the journal of chemistry vol. no. 12, biology, inc u.s.a ... · the journal of bm.ocriu. chemistry...

THE JOURNAL OF Bm.ocriu. CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 12, Issue of March 25, pp. 8762-8771, 1994 Printed in U.S.A.

Structural Studies of a Novel Type of Pentaantennary Large Glycan Unit in the Fertilization-associated Carbohydrate-rich Glycopeptide Isolated from the Fertilized Eggs of Oryzias latipes*

(Received for publication, October 18, 1993, and in revised form, December 10, 1993)

Tomohiko TaguchiS, Akira SekoS, Ken KitajimaS, Yutaka MutoS, Sadako Inoue% Kay-Hooi noon, Howard R. Morrisn, Anne Delh, and Yasuo InoueSIl From the Wepartment of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Hongo-7, Tokyo 113, the $School of Pharmaceutical Sciences, Showa Uniuersity, Hatanodai-1, Tokyo 142, Japan, and the fDepartment of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom

In a previous report (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553), we found the presence of a heavily glycosylated polyprotein, “H-hyosophorin,” isolated from the unfertilized eggs of Oryxias latipes. We now report our detailed analysis of the structure of the N-glycan chain in L-hyosophorin, the smallest repeating unit of H-hyosophorin, which was isolated from the fertilized eggs of 0. latipes and formed from H-hyosophorin upon fertilization. The N-glycan structures were defined by a combination of compositional analysis, methylation analysis, selective chemical degradation (i.e. mild methanolysis, periodate-Smith degradation, and hydrazinolysis-nitrous acid deamination), enzymatic (endo-P-galactosidase, peptideN-glycanase, and Newcastle disease virus sialidase) digestion, and instrumental analyses (one- and two-dimensional proton nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry) which revealed novel and unique features: (a ) the presence of highly branched poly-N- acetyllactosamino pentaantennary structures; ( b ) the presence of a P-galactosylated Lewis X antigenic epitope, Galpl-4 Galpl-4 (Fuccul+3)GlcNAc~l+; ( c ) the presence of a P-galactosylated sialyl Lewis X structure, Galj3l+4(Neu5Aca2+3)Gal~l~4(Fuca1-r3)GlcNAc~1+; ( d ) the presence of GalP1+4GalPl+ and GalPl-, 4Galj31+4Galpl+ as the major and minor groupings, respectively; and ( e ) the presence of the branched Gal residues, +4GlcNAcfll+3(Gal~l+4) Galpl-.

This study represents the first detailed investigation regarding the nature of highly branched complex asparagine-linked pentaantennary glycans in glycoproteins. The unique expression of such bulky multiantennary glycan units on proteins could be essential during early embryogenesis.

One of the glycan structures carrying a number of differen- tiation- and development-associated antigens is the glycoprotein-bound large carbohydrate unit comprised of poly-N-acetyllactosaminoglycan chains, i.e. (-3Galpl+4Gl~NAcpl-)~. Repeating GalP1-4 GlcNAcp1+3 sequences bearing different types of antigenecity or immunogenecity have been identified

search 02455022 (to S. I.) and 04453160 (to Y. I.) from the Ministry of * This research was supported by Grant-in-Aids for Scientific Re-

Education, Science, and Culture of Japan, a Medical Research Council Programme grant, Wellcome Trust Grant 030826 (to H. R. M. and A. D.), and Prize Fellowship 036485/2/92/2 (to K. H. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed. Fax: 81-3-5684-2394.

in surface glycoproteins of various animal cells including human granulocytes (1, 2), erythrocytes (3, 41, eggs or oocytes (zona pellucidae, Ref. 51, embryonal carcinoma cells (6, 71, leu- kocytes (leukosialin, Ref. 81, and lysosomes (9, 10). A vast amount of experimental evidence showing directly or indirectly the spatio-temporal expression of diverse forms of carbohydrate markers on poly-N-acetyllactosamine structures have been accumulated with special reference to oncogenesis, embryogenesis, fertilization, immune system, and cell differentia- tion ( 1 4 , 11-17). The unique features of such glycans as antigenic determinants of glycoproteins can be briefly summarized as ( a ) multiple forms of multivalent epitope structures can be generated by branching of linear poly-N-acetyllactosamine chains as in the case of I antigenic determinants (3) and ( b ) different types of multivalent antigenic structures can be expressed on poly-N-acetyllactosaminyl glycan chains by multiple substitution with fucose andor sialic acid residues leading to the formation of the SSEA-1 (or Lewis X) antigen, i.e. al-3 fucosylated N-acetyllactosamine and sialyl Lewis X, i.e. Neu5Aca2+3Galpl+4(Fucal+3) G1cNAcpl-t. These antigens are known to be ligands for cellular adhesion molecules (18, 191, and multivalent forms would be expected to regulate the strength of interaction with receptor molecules (20).

