The Occurrence of Novel 9- O -Sulfated N -Glycolylneuraminic Acid-capped (cid:97) 2 3 5- O glycolyl -linked Oligo/PolyNeu5Gc Chains in Sea Urchin Egg Cell Surface Glycoprotein IDENTIFICATION OF A NEW CHAIN TERMINATION SIGNAL FOR POLYSIALYLTRANSFERASE*

We report the isolation and structural characteriza- tion of an oligo/polysialic acid-containing glycopeptide fraction (designated ESP-Sia) prepared from the egg cell surface complex of the sea urchin, Hemicentrotus pulcherrimus , by exhaustive pronase treatment. The carbo- hydrate chains isolated from ESP-Sia were shown to consist of O -linked oligo/polysialic acid-containing gly- can units and N -linked carbohydrate chains. The present studies have revealed that the O -linked oligo/polysi- alic acid-containing glycan chains derived from the ESP-Sia were similar to those present in egg jelly coat polysialylated glycoprotein in being composed of tan-dem repeats of N -glycolylneuraminic acid (Neu5Gc) gly- cosidically linked in a novel fashion through the glycolyl group, ( Spectrometry atom bombard-ment-mass spectrometry spectra were recorded using a VG Analytical ZAB-2S.E. FPD mass spectrometer fitted with a cesium ion gun oper-ated at 20–25 kV. Data acquisition and processing were performed using VG Analytical Opus software. The peracylated samples were aliquoted in methanol, and monothioglycerol was used as matrix. -sulfated resistant to sialidase Monosulfated disialyl oli-gosaccharide alditol, by -elimination of ESP-Sia resistant to sialidase action These results strongly suggested that sulfation occurred exclusively on the nonreducing terminal Neu5Gc residue of oligo/polySia structures in ESP-Sia. To further estimate the ratio of sulfated Neu5Gc to Neu5Gc in ESP-Sia, we performed mild (0.1 N trifluoroacetic 80 1 h) of the ESP-Sia sample, under which conditions no desulfa-tion occurred. The hydrolysates were analyzed by TLC to con-firm that all Sia residues had been converted to free Sia. Then, Neu5Gc and Neu5Gc the sample were fractionated by HPLC on a Mono-Q column, and the elution profile was monitored by the resorcinol method. The result showed that the 3 in a total Sia residue in ESP-Sia was about 24%. Because the average DP of oligo/ polySia ESP-Sia about 3 as described about 73% of the nonreducing terminal Neu5Gc residues in oligo/polySia chains

Polysialic acid (polySia) 1 is the general name for sialic acid polymers, and the most commonly occurring of these is a linear polymer of sialic acid in ␣238 linkage expressed on cell surface glycoconjugates in a wide variety of animal species ranging from neurotropic bacteria to man (1)(2)(3)(4)(5)(6)(7). There are a number of reports demonstrating that ␣238-linked polySia chains are involved in regulation of cell-cell interactions, cell adhesion, and cell recognition (3, 8 -12).

EXPERIMENTAL PROCEDURES
Materials-H. pulcherrimus were collected at Cape Manazuru, Japan, or were obtained from Tsushima courtesy of Drs. I. Yasumasu (Waseda University) and I. Mabuchi (University of Tokyo). Artificial seawater was purchased from Jamarin (Osaka, Japan). C. perfringens sialidase was purchased from Sigma. Actinase E or pronase (specific activity; 1,000,000 Tyr units/g) was purchased from Kaken (Tokyo, Japan). Sea urchin egg jelly sialic acid-rich glycoprotein was prepared as described previously (13). Trout egg L-polysialoglycoprotein used as a molecular mass marker (9 kDa) was prepared as described previously (17)(18)(19).
