2 Department of Biological Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama, Japan
3 Department of Chemistry, School of Science, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, Japan
4 Laboratory of Developmental and Reproductive Biology, Department of Biosciences and Informatics, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama, Japan
Received on January 18, 2003; revised on March 12, 2003; accepted on March 13, 2003
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Abstract |
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Key words: acrosome reaction / ARIS / CID MS-MS / egg jelly coat / sulfated glycans
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Introduction |
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Early studies performed in our laboratory indicate that the AR-inducing activity of ARIS significantly remains even after removing about 50% of proteins by pronase digestion. Nevertheless, this activity is susceptible to periodate oxidation as well as desulfation (Koyota et al., 1997). These results suggest that the intact sulfated sugar chains are mostly responsible for the biological function of ARIS. One of the sugar chains of ARIS with biological activity is composed of a long linear polysaccharide chain consisting of the pentasaccharide repeating units as [
4]-ß-D-Xylp-(1
3)-
-D-Galp-(1
3)-
-L-Fucp-4(SO3-)-(1
3)-
-L-Fucp-4(SO3-)-(1
4)-
-L-Fucp-(1
)n (Koyota et al., 1997
). This protein-free sugar fragment was isolated as fragment 1 (Fr. 1) from the sonicated products of pronase digest of ARIS (P-ARIS) on anion exchange chromatography. In this chromaotography, another fragment, which was named Fr. 2 and also shown to have AR-inducing activity to a certain extent, was obtained. This sugar portion has been demonstated to retain the peptide part so that the sugar chains of Fr. 2 are thought to derive from the inner core region of ARIS. In this article, we describe the biochemical characterization of this biologically active core region sugar portion of ARIS called Fr. 2 en route to the complete structural elucidation of ARIS.
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Results |
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Reductive elimination
Almost all the sugar portion of Fr. 2 was released by reductive ß-elimination, indicating that the oligosaccharides of Fr. 2 were O-linked. The gel filtration of the mixture of ß-eliminated products of Fr. 2 on a Sepharose CL-6B resulted in one major sugar peak, which consisted of the released O-glycans of Fr. 2. Then the major fractions were collected and chromatographed on a DEAE-Toyopearl column to find whether there were further separations. However, further fractionation was not observed in this chromatography, suggesting that released glycans would be homogeneous as far as ionic character concerns. Thus the fractions of this peak were collected, dialyzed against water, and subjected to biochemical analysis. The results of ß-elimination of native ARIS also showed that the major glycans derived from ARIS were O-linked.
Sugar composition analysis
Table I shows the results of sugar composition analysis. Sugar compositions of native Fr. 2 and its released major sugar chain were shown to be identical, indicating that all the sugar chains of Fr. 2 were released by ß-elimination. Though Fr. 1 does not contain amino sugars, Fr. 2 was shown to possess GalNAc and GlcNAc in addition to Fuc, Gal, and Xyl. The data show that Xyl amount of Fr. 2 is one third of Fr. 1. The result suggested the presence of novel saccharide structures of ARIS. The most abundant sugar in both Fr. 1 and Fr. 2 was observed to be Fuc, indicating that Fuc is the major sugar residue in the carbohydrate portion of ARIS. The data of Fr. 2-H1 will be discussed in the following sections.
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Identification of partially methylated alditol acetates (PMAAs) was made by gas chromatography mass spectrometry (GC-MS). The results are summarized in the Table II. The quantitative data shown in the table were approximately calculated from the area of each peak corresponding to each sugar alditol of the GC profile together with sugar composition data of Fr. 2. The results clearly revealed abundant occurrences of 4- and 2-O-sulfated Gal residues, 4-O-sulfated Fuc residue, and 6-O-sulfated GlcNAc residue. Glycosidic linkages of GlcNAc were shown to be only 4-linked, whereas those of GalNAc are either 4- or 6-linked. A relatively low amount of Gal terminal residues was detected as 2,4-O- and 2,3-O-disulated and 4-O-monosulfated. Terminal GalNAc residues appeared only in the desulfated product, indicating that desulfation might cause some partial degradation of sugar chains of Fr. 2. Furthermore, considerable amounts of terminal Gal and Fuc residues were also observed.