In recent years, we have been studying in detail the structure, biosynthesis, and function of cortical alveolar carbohydrate-rich unique glycoproteins from teleost fish eggs. One of the striking findings was the observation that there were two discrete classes of glycoproteins depending on fish order; one is the a2,8-linked oligo/polysialyl group-containing polyanionic glycan units attached to the core protein through 0-glycosidic linkages and the other contains bulky multiantennary N-linked glycan units (reviewed in Ref. 21). Recently, we have introduced the term “hyosophorin” as a collective name to de- scribe these glycoprotein molecules. Glycoproteins meeting the following criteria are referred to as hyosophorin: 1) those which are Golgi-derived secretory vesicular or cortical alveolar components; 2) those which are rich in carbohydrate (the carbohydrate content is -85-90% of the total mass); 3) those in the unfertilized eggs are made up of tandem glycopeptide repeats, and are designated high molecular weight hyosophorin (H-hyosophorin); 4) those defined as H-hyosophorin are proteolyzed a t egg activation or fertilization into the smallest repeating unit(s) which is denoted L-hyosophorin.

Recently a detailed study on the structure of the oligosaccha- rides in Oryzias melastigma (Indian medaka) hyosophorin has been performed (22). The 0. melastigma hyosophorin was found to contain novel bulky tetraantennary glycan structures and to constitute a member of the glycoprotein family bearing poly-N-acetyllactosaminoglycan units. In order to establish the precise structure for such bulky multiantennary glycan chains,

8762

Highly Branched N-Linked Pentaantennary Glycans 8763

we used not only biochemical or enzymatic procedures but also chemical (selective degradation reactions) and instrumental methods (1D' 'H NMR spectroscopy and FAl3-MS spectrometry) in the course of structural elucidation. In the present study we have included 2D lH NMR spectroscopy in addition to these methods in the structural analysis of the more complex highly branched multiantennary glycan unit present in L-hyosophorin molecules isolated from the fertilized eggs of 0. latipes, another species of Medaka fish family. We found similarity and dissimilarity in the glycan units of latipes and melastigma L-hyosophorin molecules although both of them exhibited a highly conserved amino acid sequence.

EXPERIMENTAL PROCEDURES Fertilized Eggs of 0. latipes and Isolation of L-hyosophorin from the

Fertilized Eggs-Fertilized eggs (early embryos a t blastulae stage) of 0. latipes were collected and stored at -20 "C until use. Unless otherwise specified, all isolation procedures were carried out in the cold. Fertilized eggs (31.5 g) were homogenized in 2 volumes (v/w) of 0.4 M NaC1. Fertilization envelopes were removed by filtration through Tetoron gauze, a half-volume of 90% phenol was immediately added to the filtrate, and the mixture was stirred overnight a t 4 "C. After centrifu- gation at 6000 revolutions/min for 15 min, the aqueous phase was collected, and a half-volume of 0.4 M NaCl was added to the organic phase. After stirring for several hours at 4 "C, the mixture was centri- fuged and the aqueous fraction was collected. These aqueous fractions were combined, extensively dialyzed against water, and applied to a column (1.8 x 60 cm) ofDEAE-Sephadex A-25 (Cl- form, preequilibrated with 0.01 M Tris-HC1 buffer, pH 8.0). The column was eluted by a linear gradient of NaCl (0-0.6 M) in the same buffer. The elution profile was monitored by the absorbance a t 230 nm, the TBAmethod (23,241, or the resorcinol method (25) for sialic acid, and the phenol-sulfuric acid method (26) for hexose. Sialic acid-positive fractions were applied to a Sephacryl S-200 column (1.6 x 140 cm, equilibrated, and eluted with 0.1 M NaCl in 5 m Tris-HC1 buffer, pH 8.0, fraction volume 2.6 ml). The material thus obtained was identified as L-hyosophorin (L-hyo) (27).

Chemical Analyses-Chemical analyses were carried out as previously described (22).

Ezosialidase Digestion of L-hyo-A sample of L-hyo (28 mg as Gal) was digested with 0.5 units of Arthrobacter ureafaciens exosialidase (nacalai, Kyoto) in 6 ml of 50 m sodium acetate buffer, pH 5.5, at 37 "C under toluene. After 24 h of incubation, an additional 200 pl of 2.5 unitdm1 exosialidase was added and further incubated. Sialic acid (Neu5Ac) released was determined by the TBA method (23, 24). Asialo L-hyo was separated from Neu5Ac by passage of the enzyme digests through a Sephadex (2-50 column (1.8 x 60 cm, equilibrated, and eluted with 0.1 M NaCI). The asialo L-hyo was then desalted by Sephadex G-25 column chromatography (1.3 x 130 cm, equilibrated and eluted with 5% ethanol).