Isolation of Egg Cell Surface Complex-Eggs were collected from H. pulcherrimus by injection of 0.5 M KCl, washed three times with artificial seawater, and dejellied as described previously (13,20). Egg cell surface complex containing plasma membrane and vitelline layer was prepared as described previously (21). Briefly, dejellied eggs from female H. pulcherrimus were suspended in the same volume of Ca 2ϩ -free sea water containing 10 mM EDTA and protease inhibitors (1 mM phenylmethanesulfonyl fluoride, 1 g/ml aprotinin, 1 g/ml leupeptin, 10 g/ml benzamidin, and 20 g/ml antipain) and sonicated using a Branson Sonifier 250 at 50 watts for 10 min. The egg homogenate was diluted 5-fold with ice-cold Ca 2ϩ -free sea water, and the cell surface complex was pelleted by centrifugation at 1,000 ϫ g for 3 min. The pellet was immediately suspended and incubated in 1 M sucrose containing the protease inhibitors by gentle homogenization to degrade residual cortical granules. The membrane/vitelline layer complex was collected by centrifugation at 3,000 ϫ g for 20 min.
Preparation of Sia-rich Glycopeptide Fraction from Egg Surface Complex-The egg surface complex (about 120 ml) was delipidated as described previously (22). Briefly, the egg surface complex was suspended in 3 volumes of 100 mM Tris-HCl buffer (pH 8.0) with subsequent addition of 8 volumes of methanol and 4 volumes of chloroform. The resulting mixture was stirred for 2 h at room temperature and centrifuged at 15,000 ϫ g for 40 min. The pellet was resuspended in 100 mM Tris-HCl/methanol/chloroform (3:8:4) and centrifuged as above. The residual solvents were removed from the pellet by extracting with absolute ethanol. The pellet was dried under reduced pressure and weighed. The delipidated sample (22.5 g) was suspended in 800 ml of the incubation buffer (0.1 M Tris-HCl (pH 8.0), 10 mM CaCl 2 ), and 200 mg of pronase and 3 drops of toluene were added. The mixture was incubated with moderate shaking at 37°C. After 24 and 48 h, a 200-mg portion of fresh pronase was added. After 72 h, the digests were centrifuged at 10,000 ϫ g for 15 min. The supernatant fraction was dialyzed against distilled water and applied to a DEAE-Toyopearl 650 M column (3.2 ϫ 38 cm) equilibrated with 20 mM Tris-HCl (pH 8.0). The column was eluted with a linear gradient of NaCl (0 -0.6 M) in 20 mM Tris-HCl (pH 8.0). Fractions were assayed for Sia by the thiobarbituric acid method and for oligoSia by the TLC analysis of mild acid hydrolysates (pH 4.8, 80°C, 2 h). The oligoSia-containing fractions were pooled and dialyzed against distilled water. Four volumes of chloroform/methanol (2:1) were added to the sample (about 60 ml; containing 0.1 M Tris-HCl (pH 8.0)) and stirred vigorously at room temperature. After 12 h, the aqueous phase was separated, concentrated, and dialyzed against distilled water. The sample was adjusted to 4 M NaCl concentration and applied to a butyl-Toyopearl 650 M column (1.1 ϫ 15 cm) equilibrated with 4 M NaCl in 20 mM Tris-HCl (pH 8.0). The Sia-containing fraction, which was eluted in the flow-through fraction, was concentrated and then applied to a column (1.3 ϫ 96 cm) of Sephacryl S-200 that was eluted with 0.1 M NaCl in 10 mM Tris-HCl buffer (pH 8.0). The Siacontaining fraction was designated as ESP-Sia and desalted by passage through a Sephadex G-25 column (1.0 ϫ 52 cm).
Peptide:N-Glycanase Digestion-3.6 mg (as Sia) of ESP-Sia were incubated with 2 milliunits of peptide:N-glycanase derived from Flavobacterium meningosepticum (Takara Co., Kyoto) in 1.5 ml of 0.2 M sodium phosphate buffer (pH 8.5) for 24 h at 37°C. The same amount of the enzyme was then added, and the mixture was incubated for another 24 h. The reaction mixture was chromatographed on a Sephacryl S-200 column, as described above.
