Sulfated Asparagine-linked Sugar Chains of Hen Egg Albumin*

The fraction of hen egg albumin glycopeptides mixture, which passes through a Dowex 50-H’ column, contains two sulfate-containing glycopeptides. Based on the structural studies of oligo- saccharides released from the glycopeptides by hydrazinolysis, their structures were elucidated as follows. Ovalbumin, egg white, a glycoprotein 3.5% ovalbumin sugar extremely heterogeneity sugar vestri et al. (18). The analyses were corrected for inorganic sulfate contamination by assaying unpyrolyzed samples. Desulfation of acidic oligosaccharides was performed by methanolysis reported by Slomi- any et al. (9). Sulfated oligosaccharides were dissolved in dry methanol containing 0.05 M HCl and kept at room temperature for 4 h. The solution was evaporated to dryness, and the residues were freed from HC1 by evaporation with methanol three times. Glycosidic linkages including sialyl linkages are not cleaved by the treatment. However, partial de-N-acetylation from N-acetylglucosaminitol residue is in- evitable when the method was applied to oligosaccharides with N-acetylglucosaminitol as their reducing termini. Therefore, the reac- tion product was dissolved in saturated NaHC03 and N-acetylated with acetic anhydride before paper electrophoretic analysis. Phos- phate was analyzed by the method of Chen et al. (10).

The fraction of hen egg albumin glycopeptides mixture, which passes through a Dowex 50-H' column, contains two sulfate-containing glycopeptides. Based on the structural studies of oligosaccharides released from the glycopeptides by hydrazinolysis, their structures were elucidated as follows.

HO-S-O-Manal 4Mana 1 M~1+4GlcNAc@1+4GlcNAc+Asn
G~l+4GlcNAc@l-+ZMana1/1 Ovalbumin, a major constituent of hen egg white, is a glycoprotein containing 3.5% carbohydrate. Although ovalbumin contains only one asparagine-linked sugar chain in one molecule, an extremely high heterogeneity exists in its sugar moiety as is evidenced by the complicated fractionation pattern of glycopeptides of ovalbumin by Dowex 50-H' column chromatography (1,2). By the structural study of the oligosaccharides released from the ovalbumin glycopeptides by the action of various endo-P-N-acetylglucosaminidases, structures of nine glycopeptides were elucidated (2-5).
Approximately 4% of the ovalbumin glycopeptides contain acidic oligosaccharides as their carbohydrate moieties and recovered as the pass-through fraction upon Dowex 50-H+ column chromatography (2). We have recently studied the structures of these glycopeptides by using hydrazinolysis, a chemical method to cleave specifically GlcNAc'+Asn linkage (6). About one-third of the acidic oligosaccharides were converted to neutral oligosaccharides by sialidase digestion. The *This work has been supported by grants-in-aid for Scientific Research and for Cancer Research, the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The abbreviations used are: GlcNAc, N-acetylglucosamine; XylNAc, N-acetylxylosamine. Subscript OT is used in this paper to indicate NaB3H,-reduced sugars. In the same way, subscript OH is used to indicate NaBH4-reduced sugars. All sugars mentioned in this paper were of D configuration. remaining acidic oligosaccharides were completely resistant to the second sialidase digestion. Structural study of the oligosaccharides in this sialidase-resistant fraction, as will be reported in this paper, revealed that they are two novel sulfate-containing oligosaccharides.