Defucosylation by Mild Methanolysis-To release fucose residue selectively, asialo L-hyo (600 pg as Gal) was methanolyzed in 0.5 ml of 0.05 N HCUmethanol for 45 min a t 65 "C and neutralized with 0.05 N

NaOH and then desalted by Sephadex G-25 column chromatography (1.3 x 130 cm, equilibrated and eluted with 5% ethanol).

Smith Degradation-The procedure was essentially that of Spiro (28). In brief, to 20 mg (as Gal) of asialo L-hyo were added 10 ml of 50 mM sodium acetate buffer, pH 4.5, containing 130 mg of NaIO,, and the reaction mixture was kept in the dark a t 4 "C with occasional shaking. At the end of reaction (112 h), 2.3 ml of 3% ethylene glycol was added to the reaction mixture to destroy excess periodate. After 4 h a t 4 "C in the dark, 180 mg of NaBH4 and 4 ml of 0.5 M sodium borate buffer, pH 8.0, were added to the reaction mixture, and the reduction was continued at 4 "C. After adjusting pH to neutral with 1 N acetic acid, the solution was applied to a column of Sephadex G-25 to desalt. After the eluate was evaporated to dryness, the residue was treated with 2.2 ml of 0.05 N

H,SO, at 80 "C for 1 h. After cooling to room temperature, the hydrol-

sional; FAB-MS, fast atom bombardment-mass spectrometry; NeuBAc, ' The abbreviations used are: lD, one-dimensional; 2D, two-dimen-

N-acetylneuraminic acid; 2,5-anhMan-ol, 2,5-anhydro-~-mannitol; L-hyo, L-hyosophorin, low molecular weight form of hyosophorin isolated from fertilized or parthenogenetically activated eggs; TBA, thio- barbituric acid; TLC, thin layer chromatography; NDV-sialidase, New- castle disease virus sialidase; TOCSY, total correlated spectroscopy; DQF-COSY, double quantum filtered-correlated spectroscopy; ppm, partdmillion.

ysate was neutralized with 0.05 N NaOH and chromatographed on a column of Sephadex G-50 (1.8 x 60 cm, equilibrated and eluted with 0.1 M NaCI, 1.5 ml fractions were collected). The elution profile was monitored by the absorbance a t 230 nm and the phenol-sulfuric acid method (26) for hexose.

Liberation of Glycan Chain from Glycopeptide by Peptide:N- Glycanase-2.4 mg (as Gal) of glycopeptide fraction S1 (see "Results and Discussion"), derived from periodate oxidation-Smith degradation of asialo L-hyo, was incubated with 4.0 x units of PNGase F (Takara Co., Kyoto) in 3 ml of 0.25 M phosphate buffer, pH 8.6, under toluene for 24 h a t 37 "C. The same amount of the enzyme was then added, and the mixture was incubated for another 24 h. The digest was then applied to a column (1.1 x 10 cm) of Bio-RadAGlx2 (C1- form). The flow-through fraction was concentrated and then desalted by Sephadex (3-25 column chromatography. Approximately 2.0 mg (as Gal) of this desalted fraction (Sl-f; see "Results and Discussion") was purified on Bio-Gel P-4 (1.3 x 100 cm, equilibrated and eluted with 50 m acetic acid).

Digestion with Endo-a-galactosidase-(i) 200 pg (as Gal) of free glycan fraction S1-f was incubated with 0.025 units ofEscherichia freundii endo-a-galactosidase (Seikagaku Kogyo Co., Ltd., Tokyo) dissolved in the total volume of 600 pl of 100 m sodium acetate buffer, pH 5.5, for 40 h a t 37 "C. The digests were applied to a column (1.3 x 100 cm, 50 mM acetic acid) of Bio-Gel P-4. The phenol-sulfuric acid-positive fractions were subjected to composition and methylation analysis.

(ii) Glycopeptide fraction S1 (3 mg as Gal; see "Results and Discus- sion"), derived from periodate oxidation-Smith degradation of asialo L-hyo, was incubated with 0.2 units of E. freundii endo-a-galactosidase dissolved in the total volume of 2.6 ml of 100 m sodium acetate buffer, pH 5.5, for 48 h at 37 "C. The digests were applied to a column (1.3 x 130 cm, 5% ethanol, 2 ml fractions were collected) of Sephadex G-25. The glycopeptide fraction thus obtained was subjected to 2D 'H NMR measurement.