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Besides the information about linkage positions of individual sugar residues, the methylation analysis of Fr. 2 could be used to determine the ring size of some of the sugar residues. The data of tandem mass spectrometry (MS/MS) analysis of PMAAs of Fr. 2 showed the presence of 1,3,5-tri-O-acetyl-(1-deuterio)-2,4-di-O-methyl fucitol (m/z 234, 131, 118) and 1,3,5-tri-O-acetyl-(1-deuterio)-2,4,6-tri-O-methyl galactitol (m/z 234, 161, 118, 45), which can only originate from the corresponding alditols of pyranose form of 3-Fuc and 3-Gal, respectively (the m/z values are only the values of primary fragments). Detection of 1,5,6-tri-O-acetyl-(1-deuterio)-2,3,4-tri-O-methyl galctitol (m/z 233, 189, 162, 118), 1,5-di-O-acetyl-(1-deuterio)-2,3,4,6-tetra-O-methyl galctitol (m/z 249, 206, 205,162,161, 118), and 1,5-di-O-acetyl-(1-deuterio)-2,3,4-tri-O-methyl fucitol (m/z 175, 162, 131, 118) indicate the presence of 6-Gal, terminal-Gal, and terminal-Fuc in their pyranose ring form. Therefore, all the sugar residues, which are major neutral hexoses present in Fr. 2, are in the pyranose form. In the case of HexNAc, detection of 1,5,6-tri-O-acetyl- (1-deuterio)-(2-N-methylacetamide)-3-,4-di-O-methyl galctitol (m/z 247, 233, 203, 189, 159) and 1,5-di-O-acetyl-(1-deuterio)-(2-N-methylacetamide)-3,4,6-tri-O-methyl galctitol (m/z 247, 205, 203, 161, 159, 45) indicate the presence of 6-GalNAc and terminal GalNAc, respectively, in their pyranose form in Fr. 2. However, the alditols of GlcNAc and other linked GalNAc could not be identified whether they derived from pyranose ring form by using MS/MS analysis of PMAAs of Fr. 2 because both C-4 and C-5 are acetylated in suggested 4-linked HexNAcs. According to the facts that the naturally occurring HexNAc generally exists in pyranose form and 5-linked HexNAc is unusual, it may not be irrational to assume that such HexNAcs exist as pyranose form of 4-GalNAc and 4-GlcNAc. Further, electrospray ionization (ESI) MS/MS analysis of Fr. 2-H1, as will be discussed later in this article, showed the presence of 4-HexNAcs, indicating that they should exist in pyranose form. As will be discussed later in this article, Xyl is suggested to come from remaining Fr. 1 sugar units, thus it would exist as pyranose ring form (Koyota et al., 1997).
Proton NMR spectroscopy
The proton NMR spectrum of the released sugar chains of Fr. 2 is presented in Figure 1. Much structural information was not available in this NMR analysis due to complexity of the spectrum (because of high molecular weight) with many unresolved signals. However, the spectrum revealed some of the preliminary structural information of Fr. 2. The unresolved strong signals between 1.18 ppm and 1.30 ppm represented methyl hydrogen atoms of fucosyl residues (total relative area of the signals is 14.63), which should exist in more than three different environments in the sugar chains of Fr. 2. Unresolved but prominent signals corresponding to the methyl protons of acetyl groups of amino sugars with the total area of 16.85 confirmed the presence of N-acetylated amino sugars. These NMR integrals are in good agreement with the sugar composition data. Though not exact, the signals in anomeric region revealed preliminary information of anomeric resonance of sugar chains of Fr. 2.
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ELISA
Linkage analysis and NMR analysis provided some evidence for the presence of the sugar chains similar to Fr. 1 in Fr. 2. Enzyme-linked immunosorbent assay (ELISA), using specific antibody for Fr. 1, was performed for Fr. 2 to get further evidence for this observation. Our laboratory has developed a specific ELISA for Fr. 1 (unpublished data). This antibody was previously tested to be positive for ARIS, P-ARIS, and Fr. 1, whereas these became negative after periodate oxidation or desulfation of the sugar chains, suggesting that the antibody is specific for sulfated intact sugar chains of Fr. 1. In the present study, this assay was applied to native Fr. 2, released glycans of Fr. 2 by ß-elimination, and oxidized (by periodate) and desulfated Fr. 2.