Alkaline Borohydride Treatment of ESP-Sia-The ESP-Sia (2.7 mg as Sia) was treated with 2 ml of 0.1 N NaOH containing 1 M NaBH 4 at 37°C. After 48 h, the reaction mixture was neutralized with acetic acid on ice and desalted by passage through a Sephadex G-25 column.
Treatment of Sulfated Sialyloligosaccharides with C. perfringens Sialidase-1.5 g each of sulfated and nonsulfated Neu5Gc dimers, sulfated and nonsulfated Neu5Gc trimers, and sulfated disialyloligosaccharide alditol derived from ESP-Sia were separately treated with 23 milliunits of C. perfringens sialidase at 37°C in 10 l of 50 mM sodium acetate buffer (pH 5.5). A control incubation was carried out under the same conditions except that no sialidase was present. After 10 h of incubation, the reaction mixture was analyzed by TLC, as described below.
Chemical Treatments of ESP-Sia-De-N-acylation and subsequent acetylation of Neu5Gc residues in ESP-Sia were carried out as described previously (13). The resulting reaction mixture was desalted by passage through Sephadex G-25. The periodate oxidation/borohydride reduction procedure followed that previously reported (23).
ESP-Sia sample was hydrolyzed with 50 mM sodium acetate buffer (pH 4.8) at 80°C for 2-6 h for detecting oligoSia or with 0.1 N trifluoroacetic acid at 80°C for 1 h for detecting free Sia. The free Sia or oligoSia liberated was analyzed by TLC or HPLC as described below.
Chemical Analyses of ESP-Sia-Sia residues were quantitated by the thiobarbituric acid method (24, 25) and the resorcinol method (26). Sia residues in ESP-Sia were determined by the mild acid hydrolysis/ subsequent mild methanolysis/gas-liquid chromatographical procedure as described previously (27). The hexose content was estimated by the phenol-sulfuric acid method (28). Amino acid and amino sugar analyses were carried out after hydrolysis in 6 N HCl at 105°C for 24 h under reduced pressure. Samples were derivatized with phenylisothiocyanate before column chromatography (1). Carbohydrate analysis was carried out by the gas-liquid chromatography as described previously (29). The sulfate analysis was carried out by HPLC on a TSK-gel IC-Anion PW column using a JASCO HPLC system equipped with a 980-PU pump, a 970-UV detector, and a Rheodyne injector with a 20-l sample loop. The elution pattern was monitored by measuring at 265 nm and recorded on a Chromatocorder 12 (System Instruments, Tokyo, Japan). Samples were evaporated to dryness after hydrolysis in 6 N HCl at 105°C for 12 h and dissolved in 30 -50 l of water. Samples in 1-10 l were injected and eluted isocratically with acetonitrile/n-butyl alcohol (4:1) containing 0.5 mM sodium-phthalate, 5.82 mM H 3 BO 3 , 1.3 mM Na 2 B 4 O 7 , and 0.9 mM sodium-gluconate at a flow rate of 1.2 ml/min.
Thin-layer Chromatography-TLC of oligosaccharides obtained from ESP-Sia was carried out as described previously (30).
HPLC of Sialyloligosaccharide on a Mono-Q Column-The sialyloligosaccharides were resolved by HPLC on an anion exchange Mono-Q column using an Irika HPLC system as described previously (30,31).
Fast Atom Bombardment-Mass Spectrometry-Fast atom bombardment-mass spectrometry spectra were recorded using a VG Analytical ZAB-2S.E. FPD mass spectrometer fitted with a cesium ion gun operated at 20 -25 kV. Data acquisition and processing were performed using VG Analytical Opus software. The peracylated samples were aliquoted in methanol, and monothioglycerol was used as matrix.