EXPERIMENTAL PROCEDURES
Isolation of Sulfated Oligosaccharides from Hen Egg Albumin-A mixture of glycopeptides obtained by exhaustive Pronase digestion of crystalline hen egg albumin (34.2 g) was fractionated by Dowex 50-H+ column chromatography as described before (2). The fraction (GP-F) which was not retained by the column was collected and subjected to 8 h of hydrazinolysis (6) to release the carbohydrate moieties as oligosaccharides. The oligosaccharide fraction (5 mg, dry weight) was dissolved in 0.5 ml of 0.08 N NaOH. One-tenth of the sample was reduced with NaB3H4 (50 FCi) to obtain a tritium-labeled oligosaccharide fraction, and the remaining sample was reduced with 5 mg of NaBH,. The yield of the radioactive oligosaccharides was 5.0 x lo6 cpm.
When the radioactive sample was subjected to paper electrophoresis at pH 5.4, it was separated into a major and a minor acidic peak with the same mobilities as monosialyl and disialyl biantennary complex-type oligosaccharides, respectively (Fig. lA). Oligosaccharides in the major radioactive peak were recovered from paper by elution with water and subjected to sialidase digestion. Approximately 30% of the radioactive oligosaccharides were converted to neutral oligosaccharides (Fig, 1B). The oligosaccharides which were resistant to the sialidase treatment were recovered from paper by elution with water. This fraction will be called A-1R in this paper.
Analytical Methocls-Sulfate was analyzed by the barium-rhodizonate method of Terho and Hartiala (7). Liberation of sulfate from acidic oligosaccharides was performed by pyrolysis described by Sil-vestri et al. (18). The analyses were corrected for inorganic sulfate contamination by assaying unpyrolyzed samples. Desulfation of acidic oligosaccharides was performed by methanolysis reported by Slomiany et al. (9). Sulfated oligosaccharides were dissolved in dry methanol containing 0.05 M HCl and kept at room temperature for 4 h. The solution was evaporated to dryness, and the residues were freed from HC1 by evaporation with methanol three times. Glycosidic linkages including sialyl linkages are not cleaved by the treatment. However, partial de-N-acetylation from N-acetylglucosaminitol residue is inevitable when the method was applied to oligosaccharides with Nacetylglucosaminitol as their reducing termini. Therefore, the reaction product was dissolved in saturated NaHC03 and N-acetylated with acetic anhydride before paper electrophoretic analysis. Phosphate was analyzed by the method of Chen et al. (10).
Negative fast atom bombardment mass spectrometry was performed by using a JEOL model JMS-DX 300 mass spectrometer (JEOL, Ltd., Tokyo).
Enzymes-@-Galactosidase and @-N-acetylhexosaminidase were purified from culture fluid of Dipbcoccus pneumonim according to the method of Glasgow et al. (11). a-Mannosidases were purified from Aspergillus saitoi (12) and from jack bean meal (13) according to the cited references. Sialidase purified from Arthrobacter ureafaciens (14) was purchased from Nakarai Chemicals, Ltd., Kyoto. Alkaline phosphatase (Escherichia coli, Type 111) was purchased from Sigma.

Characteristics of Oligosaccharides in A-IR Fraction and
Their Isolation-Fraction A-1R was completely resistant to sialidase digestion and alkaline phosphatase digestion. However, it was converted to neutral oligosaccharides after methanolysis followed by N-acetylation (Fig. IC). Since the last result suggested that the oligosaccharides in fraction A-1R may contain sulfate residues as their acidic constituent, analysis for sulfate in A-1R was performed. By use of pyrolysis in combination with a rhodizonate procedure, we found an average of 0.8 mol of sulfate/mol of oligosaccharides calculated on the basis of their radioactivities. No phosphate residue was found.
When the radioactive A-1R was subjected to paper chromatography, it was separated into two components as shown Paper chromatography of the radioactive A-1R fraction. Sample was spotted on Whatman No. 3MM paper and developed with ethyl acetate/pyridine/acetic acid/water (5:5:1:3) for 93 h. Arrow indicates the position where authentic Manal+ 6 ( M a n a l + 3 ) M a n a l -P 6 ( M a n a l + 3 ) M a n @ l -+ 4 G l c N A c a l + 4GlcNAco~ migrated. a n a l + 6 ( M a n a l + 3 ) M a n a l -6 ( M a n a l + 3 ) M a n @ l + 4GlcNAc@1+ 4GlcNAco~; 3, M a n a l -t 6 M a n~l~4 G l c N A c~l + 4 X y~~A c~~;