We observed positive results for both the native and released glycans of Fr. 2 and negative results for the oxidized and the desulfated products. These results provided further evidence of the presence of some Fr. 1 repeating units as [4]-ß-D-Xylp-(1
3)-
-D-Galp-(1
3)-
-L-Fucp-4(SO3-)-(1
3)-
-L-Fucp-4(SO3-)-(1
4)-
-L- Fucp-(1
) in Fr. 2, which is likely the source for the positive result of the ELISA for Fr. 1. As will be discussed later in this article, Fr. 1 is assumed to be located in the outermost region of ARIS sugar chains, which is linked to the protein part through the inner core region, namely, Fr. 2. Fr. 1 and Fr. 2 are generated by the sonication of P-ARIS. Thus there is a good possibility that some sugar-repeating units similar to Fr. 1 remain in the protein-free end of Fr. 2.
Partial acid hydrolysis of Fr. 2
Further structural studies of Fr. 2, especially sugar sequencing studies, were restricted by high molecular mass of the sugar chains of Fr. 2. Therefore, either specific or nonspecific degradation procedures were required to obtain easily manageable fragments from the intact chain. For this purpose, we attempted specific degradation methods, such as Smith degradation, deamination, and so on with Fr. 2 (data not shown). All of the methods, except hydrolysis with a strong acidic-ion exchanger (as will be described later in this article), failed, probably because of a number of sulfate groups associated with Fr. 2 and its largeness. The sample of released sugar chains of Fr. 2 was subjected to partial acid hydrolysis using Dowex 50W (H+ form) to obtain easily manageable fragments. In contrast to the common procedures of partial hydrolysis, this method allows cleaving of HexNAc glycosidic bounds successfully in a single step with the prevention of cleaving acetyl groups (Mark et al., 2000). Hence, the method is fit for Fr. 2 because it is composed of a significant amount of HexANc.
On the resin hydrolysis of Fr. 2, the hydrolysate fractions were isolated by spin filtrations. Because the total fractions were not amenable to mass analysis, smaller fractions were isolated as Fr. 2-H1 (containing sugar fragments <5 kDa) as described in Materials and methods. Although this hydrolysis procedure was successful in providing the fragments amenable for the mass analysis, the yield obtained per cycle of hydrolysis was very low, indicating that the reaction was still restricted by its molecular size and/or sulfates. Therefore, the hydrolysis was repeated for the remaining bigger part of sugar chains (>5 kDa) to obtain adequate amounts of the sample for further analysis. Finally, 5% of the yield was obtained with the repeating of four cycles of hydrolysis. However, the fractions obtained after the fourth cycle of repeated hydrolysis were accompanied with high noises in MS experiments. This may be due to the presence of degradative products of the resin molecules in hydrolysates.
Sugar composition
The result of sugar composition analysis of Fr. 2-H1 is tabulated in Table I. The results showed the absence of Xyl and the presence of considerable amount of HexNAc and Gal in Fr. 2-H1.
MS analysis of hydrolysates of Fr. 2
A sample of the major sugar fragments generated by the partial hydrolysis of Fr. 2 (Fr. 2-H1) was subjected to MS analysis to obtain the sequencing information pertaining to the major sugar portion of Fr. 2. This ESI-MS analysis was carried out in positive ion mode, and ions were detected as mainly sodium-adduct ions. According to the sugar composition data, Fr. 2-H1 is composed of high amounts of HexNAc and Gal and less Fuc. Though not precise, we have observed that more than 10% (w/w) of sulfate is present in Fr. 2-H1 by sulfate content analysis of Fr. 2-H1. Altogether, we could propose possible components (as tabulated in Table III) in which these components correspond to the major signals appeared in ESI spectrum of Fr. 2-H1 shown in Figure 2. In addition to the sodium adduct forms, protonated forms, though not abundant, were also observed in this ESI spectrum. The results showed that both mono- and disulfated components were predominant. However the completely desulfated components were less abundant. The highest molecular-related ion was observed at m/z 1486 as sodium adduct or 1464 as protonated form, which could be derived from the chemical species of Hex3FucHexNAc3(SO3Na)2. Because Gal was detected as the only Hex in this sample, all Hex in the table can be replaced by Gal. The molecular-related ions at m/z 1324, 1178, 1016, and 813 were shown to possess two sulfate groups. Except for the molecular-related ion at m/z 1324, all of these ions were consist of only Gal and HexNAc. Thus, the sulfate groups should attach to either Gal or HexNAc, not Fuc.