Hydrazinolysis-Nitrous Acid Deamination-A freeze-dried sample of mm-asialo L-hyo (0.5 mg as Gal; see "Results and Discussion") was reacted with 2 ml of anhydrous hydrazine together with 5 mg of hydra- zinium sulfate in a commercial hydrazinolysis apparatus, Hydraclub (Honen Corp.) at 110 "C for 24 h. After cooling to room temperature, the reaction mixture was dried overnight in a vacuum desiccator over concentrated H,SO,. 25 mg of sodium nitrite and 2 ml of 0.5 N acetic acid were then added. After reaction at room temperature for 1 h, an additional 10 mg of NaNO, was added, and deamination was continued overnight. Ethylamine (70%) was then added to destroy excess nitrite, and the solution was treated with 10 mg of NaBH4 a t room temperature for 3 h. The pH was adjusted to 5-6 with 1 N acetic acid, the reaction products were then applied to a column of Dowex 50 (H+ form, 1.4 x 10 cm) in the cold, and the flow-through fractions were collected and chromatographed on a column (1.3 x 100 cm, 50 m acetic acid) of Bio-Gel P-4. The eluant was monitored for neutral sugar by the phenol-sulfuric acid method (261, and the phenol-sulfuric acid method-positive fractions were collected and analyzed.

Methylation Analysis-Methylation analysis was performed as previously described (22).

Thin Layer Chromatography (TLC)-TLC was carried out as previously described (22).

NDV-Sialidase Digestion4i) U3'-1, U3'-3 (29) (100 pg as Neu5Ac) were incubated individually with Newcastle disease virus sialidase (0.01 units, Oxford Glyco Systems, United Kingdom) in 500 pl of 50 m sodium acetate buffer, pH 5.5, a t 37 "C. Release of free sialic acid was estimated a t 30, 60, 130, and 250 min by the TBA method (23, 24).

(ii) Intact L-hyo (400 pg as Neu5Ac) was incubated with NDV-sialidase (0.04 units) in 1.2 ml of 50 m sodium acetate buffer, pH 5.5, at 37 "C for 14 h. Then, an additional 0.01 units of enzyme (suspended in 100 pl of 50 m sodium acetate buffer, pH 5.5) was added and further incubated for 9 h. Free sialic acid released was separated by passage through Sephadex G-25 column (1.3 x 130 cm, 5% ethanol). NDV-sialidase-digested L-hyo thus obtained was subjected to sugar composition analysis.

FAB-MS Spectrometry-N-Glycans were released from the L-hyo glycopeptide by peptidefl-glycanase F and permethylated as described (30). FAB spectra were recorded using a VG Analytical ZAB-2SE FPD mass spectrometer fitted with a cesium ion gun operated a t 20-25 kV. Data acquisition and processing were performed using the VG Analyti- cal Opus@ software. The permethylated glycans were aliquoted in methanol and monothioglycerol was used as matrix.

'HNMR Measurement-1D 400 MHz 'H NMR spectra were recorded with the apparatus and by methods described previously (31). 2D 500 MHz 'H DQF-COSY and TOCSY spectra of Sl-a (see "Results and Discussion") were recorded using a Bruker "500 NMR spectrom-

8764 Highly Branched N-Linked Pentaantennary Glycans eter at 60 "C. For the TOCSY spectrum, a MLEV-17 mixing sequence of 100 ms was used. Proton chemical shifts are expressed in parts/million relative to the methyl proton signal of sodium 3-(trimethylsilyl) propi- onate-2,2,3,3d4 set equal to 0.00 ppm.

RESULTS AND DISCUSSION

Isolation a n d Purification of L-hyosophorin from the Fertilized Eggs of 0. latipes

As described under "Experimental Procedures," the phenol- treated crude material obtained from 31.5 g of the fertilized eggs of 0. latipes was chromatographed on DEAE-Sephadex A-25. Analysis of the fractions for sialic acids and neutral sug- ars by the TBA and phenol-sulfuric acid procedures gave a peak eluting at 0.05 - 0.18 M NaC1. The fractions eluted with NaCl concentration between 0.05 and 0.18 M were further purified by gel filtration on the Sephacryl S-200 column. Material eluted in fraction 62-78 was assigned as L-hyosophorin, labeled L-hyo (yield, 41 mg as Gal), and used for structural studies. L-hyo was a glycononapeptide in which a large glycan chain is linked to the asparagine residue (27): Asp-Ala-Ala-Ser-Asn(CH0)-Gln- Thr-Val-Ser.