500-MHz 1 H NMR Spectroscopy-1 H NMR spectra of oligoSia were determined in D 2 O at 23°C with a Bruker AMX-500 spectrometer. Proton chemical shifts were expressed in parts/million relative to the methyl signal of sodium 3-(trimethylsilyl)propionate-2,2,3,3-d 4 . NMR measurements were generously carried out by Drs. Y. Muto, T. Niimi, Y. Takeda, and S. Watanabe in Prof. Yokoyama's laboratory at the University of Tokyo.

Preparation of ESP-Sia Fraction from Egg Surface Complex of Sea Urchin Eggs
The pronase digests of egg surface complex from 600 female H. pulcherrimus (600 ml of packed, dejellied eggs) were found to contain about 60 mg of Sia and were chromatographed on a DEAE-Toyopearl 650 M column (Fig. 1). TLC analysis of mild acid hydrolysates of Sia-containing fractions eluted from the columns was carried out to detect oligoSia-containing fractions. Sia-containing fractions eluting at 0.25-0.45 M NaCl, which were positive for oligoSia on TLC analysis of mild acid hydrolysates, were collected and dialyzed against distilled water.
Because the sample thus obtained was found to be contaminated by lipidic material, it was subjected to delipidation. Resultant insoluble material between the aqueous and organic phases did not contain Sia and was discarded. The aqueous phase was dialyzed against water, adjusted to 4 M NaCl, and chromatographed on a butyl-Toyopearl column. More than 82% of the Sia-containing glycopeptide was recovered in the flowthrough fraction and designated as ESP-Sia to denote egg surface complex-pronase digests. The glycopeptides, ESP-Sia, had an average molecular weight of approximately 9,000 as determined by gel filtration chromatography over the Sephacryl S-200 column.

Chemical Compositions of ESP-Sia
The chemical composition of ESP-Sia is shown in Table I. The carbohydrate content of ESP-Sia was about 89% by weight. It should be noted that ESP-Sia contained high amounts of sulfate. The Sia residue in ESP-Sia was identified as N-glycolylneuraminic acid (Neu5Gc). The presence of Man, GalNAc, and GlcNAc indicated the presence of both N-and O-linked glycan chains. However, peptide: N-glycanase digestion of ESP-Sia did not liberate any N-glycans, probably either because glycosylated asparagine residues were located at N-termini and/or C-termini (32) or because N-linked glycan chains had peptide: N-glycanase-resistant structures. After alkaline borohydride treatment of ESP-Sia, on average, one Thr per molecular weight of 9,000 was converted to ␣-aminobutyric acid, and GalNAcol appeared at the expense of GalNAc. Only a trace amount of the Ser residues, if any, was glycosylated. The results indicated that at least one O-linked carbohydrate chain was attached to a Thr residue in the peptide core of ESP-Sia.

Structural Elucidation of Sulfated Oligo/polySia Chains in ESP-Sia
Isolation and Characterization of oligoSia Prepared from ESP-Sia by Mild Acid Hydrolysis-After mild acid hydrolysis of ESP-Sia (pH 4.8, 80°C, 2 h), a series of sialyloligomers visualized by the resorcinol reagent were detectable on a thinlayer chromatogram (Fig. 2). The result, shown in Fig. 2, indicated for the first time the presence of oligo/polySia residues of DP Ͼ 7 in ESP-Sia. G1 in lane 4 migrated faster than Neu5Gc monomer and seemed to be a new component, which was shown to be converted into Neu5Gc monomer when heated under more acidic conditions with 0.1 N trifluoroacetic acid for 5 h at 80°C (data not shown). As shown in Fig. 2 (lane 4), the migration rates of G2 and G3 did not correspond to those for ␣235-O glycolyl -linked di-and tri-Neu5Gc (lane 2) or ␣238-linked oligoNeu5Gc (lane 1).