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The results of collision-induced dissociation (CID) MS/MS of all these three ions, to be discussed, suggested that the sequence corresponding to all three would be HexNAc-Gal-HexNAc, which may represent the reducing-end terminal sequence, the ion at m/z 813 being disulfated. According to the data of methylation analysis of Fr. 2-H1, discussed later in this article, the sample contained monosulfated Gal and monosulfated GlcNAc residues. We have already stated that HexNAc2(SO3Na) must be present in the sugar sequence. Taking all these observations into account, the reducing-end terminal sequence would be HexNAc-HexNAc(SO3Na)-Gal(SO3Na)-HexNAc. The molecular-related ion at 610 can only be derived from the formula of HexNAcGal(SO3Na)2, indicating that sulfated HexNAc and sulfated Gal must be linked to each other. This observation is a positive clue for the presence of the reducing terminal sequence. Taking all the facts discussed into consideration, we could assume that the sugar sequence corresponding to the ion at m/z 1486 is Gal-Fuc-Gal- HexNAc-HexNAc(SO3Na)-Gal(SO3Na)-HexNAc. Except for the Gal-Fuc sequence at nonreducing terminal of this sequence, the rest was confirmed by the CID MS/MS analysis of corresponding molecular ions as follows.
CID MS/MS analysis of major precursor ions
The CID MS/MS spectra of the major molecular ions were dominated mainly by abundant, mainly Y- and B-type ions (nomenclature of Domon and Costello, 1988). Under general conditions, particularly at low collision energy, Y- and B-type fragmentations are predominant in CID MS/MS analysis of oligosaccharides (Harvey, 2000a
,b
). The CID MS/MS spectrum of the precursor ion at m/z 508 and the schematic explanation of the fragmentation of the ion are shown in Figures 3a and 3b, respectively. The ions at m/z 244 and 287 correspond to Y- and B-type glycosidic cleavages, respectively. The product ion at m/z 407 is attributable to the removal of 101 units from the reducing-end HexNAc by 0,2A cross-ring cleavage. It can be assumed that this type of cleavage is observed in a sugar sequence, which is terminated with -(1
4)-HexNAc residue at the reducing end. The results of methylation analysis of Fr. 2-H1 (discussed later in this article) have indicated that all the HexNAcs of the suggested sugar chain for Fr. 2-H1 are 4-linked. Thus such type of cross-ring cleavage can be observed from the sugar chains with -(1
4)-HexNAc residue in ESI CID MS/MS mode, much like in matrix-assisted laser desorption/ionization-post source decay as previously reported by Yamagaki and Nakanishi (2000)
.
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According to all the product ions, the sequence corresponding to this precursor ion must be Gal(SO3Na)-HexNAc. The CID MS/MS spectrum of the precursor ion at m/z 609 and the schematic explanation for the cleavages are shown in Figure 4. All the Y- and B-type glycosidic cleavages, as well as cross-ring cleavages from the reducing terminal, were observed from this precursor ion. Two types of cross-ring cleavages at m/z 508 (0,2A) and 448 (2,4A) resulted from the reducing end clearly indicated that reducing sugar of this sequence should be 4-linked HexNAc. A presence of product ions at m/z 406 (Y2) and 226 (B1), both sodiated, indicated that the nonreducing sugar would also be HexNAc; thus the sequence corresponding to this ion should be HexNAc-Gal-HexNAc. The CID MS/MS spectrum of precursor ion at m/z 711 showed (the MS/MS spectrum is not shown) that its sequence was in accord with that of the ion at m/z 609 but in monosulfated form. Other expected cleavages were observed in the MS/MS results of this ion. Among them, the presence of product ion at m/z 328 originated by B1 (sodiated) cleavage implied that the sulfate group would be located in nonreducing end HexNAc residue. The CID MS/MS spectrum of ion at m/z 813 and the schematic explanation for the formation of the product ions are shown in Figure 5. In addition to the removal of sulfate group as -NaHSO4 and (-SO3Na+H+), the removal of Na2S2O7 (222 units) was observed in this spectrum, indicating that the ion consists of two sulfate groups. These sulfate removals were observed from the precursor ion itself and the product ion at m/z 712. Other sulfate removal routes are shown in the Figure 5b. The product ion at m/z 712 indicated ring cleavage fragmentation of 0,2A from reducing HexNAc residue.