Structural Elucidation of the Large N-Glycan Unit of L-Hyosophorin

The structure for the N-linked large glycan chain of L-hyo was deduced from the data obtained from carbohydrate composition analysis, methylation analysis, mild methanolysis, periodate Smith degradation, endo-P-galactosidase digestion, hydrazinolysis-nitrous acid deamination, NDV-sialidase digestion, FAB-MS spectrometry, and lH NMR spectroscopy.

Carbohydrate Composition of L-hyosophorin-The intact L- hyo contained FucfMan/GaVGlcNAc/Neu5Ac in a molar ratio of 1.9:3.0:14.1:8.9:4.6. The sialic acid residues were exclusively Neu5Ac.

Methylation Analysis of the Intact L-hyosophorin and Asialo L-hyosophorin-The native L-hyo and asialo L-hyo were subjected to methylation analysis (Fig. 1). A number of partially methylated alditol and hexosaminitol acetates were identified and quantified using gas-liquid chromatography by comparison of their relative retention times with the values for authentic samples (22). The data from the asialo glycopeptide showed the presence of 2,3,4-tri-O-Me-Fuc, 2,3,4,6-tetra-O-Me-Gal, 2,3,6- tri-0-Me-Gal, 2,6-di-O-Me-Gal, 3,6-di-O-Me-Man, 2,4-di-O-Me- Man, 3-0-Me-Man, 3,6-di-O-Me-GlcNAc, and 6-0-Me-GlcNAc. These data indicated the presence of terminal Fuc, terminal Gal, 4-0-substituted Gal, 3,4-di-O-substituted Gal, 2,4-di-0- substituted Man, 3,6-di-O-substituted Man, 2,4,6-tri-O-substituted Man, 4-0-substituted GlcNAc, and 3,4-di-O-substituted GlcNAc. On comparison of these data for asialo L-hyo with those for the intact L-hyo, the substitution patterns of Man and GlcNAc residues were identical with each other, and the nature of the substitution of the Gal residues was the only difference found (Table I). On removal of Neu5Ac residues from L-hyo, the proportion of the terminal Gal and 4-0-substituted Gal residues increased at the expense of the 3-0-substituted Gal and 3,4-di-O-substituted Gal, respectively. These results indicated that two types of Neu5Ac residues were present; one was origi- nally attached to 0 -3 of the terminal Gal residue(s) and the other was attached to 0-3 of the internal 4-0-substituted Gal residue(s). The relative amounts of these two types of Neu5Ac residues in L-hyo were estimated to be almost identical in agreement with the estimation based on the intensity of H-3,, proton signals due to different types of the Neu5Ac residues in the 'H NMR spectrum of L-hyo (see below). The presence of 24 - and 3,6-di-O-substituted Man residues and 2,4,6-tri-O-substituted Man residue in a ratio of 1:l:l strongly indicated pentaantennary core glycan structure for the L-hyo molecule.

8 I

I I I 5 10 15 20 25 30

Rclcntion limc (nlin)

FIG. 1. Gas-liquid chromatogram of partially methylated aldi- to1 acetates originated from asialo L-hyo. The following peaks were identified as alditol acetates of: 1 , 2,3,4-tri-O-methyl Fuc; 2, 2,3,4,6- tetra-0-methyl Gal; 3,2,3,6-tri-O-methyl Gal; 4,2,6-di-O-methyl Gal; 5, 3,6-di-O-methyl Man; 6, 2,4-di-O-methyl Man; 7, 3-0-methyl Man; 8, 3,6-di-O-methyl GlcN(Me)Ac; 9, 6-0-methyl GlcN(Me)Ac.

TABLE I Molar ratios of permethylated galactose residues derived

from L-hyo and asialo L-hyo

Alditol acetates derived from L-hyo ksialo L-hyo

2,3,4,6-Tetra-O-Me-Gal 0.8 1.3 2,3,6-Tri-O-Me-Gal 0.6 1.0" 2,4,6-Tri-O-Me-Gal 0.4 2,6-Di-0-Me-Gal 0.9 0.4

lactitol of asialo L-hyo taken as 1.0. Molar ratios are relative to 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl ga-

Defucosylation of Asialo L-hyosophorin by Mild Metha- nolysis-To remove the fucose residues selectively, mild methanolysis of asialo L-hyo was carried out as described under "Experimental Procedures." The reaction product was desalted and named as mm-asialo L-hyo. The mm-asialo L-hyo was found to have a carbohydrate composition of Fuc/Man/GaV GlcNAc = 0.65:3.0:12.9:8.9, showing that approximately 70 and 8.5% of fucose and galactose residues were lost, respectively. An increase of about 1.3 residues of 4-0-substituted GlcNAc at the expense of about 1.3 residues each of the unsubstituted Fuc and the 3,4-di-O-substituted GlcNAc was observed on methylation analysis (Table 11). These data demonstrated that the Fuc residue was not linked to the Gal residue but attached to the 0 -3 of GlcNAc residue. Supportive evidence for this was also obtained from the 'H NMR spectral measurement (see below).