Strong Alkali Treatment and Subsequent N-Acetylation-Strong alkali treatment was employed to show that the interketosidic linkages in oligo/polySia chains of ESP-Sia are ␣235-O glycolyl . ESP-Sia was treated with 2 N NaOH as described previously (13,33), and after N-acetylation the reaction product was analyzed by TLC. Two spots appeared by visualization of the TLC plate using the resorcinol reagent. The lower spot corresponded to Neu5Ac␣23 O-CH 2 COOH by TLC mobility and elution position on Mono-Q HPLC (data not shown). The upper one (R f ϭ 1.23 relative to Neu5Ac␣23 O-CH 2 COOH) eluted from Mono-Q HPLC at retention time similar to Neu5Ac␣23 O-CH 2 COOH, and the molar ratio of the upper to the lower one was estimated to be 1:2 by the resorcinol method. As is described below, ESP-Sia was verified to contain the nonreducing terminal 9-O-sulfated Neu5Gc residues on the oligo/polySia chains with average DP of 3. Thus, the upper component was tentatively assigned to desulfated  substituent. When G1 was subjected to Mono-Q HR5/5 column, as described under "Experimental Procedures," it eluted at higher NaCl concentration than the Neu5Gc monomer (Fig. 3A). This finding indicated that G1 had an additional anionic group.  . 34 and 35)). The largest downfield shift was observed for H-9, and the spectrum of G1 was different from that of 8-O-sulfated Neu5Gc, previously identified in Anthocidaris crassipina (36), where H-8 signal resided at the lowest magnetic field. To further examine the position of sulfate ester on Neu5Gc residue, periodate oxidation/borohydride reduction of ESP-Sia was carried out (23). The GLC analysis of the reaction products showed that all of the Sia residues in ESP-Sia were converted into 2-keto-3,5-deoxy-D-galacto-5-N-glycolylheptonic acid, the C 7 analogue of N-glycolylneuraminic acid. Based on these data, it was concluded that G1 was 9-O-sulfated Neu5Gc (Neu5Gc9HSO 3 ). This is the first example of a sulfate ester at the C-9 position of sialic acid.
Separation of Sulfated oligoSia and Nonsulfated oligoSia by Mono-Q HPLC-G2 and G3 were separately subjected to HPLC on Mono-Q HR5/5 column. Unexpectedly, G2 gave two major peaks (Fig. 3B). One of them (designated as (Gc) 2 ) eluted at almost the same position as that of the ␣238-linked Neu5Gc dimer chromatographed as a reference. The other one (designated as GX2) was eluted at a higher NaCl concentration than the Neu5Gc dimer. G3 also gave two major peaks (Fig. 3C). One of them (designated as (Gc) 3 ) eluted at the same position as the ␣238-linked Neu5Gc trimer used as a reference, and the other (GX3) eluted at a higher NaCl concentration than the Neu5Gc trimer. Compositional analysis of GX2 and GX3 revealed that molar ratios of Neu5Gc to sulfate were 2:1 for GX2 and 3:1 for GX3, indicating that these were mono-O-sulfated oligomers.