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The CID MS/MS analyses for weak ions of m/z 1486 and 1324 could not be carried out. Therefore the presence of GalFuc sequence is yet to be confirmed. Though we have attempted to carry out MS/MS analyses of weak ions at m/z 1486 and 1324, satisfactory results have not been obtained. However, according to the ESI spectrum of Fr. 2-H1, this GalFuc would be at a nonreducing terminal. This is because no molecular ions corresponding to reducing terminal fragments with this sequence (such as HexNAcFuc/Gal or Gal(SO3Na)-HexNAc Fuc/Gal) were observed in the ESI spectrum, whereas other reducing terminal species (such as HexNAc(SO3Na)-Gal(SO3Na)-HexNAc or Gal(SO3Na)-HexNAc) were abundant. Therefore we can suggest a sugar sequence for the biggest fragment in Fr. 2-H1 as depicted in Figure 8a.
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Taking all the data of MS and the methylation analysis into account, we propose a sugar structure for the major fragment present in the hydrolysate as depicted in Figure 8a. The total yield of Fr. 2-H1 obtained from four repeating cycles of hydrolysis was nearly 55% (w/w) and the same results in the mass analysis for the fragments generated from every consequent hydrolysis were observed, evoking conclusions that new sugar sequence seems to represent the major part of Fr. 2. As discussed previously in this article, some part of the sugar chains of Fr. 2 could be composed of the similar sugar-repeating units of the Fr. 1. Providing that all the Xyl residues of Fr. 2 are attributable to this part of sugar chain, approximately a fourth of sugar residues of Fr. 2 should be composed of Fr. 1 repeating units (nearly 100 units). If this assumption is the case, the other sugar portion of Fr. 2 would be composed of Fuc:Gal:GalNAc:GlcNAc in a molar ratio of 1:2:2:1. Unlike Fr. 1, these new sugar chains were shown to contain mono- and disulfated Gal, 6-linked Gal, sulfated GlcNAc, and GalNAc besides Fuc. The suggested sugar structure (Figure 8a) represents the major linkages shown, indicating that it consists of the major part of Fr. 2. The results shown in Table II indicated that more than half of GlcNAcs were sulfated in intact sugar chains of Fr. 2. Provided that all this amount of sulfated GlcNAc is assigned to the proposed new sugar chain of Fr. 2, sugar chains of Fr. 2 would be composed of at least 120 units of suggested heptasaccharide domains.
As stated previously, Fr. 2 is assumed to consist of partly the Fr. 1 repeating units. However, until recently it was not clear whether they are derived from one single sugar chain of ARIS or not. In the current study, we have obtained a quite homogenous mixture of sugar chains after ß-elimination by means of size and acidity, suggesting that Fr. 1 repeating units are derived from the same sugar chain of Fr. 2. Furthermore, Fr. 1 was observed to be protein free, whereas Fr. 2 was shown to retain peptides. Based on all the results discussed so far and with some assumptions, the major glycans of ARIS can be suggested as follows: (P)m>200 (Q) (R)n>100 (S) Ser/Thr (Protein), where P is [4)-ß-D-Xylp-(1
3)-
-D-Galp-(1
3)-
-L-Fucp-4(SO3-)-(1
3)-
-L-Fucp-4(SO3-)-(1
4)-
-L-Fucp-(1
and R is
3)-Galp-(1
3)-Fucp-(1
3)-Galp-(1
4)-GalNAcp-(1
4)-GlcNAcp-6(SO3-)-(1
6)-Galp-4(SO3-)-(1
4)-GalNAcp-(1]
. The structures of parts Q and S have yet to be revealed. Furthermore, the anomeric information is still not available for the suggested new sugar sequence. In fact, we have attempted with glycosidase enzyme digestion to reveal this information. Because of the limited sample availability, we have not yet obtained fragments from these reactions amenable for mass analysis.