400 MHz ' H NMR Spectroscopy of L-hyosophorin-Fig. 2 shows the 400 MHz 'H NMR spectrum of the intact L-hyo. The anomeric configurations of all of the Gal and GlcNAc residues in L-hyo were concluded to be P by comparing the observed chemical shift values for their H-1 protons with the corresponding literature values (22, 32). As anticipated, two pairs of dis- tinct resonances (H-3,,, 1.78 and 1.81 ppm; H-3,,, 2.73 and 2.76 ppm) of roughly equal intensity were observed for the


sialic acid residues. By comparison with the reported spectral data for Neu5Aca2-3Ga1~1+4GlcNAcpl+ (29) and Neu5- Aca2-.3Galp1-4Galpl-. (33), the chemical shift values of 1.78 ppm for H-3,, and 2.76 ppm for H-3, were assigned to the Neu5Ac residue a2h3-linked to the terminal Gal residue present in L-hyo. The remaining pair of the signals at 1.81 and 2.73 ppm were virtually the same as those previously shown for Neu5Aca2+3(Gal~l--.4)Gal~1~4GlcNAc~l- (29), indicating the presence of the Neu5Ac residue which is a2 -33-linked to the internal 4-0-substituted Gal residue. The methylation analysis (Table I) was also consistent with the presence of these two types of sialic acid substitution patterns in equimolar pro- portions in L-hyo. Two proton signals a t 1.18 and 5.09-5.10 ppm were assignable to the methyl proton of the Fuc residue and the anomeric proton of the Fuc residue al43-linked to GlcNAc residue, respectively (cf. 32, 34). These data are consistent with the results of methylation analysis of asialo L-hyo and mildly methanolyzed asialo L-hyo (mm-asialo L-hyo) given in Table 11. Attachment of a Fuc residue a1-3-linked to the proximal GlcNAc residue is known to cause an upfield shift of the GlcNAc H-1 proton signal by about 0.03 ppm (GlcNAcpl- 2Manal-3(Manal-6)(Xyl~l-~2)Man~l+4GlcNAc~l- 4GlcNAc versus Manal+3(Manal-6)(Xylpl .2)Manpl+ 4GlcNAc~l-4(Fucal-3)GlcNAc (35, 36)). The appearance of a signal for the proximal GlcNAc H-1 proton a t 5.03 ppm, which was also previously observed for 0. melastigma L-hyo (22), was indicative of the absence of the Fuc residue on the proximal GlcNAc of 0. latipes L-hyo.

Periodate Oxidation-Smith Degradation of Asialo L-hyosophorin-The asialo L-hyo was subjected to periodate oxidation- Smith degradation as described under "Experimental Proce- dures.'' The reaction products were applied to a column of Sephadex G-50. Eluted fractions were monitored by the phenol- sulfuric acid method and absorbance at 230 nm, and material under fraction 50-79 (phenol-sulfuric acid-positive) was pooled

TABLE I1 Molar ratios of permethyluted Fuc and GlcNAc residues derived

from asiulo L-hyo and mm-asialo L-hyo

Alditol acetates derived from Asialo L-hyo mm-Asialo L-hyo

2,3,4-Tri-O-Me-Fuc 1.9" 0.65" 3,6-Di-O-Me-GlcNAc 6.8 8.1 6-OMe-GlcNAc 2.1 0.81

" For asialo L-hyo, 2,3,4-tri-O-Me-Fuc is set equal to 1.9, and for mm-asialo L-hyo, it is taken as 0.65.

and designated S1. No carbohydrate components characteristic of L-hyo were detected in other fractions. The carbohydrate composition of S1 is presented in Table 111. The periodate- Smith degradation caused the destruction of Gal and Fuc residues whereas no loss of GlcNAc was detected. A part (2.4 mg as Gal) of S1 was treated with PNGase F, and the free oligosaccharide fraction thus liberated was denoted as S1-f.