Determination of the Position of Sulfated Neu5Gc Residue in Sulfated oligo/polySia Structures-(Gc) 2 , GX2, (Gc) 3 , and GX3 were separately treated with C. perfringens sialidase (Fig. 5). The (35-O glycolyl Neu5Gc␣23) n chains are known to be poor substrates for C. perfringens sialidase (14), but under the reaction conditions employed Neu5Gc␣235-O glycolyl Neu5Gc, (Gc) 2 , and Neu5Gc␣235-O glycolyl Neu5Gc␣235-O glycoly Neu5Gc, (Gc) 3 4 and lane 8). Previously, gangliosides containing 8-O-sulfated Neu5Gc and Neu5Ac residues had been shown to be resistant to sialidase (36,37). Monosulfated disialyl oligosaccharide alditol, which was liberated by ␤-elimination of ESP-Sia was also found to be resistant to sialidase action (data not shown). These results strongly suggested that sulfation occurred exclusively on the nonreducing terminal Neu5Gc residue of oligo/polySia structures in ESP-Sia. To further estimate the ratio of sulfated Neu5Gc to Neu5Gc in ESP-Sia, we performed mild acid hydrolysis (0.1 N trifluoroacetic acid, 80°C, 1 h) of the ESP-Sia sample, under which conditions no desulfation occurred. The hydrolysates were analyzed by TLC to confirm that all Sia residues had been converted to free Sia. Then, Neu5Gc and sulfated Neu5Gc in the sample were fractionated by HPLC on a Mono-Q column, and the elution profile was monitored by the resorcinol method. The result showed that the content of Neu5Gc9HSO 3 in a total Sia residue present in ESP-Sia was about 24%. Because the average DP of oligo/ polySia in ESP-Sia was about 3 as described below, about 73% of the nonreducing terminal Neu5Gc residues in oligo/polySia chains were sulfated. 500-MHz 1 H NMR Spectroscopy of Neu5Gc9HSO 3 ␣2(35-O glycolyl -Neu5Gc␣23) n Structures-The 500-MHz 1 H NMR spectra of (Gc) 2 , GX2, (Gc) 3 , and GX3 are reproduced in Fig. 4  (B, C, D, and E). Comparison of the 500-MHz 1 H NMR spectral data led us to identify (Gc) 2 and (Gc) 3 as Neu5Gc␣235-O glycolyl Neu5Gc and Neu5Gc␣235-O glycolyl Neu5Gc␣235-O glycolyl Neu5Gc, respectively (13). In the 1 H NMR spectrum of GX2 (Fig. 4C), the one-proton signal at 4.46 ppm was assignable to one of the two protons at C-9 of the 9-O-sulfated Neu5Gc residue as described above (Fig. 4A). The resonances at 1.82 ppm and 2.20 ppm were assigned to H-3 ax and H-3 eq protons of the reducing terminal ␤-Neu5Gc residue, respectively, by comparing with the 1 H NMR spectrum of Neu5Gc␣235-O glycolyl Neu5Gc (Fig. 4B). The resonances at 1.77 and 2.68 ppm were respectively assignable to H-3 ax and H-3 eq of the nonreducing terminal 9-O-sulfated Neu5Gc residue. Other resonances observed in Fig. 4 (B and C) were superimposable. These results indicated that GX2 is Neu5Gc9HSO 3 ␣235-O glycolyl Neu5Gc. The 1 H NMR spectrum of GX3 showed that one of the H-9 protons was again shifted down-field due to sulfation on the nonreducing terminal Neu5Gc residue (Fig.  4E). The resonance at 1.81 ppm was assigned to overlapped H-3 ax protons of reducing terminal and internal ␣235-O glycolyllinked Neu5Gc residues by comparison with the 1 H NMR spectrum of (Gc) 3 , Neu5Gc␣235-O glycolyl Neu5Gc␣235-O glycolyl Neu5Gc (Fig. 4D). The resonances at 2.20 and 2.78 ppm were respectively assigned to H-3 eq of the reducing terminal and internal ␣235-O glycolyl -linked Neu5Gc residues, respectively. The resonances at 1.76 and 2.67 ppm were respectively thought to be the down-field shifted H-3 ax and H-3 eq of the sulfated nonreducing terminal Neu5Gc residue. Other resonances observed in Fig. 4 (D and E) were again superimposable. Based on these results, GX3 was determined to be Neu5Gc9HSO 3 ␣23 5-O glycolyl Neu5Gc␣235-O glycolyl Neu5Gc.
Oligo/Polysialylated Glycan Chains-The carbohydrate composition of ESP-Sia suggested the presence of both N-and O-linked glycan chains. The ESP-Sia sample treated with alkaline borohydride was subjected to preparative TLC as described under "Experimental Procedures." Analysis of the TLC showed that almost all Sia-containing components migrated from the origin, suggesting that oligo/polysialyl groups were attached to the liberated O-linked glycan chains. The N-glycan chains, still linked to peptide, remained at the origin in this TLC system (data not shown). Detailed structural analysis of the core glycan chains is now under way.