According to this proposed structure, the sugar chain of Fr. 1 is located in the outermost region of ARIS, which is linked to the peptide part through the sugar chain of Fr. 2, called inner sugar chain. Therefore the sugar chains in outermost region of ARIS entirely differ from the inner sugar chains in terms of sugar compositions and linkages as well as sulfate substitutions. Although we have yet to reveal the structural information of sugar chains of Q and S in Fr. 2, the major sugar chains derived from ARIS (Fr. 1 and major sugar part of Fr. 2) were shown to be linear and very long as well. Thus ARIS is likely to be a proteoglycan-like molecule.
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Discussion |
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The current study confirms that sulfate groups are solely liable to the anionic character of ARIS, which are shown to be essential in the induction of the AR. Our previous study has shown that the removal of even one sulfate group from a repeating unit of Fr. 1 entirely destroys its biological activity. As discussed earlier, sea urchin AR-inducing molecules are also highly sulfated. The sulfation of sea urchin egg jelly fucans was known to be essential for the spermegg interactions (Hirohashi and Vacquier, 2002). In the ascidian Halocynthia roretzi, spermegg binding was shown to be mediated through sulfated glycans present in the vitelline coat (Baginski et al., 1999
). In not only echinoderms but also mammals sulfate has been implicated in spermegg interaction, including induction of the AR. The sulfated glycans present in porcine zona pellucida (ZP), for example, were shown to participate in spermegg interactions (Yurewicz et al., 1991
; Noguchi et al., 1992
). It has been shown that the murine ZP3 (mZP3) is also composed of sulfated N- and O-glycans, although Wassarman and co-workers showed that sulfation is not directly involved in the binding of sperm to mZP3 (Liu et al., 1997
). In human, the initial binding of the sperm to eggs has been shown to be blocked by fucoidan, a sulfated fucan polymer (reviewed by Dell et al., 1999
). Thus in most animals it seems that the sperm recognizes the spatial arrangement of sulfate moieties in the egg jelly coat glycans to undergo spermegg interactions, including AR.
One of the other important observations in this study is that the major sugar chains of ARIS were found to be O-linked, presumably via Ser or Thr residues. These released O-glycans of ARIS, which have an admirable biological activity, would be used as good ligands to identify ARIS receptors. Our group has made several attempts to isolate the receptors for ARIS using Fr. 1 as a ligand, which made it easier to handle in comparison with ARIS or P-ARIS as a whole, although it is less active compared to ARIS or P-ARIS. The efforts resulted in only partial isolation of some sperm proteins of 5060 kDa, which were likely to be some component related to ARIS receptor (Kawamura et al., 2002).
In addition to the starfish, O-linked glycans in many animals have also been documented to carry considerable biological activity in fertilization events. The O-linked glycans isolated from the vitelline coats, for example, were reported to carry a key function in spermegg interactions of the mollusk Unio elongatulus (Focarelli and Rosati, 1995) and the ascidian Halocynthia roretzi (Baginski et al., 1999
). In the amphibian Xenopus laevis, the spermegg binding process was shown to be involved in O-linked glycans (Tina et al., 1999
). Though the analysis of porcine ZP glycans yields conflicting results, some studies find that O-linked glycans present in porcine ZP are responsible for spermegg binding (Yurewicz et al., 1991
, 1993
). The presence of O-linked glycans in the glycoprotein mZP3, which plays an important role in the induction of the AR in mice, has been reported (Florman and Wassarman, 1985
; Wassarman, 1988
; Easton et al., 2000
). All the facts discussed suggest that sulfation and O-glycosylation would be essential modifications of the glycosilated proteins involved in spermegg interactions. Moreover, it seems that the fucosylation could also be important for the activity of such glycans. Our studies have already shown that starfish ARIS possess all these three features. According to these intrinsic features of ARIS and the other factorssuch as obtaining ample quantities of eggs and sperms on demand, easily detectable morphological changes during AR and so onthe starfish could be used as a very good model for understanding the comprehensive mechanisms of spermegg interactions, including the AR.