Digestion of Sl-f with Endo-P-galactosidase-The products derived after exhaustive digestion of S1-f (200 pg as Gal) with endo-0-galactosidase were separated by gel chromatography on Bio-Gel P-4 (Fig. 3). Two discrete carbohydrate fractions, designated S1-fa and S1-fb, were obtained. The carbohydrate composition of S1-fa given in Table I11 showed Man3GlcNAc7, and Gal was no longer present. S1-fa gave 3,6-di-O-methyl Man, 2,4-di-O-methyl Man, 3-0-methyl Man, 3,4,6-tri-O-methyl GlcNAc, and 3,6-di-O-methyl GlcNAc in a molar ratio of 1:1:1:5:2 on methylation analysis consistent with S1-fa having a pentaantennary structure (see Fig. 5). Based on the carbohydrate composition for S1-fb (Table 111) together with the known substrate specificity of endo-0-galactosidase used, we concluded that S1-fb had the structure GlcNAcpl~+3Gal. These results, when combined with the carbohydrate composition of S1, led us to consider that S1 had the structure in which an average number of 1.9 chains of GlcNAcPl .3Galp1+4 were attached to S1-fa.

Gel Chromatography of Sl-f on Bio-Gel P-4"The fraction S1-f (-2.0 mg as Gal) was subjected to gel chromatography on Bio-Gel P-4. The elution profile (Fig. 4) showed four carbohydrate peaks labeled S1A through S1D. These subfractions were rechromatographed on the same column and the yield and carbohydrate composition were given in Table IV. Consideration of the data shown in Table IV and the results obtained from endo-0-galactosidase digestion of S1-f permitted us to draw a conclusion that (i) the structure of S1D was identical with that of SI-fa and (ii) SIC, SlB, and S1A were made up of S1-fa to which 1,2, and on average 4.2 chains of GlcNAcpl~ -3Gal were attached by p l ~ .4-linkages, respectively (Fig. 5). To distinguish between the possibilities which the 2,4,6-tri-O-substituted Man or the 2,4-di-O-substituted Man were a1 *3- or a1 .6-linked to the p-Man residue, the two-dimensional 'H NMR spectral measurement was performed as described below.

Consequently, the periodate-Smith degradation of asialo L- hyo resulted in the products having the structures in which a varying number (from zero to more than five) of GlcNAcpl-3Galpl 34 chains were linked to the pentaanten-

r l f t t

ppm 1 1 1 I I I I I I I I I I

5.2 5.0 4.8 2.5 2.0

FIG. 2. Parts (1.7-2.8 ppm and 4.7-5.2 ppm) of 400 MHz 'H NMR spectrum of intact L-hyo in DzO at 60 "C. The residue numberings are given using the triomannosido-di-N-acetylchitobiose structure.

8766 Highly Branched N-Linkec TABLE I11

Carbohydrate compositions of the fractions SI , S1-fa, and Sl-fb

d Pentaantennary Glycans

TABLE IV Carbohydrate compositions of the oligosaccharide fractions SIA-SID

s1 S1-fa s1-fll Man Gal GlcNAc

3.0a 3.0" 1.9 1.0" 8.9 6.9 1.1

a For S1 and S1-fa, Man is set equal to 3.0, and for S1-fb Gal is set equal to 1.0.

0.6 , 7

0.5 1 - 0.4 t I

Fractlon Number

FIG. 3. Gel chromatography on Bio-Gel P-4 of the products after exhaustive digestion of S1-f with endo-P-galactosidase. The phenol-sulfuric acid profile of the material is shown. The column (1.3 x 100 cm) was equilibrated and eluted with 50 m acetic acid, and 1.2-ml fractions were collected. The bars indicate fractions pooled to give S1-fa and S1-fb.

*'O li,I 1.5 -

1.0 -

0.5 -

S1 A

0.0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

Fraction number

FIG. 4. Gel chromatography on Bio-Gel P-4 of fraction S1-f. The phenol-sulfuric acid profile of the material is shown. The column (1.3 x

fractions were collected. The fractions, S l A through SlD, indicated by 100 cm) was equilibrated and eluted with 50 mM acetic acid, and 1.0-ml

the bars were pooled and rechromatographed on the same column. V,,, void volume.

nary core, S1-fa. Two-dimensional ' H NMR Spectral Analysis of S l - p a t 500

MHz-A glycopeptide fraction S1 (3 mg as Gal) was digested exhaustively with endo-@-galactosidase as described under "Experimental Procedures," the glycopeptide product was desalted and designated as Sl-p. The results of the compositional and methylation analyses were identical with those of S1-fa. The 2D 500 MHz 'H DQF-COSY and TOCSY spectral measure- ments were carried out for Sl-p. The resonance signals at 4.22, 4.05, and 3.64 ppm were, respectively, assigned to H-2, H-3, and H-4 protons of the Man(4) residue, which is attached a 1 3 3 to the P-Man residue, by connecting cross-peaks due to magneti- zation transfer from the anomeric proton resonance of this residue appearing at 5.13 ppm. These values of chemical shifts are essentially identical to those reported for the corresponding

~

S 1A S1B s1c S1D

Man 3.O0 3.0" 3.0" Gal

3.0" 4.2 2.2 1.1

GlcNAc 10.9 8.8 8.3 6.9

Yieldb 400 620 380 270

" Man is taken as 3.0. Expressed in micrograms as recovery of neutral sugar.