DISCUSSION
Polysialylated glycoproteins from a wide variety of sources have been studied in recent years, but the contributions of polysialylation to glycoprotein functions are only beginning to be understood (2,3). The discovery of the new types of oligo/ polySia residues in different biological materials is expanding the long list of naturally occurring oligo/polySia chains. Indeed, the recent discovery of unusual structural features in the oligo/ polySia chains containing (35-O glycolyl -Neu5Gc␣23) n units having a mean chain length of approximately 20 Neu5Gc residues in a sialic acid-rich glycoprotein isolated from the jelly coat of sea urchin eggs (13) led us to search for related proteins on the egg cell surface.
In this study, we first showed that the sea urchin egg cell surface glycopeptides (ESP-Sia) released by exhaustive pronase digestion of the cell surface complex have O-linked oligo/ polySia-glycan chains and (35-O glycolyl -Neu5Gc␣23) n common basal structures that were characterized thoroughly by chemical and physical methods. The main differences between jelly coat-derived oligo/polySia chains and egg cell surface-derived oligo/polySia chains appear to be (a) the lower degree of polymerization of the latter (average DP was about 3) and (b) the presence of sulfate groups on (35-O glycolyl -Neu5Gc␣23) n chains in the latter. Perhaps the most prominent feature of oligo/polySia chains found in ESP-Sia is that the ␣235-O glycolyl -linked oligo/polyNeu5Gc chains are capped at their nonreducing termini by the 9-O-sulfated Neu5Gc residues, i.e. Neu5Gc9HSO 3 ␣23(35-O glycolyl Neu5Gc␣23) n . This appears to be the first report of the occurrence of sulfated polySia groups in a glycoprotein. Although generally present in relatively very low amounts, O-sulfated sialic acids are not uncommon in nature, particularly in gangliosides. It has been reported also that they are constituents of gangliosides from lower animals and of 8-O-sulfated Neu5Gc and 8-O-sulfated Neu5Ac in Echinocardium cordatum (39) and A. crassipina (36). A number of modified sialic acids have previously been isolated from sialoglycoconjugates, but this is the first report of the occurrence of 9-O-sulfated N-glycolylneuraminic acid in nature.
The Neu5Gc9HSO 3 residues in oligo/polySia-glycoprotein of sea urchin eggs are not randomly incorporated into the oligo/ polySia chains; they occur exclusively as the oligoSia from nonenzymatic mild acid hydrolysates of ESP-Sia that was consistent with the structures in the form of Neu5Gc9HSO 3 ␣23(35-O glycolyl Neu5Gc␣23) n (n ϭ 1, 2, and 3) because they were totally resistant to sialidase; and (c) the absence of disaccharides such as Neu5Gc9HSO  A pertinent question concerns the origin and composition of ESP-Sia prepared from the cell surface complex. This complex, when prepared from S. purpuratus, was shown to consist of the plasma membrane, vitelline layer, and cortical granules. ESP-Sia is a mixture of glycopeptides released by pronase digestion of the egg cell surface complex. The glycopeptides were shown to contain both sulfated oligo/polySia-containing O-linked glycan and N-linked sulfated glycan chains. However, it is important to note that ESP-Sia is structurally distinct from the sialic acid-rich glycoprotein of the jelly coat. In fact, ESP-Sia is more abundant than the egg jelly sialic acid-rich glycoprotein, being present at a molar level that is 100 times greater per egg.
The presence of a sulfated glycoprotein, from which ESP-Sia was derived, on egg cell surface raises the possibility that the egg receptor for sperm is a 350-kDa glycoprotein (21, 40 -44) containing both N-and O-linked chains. However, the structure of these N-and O-linked oligosaccharides has yet to be determined. It is quite possible that the sulfated O-linked oligosaccharide chains of the receptor (44) are involved in its function, as is the case for other well characterized receptors. Although at present time the origin of ESP-Sia is obscure, preliminary results suggest that ESP-Sia could constitute a glycopeptide fragment of the egg receptor for sperm. Studies to understand the origin of ESP-Sia and to test the possibility of its functional role in fertilization are currently in progress in our laboratories.