Although they have very complex structures, sugar chains of ARIS possess some unusual sugar structural features. When Fuc is present in glycoprotein with other sugar residues, it is commonly located at the terminal or as a short branch. However peripheral as well as core region sugar chains of ARIS were shown to have in-chain Fuc residues, suggesting that fucosylation is associated with elongation, not termination, of the glycans in the starfish egg coats. The other unusual feature of ARIS sugar chain is that -Gal is in in-chain, which is unexpected. Generally,
-Gal is present as a terminal sugar residue of many of glycans, for example, O-glycans from the jelly coat of amphibian eggs terminate with Gal
1
4(Fuc
1
2)Galß (Strecker et al., 1992
, 1995
), blood group P1 antigen Gal
1
4Galß1
, and blood group B antigen Gal
1
3(Fuc
1
2)Galß. It has previously been shown that Gal in Fr. 1 is
-linked (Koyota et al., 1997
). Therefore,
-Gal residues can also be present in Fr. 2, as Fr. 1 sugar chains were shown to remain in Fr. 2. These
-Gal residues should be in-chain as Fr. 2 represents inner core sugar structure of ARIS (between Fr. 1 and peptide part). Other than the sugar chains of starfish ARIS, very few cases having such an unusual property of naturally occurring sugar chains were previously reported in N-glycans isolated from the eggs of flounder, Paralichthys olivaceus (Seko et al., 1989
), and O-glycans derived from cercarial glycocalyx of Schistosoma mansoni (Khoo et al., 1995
).
Although a significant activity was observed in the sugar chains of ARIS, the best activity was always noted in the intact ARIS, hinting that protein portion is also somewhat important in triggering the AR. This may be attributable to the clustering effect (Lee, 1992) for which we do not have direct evidence so far. Recently we have isolated the protein portion of ARIS using reductive-elimination reaction described by Gerken et al. (1992)
. Its size seems to be over 100 kDa. The molecular cloning of this isolated protein is currently under progress. The targets of our current study are to figure out (1) the anomeric information of suggested sugar chains of Fr. 2, (2) the rest of the sugar structures of ARIS with reducing terminal information, and (3) the primary structure of protein part of ARIS.
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Materials and methods |
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Preparation of core region sugar fragments of ARIS (Fr. 2)
Preparation of Fr. 2 was carried out essentially as described by Koyota et al. (1997). Breifly, P-ARIS was prepared by actinase E digestion of ethanol precipitation of the egg jelly solution of the starfish A. amurensis followed by Sepharose CL-4B gel filtration and DEAE-Toyopearl 650 M ion exchange chromotography, respectively. ARIS was prepared in the same way using undigested egg jelly solution instead of ethanol precipitation. The solution of the acidic fraction containing sugar and protein (P-ARIS) was sonicated for 30 min using a Bransonic ultrasonic apparatus (Danbury, CT), followed by DEAE Toyopearl 650 M anion exchange chromatography. The column was eluted with a linear gradient of 01 M NaCl. Fr. 1 was recovered in 0.70.8 M NaCl solution, whereas Fr. 2 was recovered in
0.9 M NaCl concentration. The fractions were dialyzed and lyophilized to give purified Fr. 1 (
45%) and Fr. 2 (
14%).
Reductive ß-elimination
ß-Elimination was performed as described by Chaplin and Kennedy (1994). Each sample (1 mg) was treated with 0.05 M NaOH-containing 1 M NaBH4 (1 ml) at 45°C, terminating the reaction with 50% acetic acid after 48 h. Released sugar chains were isolated by gel filtration on a Sepharose CL-6B column (2.2 x 90 ml) using the eluent of 100 mM ammonium acetate buffer (pH 5.5) followed by dialysis and lyophilization.
Sugar composition analysis
Sugar composition analysis was performed by 1-phenyl-3-methyl-5-pyrazolone (PMP) derivertization method described by Fu and O'Neill (1995). Each sample (50 µg) was hydrolyzed using 4 M trifluoracetic acid at 120°C for 2 h. Hydrolyzed samples were directly labeled with PMP, extracted, and separated with a C18 reverse-phase HPLC column (4.6 x 250 mm, 5C18-AR, Waters, Nacalai Tesque), essentially as described by the original procedure.
