varian spectrometer. The polysaccharide sample (~15 mg) was converted to the sodium salt by passage through a column 10 ×1 cm of DOWEX 50-XS Na+ form and all samples were dissolved in approximately 0.7 ml of 99.8% D2O. The spectra were recorded at 60°C with suppression of the HOD signal by presaturation. 13C spectra were recorded with full proton decoupling. Two-dimensional double-quantum filtered COSY, TOCSY, and NOESY experiments were performed using pulse sequences supplied by Varian. TOCSY spectra were run with a spin-lock field of about 10 kHz and a mixing time of 80 ms. NOESY spectra were run with a mixing time of 100 ms. All chemical shifts were relative to internal or external trimethylsilylpropionic acid.

Sulfated fucans as inducers of the sperm acrosome reaction. Sperm 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 induction by sulfated polysaccharides were done as described previously (Vacquier, 1986; Vacquier and Moy, 1997).

Acknowledgments

We are grateful to Adriana A. Eira for technical assistance.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), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and NIH Grant HD 12986 (to V.D.V.).

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