In previous studies, ␣235-O glycolyl -linked oligo/polyNeu5Gc chains were shown to be relatively stable to mild acid hydrolysis at pH levels of more than 3.8 (14). In contrast to this we found that sulfated oligo/polySia chains in ESP-Sia were labile to mild acid hydrolysis (pH 4.8, 80°C, 2 h). This was confirmed by parallel experiments in which (35-O glycolyl Neu5Gc␣23) n and Neu5Gc9HSO 3 ␣23(35-O glycolyl Neu5Gc␣23) n were hydrolyzed with acid. It is particularly noteworthy that free Neu5Gc9HSO 3 was readily formed from ESP-Sia upon mild acid treatment or even by repeated freeze and thawing (data not shown). Thus, care must be exercised to avoid acidic pHs and multiple freeze-thawing procedures.
In light of the universal occurrence of oligo/polySia chains in animal glycoconjugates, one of the central unsolved questions is the molecular mechanism of their synthesis in vivo. Remarkable progress has been made in the last few years in understanding the mechanism of synthesis of ␣238-linked oligo/ polySia chains during post-translational modification by identification of at least two or three discrete sialyltransferases that function sequentially in the initiation and elongation reactions (16). Likewise it will be most interesting to understand how (35-O glycolyl Neu5Gc␣23) n chains are formed in vivo. It also remains an important challenge to determine how the control of the final oligo/polySia structure, i.e. the average length of oligo/polySia chains, is achieved and how far it is correlated with the functional requirements of the oligo/polySia-glycoconjugates.
Another fundamental question that needs to be answered in future is the manner by which elongation of oligo/polySia chains is terminated. It was presumed that the incorporation of an unusual component at the nonreducing terminus of a given oligo/polySia chain can function as a terminating signal, preventing further chain elongation by polysialyltransferase. The isolation and characterization of a minor 9-O-sulfated Neu5Gc from ESP-Sia raised our interest in the chemistry and biological implications of the possible termination mechanism for biosynthesis of oligo/polySia chains. Perhaps one of the most prominent findings in the current study is the occurrence of the capped structures of oligo/polySia chains, Neu5Gc9HSO 3 ␣23(35-O glycolyl Neu5Gc␣23) n . This together with our previous finding (45) and more recent study on the biosynthetic mechanism (46) of the KDN-capped structure of ␣238-linked oligo/polyNeu5Gc, i.e. KDN␣23(38Neu5 Gc␣23) n , in salmonid fish egg polysialoglycoproteins give insight into the diverse nature of termination of oligo/polySia chain elongation in vivo. Thus, the current findings, coupled to earlier studies (16), lead us to propose the following scheme for multiple stages in polySia chain synthesis (R represents core glycan unit): Initiation ͑diSia formation͒: SiaϪR ϩ CMPϪSia* 3 Sia*ϪSiaϪR REACTION 1.
To date, however, only limited information is available about the termination mechanism of polysialyl chain elongation. The present finding of 9-O-sulfation of the nonreducing terminal sialic acid residues of oligo/polySia chains provides some insight into the mechanism of termination of the oligo/polySia chains in the sea urchin egg surface glycoprotein.
Termination ͑substitution of stopper signal͒:  In the case of KDN-and sulfated Sia-containing oligo/polySia, great resistance was observed toward hydrolytic removal of their sialyl residues by the bacterial exosialidases tested. Thus one attractive possibility is that these signals serve as a protective mechanism against attack by bacteria. Another possibility, based on the recent finding of sulfated sialic acid as a component of the egg receptor for sperm (unpublished observations), is that this sulfated sugar residue is involved in sperm-egg recognition.