ELISA
Dried samples (50500 ng) in a microtiter plate were incubated with 1% human serum albumin in phosphate buffered saline (PBS) for 30 min. After removing the blocking buffer, the samples were incubated with 50 µl mouse anti-Fr. 1 antibody (IgG) (prepared by our laboratory) in PBS for 1 h followed by washing each well with PBS. Each sample was then treated with 200-fold diluted horseradish peroxidaseconjugated goat anti-mouse IgG antibody (CHEMI CON, Temecula, CA) in PBS (50 µl) for 30 min, followed by washing with PBS to remove the excess antibody. Then the samples were labeled with freshly prepared 200 µl o-phenylenediamine (4 mg o-phenylenediamine in 10 ml 0.1 M citric acid buffer, pH 5.0) and 4 µl 30% hydrogen peroxide for 20 min. The reaction was terminated by adding 20 µl 5 N sulfuric acid. The plate was read at 492 nm.
Desulfation
A solution of Fr. 2 was dialyzed against 0.1 M pyridinium acetate buffer (pH 5.4) for 2 days. The resulting pyridinium salt (2 mg) was treated with 1 ml 10% methanol in dimethyl sulfoxide or 5 h at 80°C, concentrated to dryness, and passed through a 10-ml column of Sephadex G-25 (PD-10) (Nagasawa et al., 1979). Thoroughly desulfated sample was obtained by passing through a Dowex 1 x 8 column equilibrated with 1 M acetic acid. The desulfated sample (1.2 mg) was finally subjected to methylation analysis. In the case of small fragments of Fr. 2 generated by resin hydrolysis, the pyridinium salt was prepared by passage through a microcolumn of Dowex 50 (x8) in the pyridinium form because of its limited sample availability.
Proton NMR spectroscopy
A sample of released sugar portion by ß-elimination was repeatedly exchanged in D2O (99.96%, Aldrich, St. Louis, MO), with intermediate lyophilization. Lyophilized sample was dissolved in 150 µl 99.996% D2O (Aldrich) and transferred to an NMR tube (3 mm x 18 cm). The spectra were recorded at 70°C. Chemical shifts were reported in ppm using HDO signal in D2O (
4.35 ppm at 70°C) as the internal reference.
Preparation of PMAAs
Prior to permethylation, samples were reduced with 1% (w/v) NaBH4 for 2 h at room temperature. In the case of sulfated sugars, reduced samples were converted into triethylammonium salts by passage through a microcolumn of Dowex 50 (x8) in the triethylammonium form (Stevenson and Furneaux, 1991). Then PMAAs were prepared as described by Anamula and Taylor (1992)
with consequent methylations for three cycles.
GC-MS analysis
PMAAs were dissolved in chloroform prior to injection. An aliquot of the sample was applied on a HP-5 column (5% phenylmethylsilicone, 0.25 mm x 30 mm, Hewlett-Packard, Wilmington, DE) at 100°C. This temperature was held for 2 min and then increased to 250°C over 60 min at 2.5°C/min.
Partial acid hydrolysis of Fr. 2 and isolation of hydrolysates
A sample of released sugar portion of Fr. 2 (300 µg) was treated with H+ form of Dowex 50WX2-400 strong acidic ion exchange resin at 75°C for 24 h as described by Mark et al. (2000). The oligosacchraide fragments were isolated by centrifuging the resin-containing solution through a 100,000 MWCO spin-filter at 3500 x g. Because the resulted hydrolysate was shown not to be amenable to mass analysis, the mixture was further separated by centrifuging through a 5000 MWCO spin-filter at 3500 x g followed by a gel filtration on a Superdex Peptide PC 3.2/30 column on a SMART system (Pharmacia, Uppsala, Sweden) using water as the eluent to remove the small molecules as the total volume. The other major fractions, named Fr. 2-H1, were collected (
160 µg,
55%) and subjected to sugar composition and mass and methylation analysis. The procedure was repeated for the remaining part obtained after the second filtration (>5 kDa) to give the adequate amount of sample for the further analyses.
MS measurements
ESI quadrapole/time-of-flight MS was used for the sequencing analysis of the small fragments of Fr. 2 generated from the resin hydrolysis. An aliquot of each sample was dissolved in methanolwater (1:1, v/v) and mixed with equal amount of 1 mM NaCl in 1 mM acetic acid water solution. An aliquot of this sample solution (8 µl) was deposited in a metal-coated borosilicate nanoelectrospray tip (F type tips, Micromass). The needle voltage was 1000 V, and the ion source was maintained at 80°C.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: hoshim{at}bio.keio.ac.jp
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References |
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