G~C N A C @~~X~I N A C O T .
A , the radioactive peak (a-N) in Fig. 1D; B, the radioactive peak (b-N) in Fig:lE; C, the radioactive peak in A incubated with @-galactosidase (0.8 milliunits/50 pl at 37 "C for 18 h); f), the radioactive peak in B or C incubated with dipbeoccal P-Nacetylhexosaminidase (6 milliunits/50 pl at 37 'C for 18 h); E, the radioactive peak in D subjected to periodate oxidation; F, the radioactive peak in E subjected to periodate oxidation.  Fig. 2. They were recovered from paper by elution with water and named components a and 6.
Structural Studies of Components a and b-By methanolysis followed by N-acetylation, both components a and b were completely converted to neutral oligosaccharides (Fig. 1, D  and E ) . These neutral oligosaccharides were named a-N and b-N, respectively. Upon Bio-Gel P-4 column chromatography, a-N gave a single radioactive peak with mobility of 12.3 glucose units (Fig. 3A) and 6-N also gave a single peak with mobility of 11.1 glucose units (Fig. 3B). The radioactive component in Fig. 3A was converted to a radioactive oligosaccharide with mobility of 11.1 glucose units releasing a galactose residue by 8-galactosidase digestion (Fig. 3C). Sequential exoglycosidase digestion of the radioactive components in Fig.  3, B and C, and methylation analysis of their nonradioactive counterparts gave exactly the same results as obtained by the structural study of oligosaccharide C liberated from bovine rhodopsin (17) (data not shown). Therefore, b-N and degalactosyl a-N should have the following structure. Mancul 6 Manu1
Comparative methylation analysis of components a-N and 6-N revealed that a 3,4,6-tri-0-methyl2-N-methylacetamido-2-deoxyglucitol €ound in 6-N was not detected in a-N (Table  I) A ) and a ( B ) . The acidic oligosaccharide samples (0.1 mg each) were dissolved in 10 ~1 of dimethyl sulfoxide, mixed with 10 gl of glycerol, and then analyzed by a mass spectrometer.
Components a and b are sulfate derivatives of a-N and b-Ny respectively. Fast atom bombardment mass spectrometry of components a and b gave m/z 1680 and 1518, respectively, as negative molecular ions: (M -H)- (Fig. 4, B and A ) . These results indicated that only one sulfate group is included in both components.
Methylation analysis of components a and b gave a 2,3,6tri-0-methylmannitol which was not found in a-N and b-N (Table I). Therefore, the structures of components a and b should be as shown in Fig. 5.
An attempt to determine by exoglycosidase digestion whether the sulfate residue is linked to a particular a-man-nosy1 residue or evenly distributed to the 2 a-mannosyl residues was not successful, because both acidic oligosaccharides were extremely resistant to exoglycosidase digestion.

DISCUSSION
Structurally, the carbohydrate moieties of the two sulfatecontaining sugar chains of hen egg albumin in Fig. 5 can be classified as hybrid-type sugar chains which were found for the first time in this glycoprotein (4,5 ) . However, they are different from other nonsulfated hybrid-type sugar chains in two points. The first difference is the absence of the bisecting N-acetylglucosamine residue in the sulfated sugar chains. This residue was included without exception in the nonsulfated hybrid-type sugar chains of hen egg albumin (4,5), and the evidence was used by Harpaz and Schachter (18) to explain why hybrid-type sugar chains are formed. Usually, the 2 a-mannosyl residues should be removed by the action of a-mannosidase I1 (19) located in the Golgi membrane and the sugar chains are converted to the complex-type sugar chains. This is the case for the sugar chains of bovine rhodopsin (17). Harpaz and Schachter (18) found that a-mannosidase I1 cannot remove the a-mannosyl residues from hybridtype sugar chains with the bisecting N-acetylglucosamine residue. Therefore, introduction of this residue to the processing intermediates of asparagine-linked sugar chains results in formation of bisected hybrid-type sugar chains as final products. The structural characteristics of the two sulfated sugar chains in Fig. 5 indicate that addition of a single sulfate residue to the processing intermediate may inhibit the action of a-mannosidase I1 and also inhibit the addition of the bisecting N-acetylglucosamine residue. Such control mechaism of the sugar chain maturation by sulfation might be an interesting subject for future enzymatic study.
Another difference is that the Galfil-wIGlcNAc group of the sulfated sugar chains is located at C-2 of the mannose residue linked a1-3 to the P-mannosyl residue, while the group in nonsulfated hybrid-type sugar chains of hen egg albumin is located at C-4 of the mannose (5). Possibly, introduction of the sulfate group to the hybrid-type sugar chain may inhibit the formation of the GlcNAcfil4Manalgroup, which might be a better galactosylation site than the GlcNAc/3l+PManal-group.
During the past few years, sulfated asparagine-linked sugar chains have been found in various sources such as viral coat protein (20), liver and lung of chick embryo (21), lutropins of many mammals (22,23), human vascular endothelial cells (24), and sea urchin (25). Interestingly, the structures of the sugar chains of viral coat protein, lutropins and hen egg albumin are quite different, and the sulfation seems to occur a t various sites of the sugar chains. This is a big contrast from the case of phosphorylated asparagine-linked sugar chains, in which phosphate residues are linked always to the a-mannosyl residue (26).
This evidence may indicate that a variety of sulfating enzymes exist in different tissues and play a vital role in cell differentiation and development. * 4 M a n a L 6 *wanal' ~an~1-+4GlcNAcB1-+4CIcNAcOH ?anal\ GlcNAcB1+2Manal f