GlcNAcpl y6

(GlcNAcpl~3Galpl+4) , GlcNAcplv

$Manpl-4GlcNAc~l-.4GlcNAc

FIG. 5. Proposed glycan structures for SlAthrough S1D. S1D or S1-fa, n = 0; SlC, n = 1; SlB, n = 2; SlA, n = 4.2.

*Fucal 3 Galgl GlcNAcp 1 -

4 4

3 Gal~1-4Galf31

4

3

Galpl GlcNAcPl

*Fwdf SCHEME 1.

protons of the 2,4-di-O-substituted a-Man residue linked to the D-Man residue of the largest glycan unit N4/B of human eryth- ropoietin expressed in recombinant BHK-21 cells (H-1, 5.13 ppm; H-2, 4.22 ppm; H-3, 4.04 ppm; H-4, 3.61 ppm; Ref. 37), showing the presence of the 2,4-di-O-substituted al-3-linked Man arm, i.e. R1-4(R2-2)Manal+3Manpl+ in Sl-p. Thus, the results of methylation analysis can be taken as the evidence for the presence of 2,4,6-tri-O-substituted al+G-linked Man arm in Sl-p.

HydrazinoLysis-Nitrous Acid Depolymerization of mm-Asialo L-hyosophorin-Further structural investigation on the peripheral portions of the asialo glycan unit of L-hyo was performed. The products formed by hydrazinolysis-nitrous acid deamination of mm-asialo L-hyo followed by NaBH4 reduction were applied to a column of Bio-Gel P-4. The neutral sugar analysis for the column effluents by the phenol-H2S0, method gave a profile having a single major peak with a shoulder at the excluded volume side (not shown). The lower molecular weight fractions (designated fraction HN) were pooled and subjected to structural analysis. The recovery of Gal in the fraction HN amounted to -80% of the mm-asialo L-hyo. The higher molecular weight shoulder fraction was found to contain a high proportion of Man, and no further structural study was made for this material. The fraction HN was found to have a sugar composition of 2,5-anhydro Man-ol and Gal in a ratio of 1.0:2.0 and to give 4-0-acetyl-1,3,6-tri-O-methyl2,5-anhydromannitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl galactitol, and 1,5-di-0- acetyI-2,3,4,6-tetra-O-methyl galactitol in 1.2:l.l:l.O ratio on methylation analysis. No Fuc residue was detected in this fraction. The homogeneity of the fraction HN was examined by TLC, and two minor spots in addition to the major spot were exhibited by orcinol-HzSO4 spray: one is the faster moving spot and the other is the slower moving one, relative to the major component. The above results and the FAB-MS spectral data (see below) were consistent with the major component having the structure Ga11-4Ga11+4(2,5-anhydromannitol) and with the structures Gall-.4(2,5-anhydromannitol) and Gall- 4Ga11-4Ga11+4(2,5-anhydromannitol) for the faster and slower moving minor components, respectively. The 'H NMR spectrum of L-hyo (Fig. 2) revealed that the anomeric configu-


30 20 10

100 90 EO 7 0 60 50 40 30 20 10

6

668

636 I

1321

x5.00

70 1974 60 50 40 3 0 20 10 0

U 3 1 3 " ' I " ' , 1 ~ ~ ~ ' I

2100 2 1'50 2 2'0 0 2250 2 3'0 0 2350 2 4'0 0 2 4'5 0 M/ 2

undermethylated counterparts. Loss of a MeOH moiety (32 units) was observed for the major A-type ions. The prominent ions at m / z 246 FIG. 6. FAB-spectrum of permethylated asialo glycan released from L-hyo. Ions at 14 mass units below those assigned correspond to their

((HO),(GlcNAc),)' and 450 ((HO),(Gal),(GlcNAc),)+ are probably derived from the abundant ion at m / z 668 ((Gal),(GlcNAc),)+ after secondary B-cleavage. Incomplete desialylation yielded the ((Neu5Ac),(Gal),(GlcNAc),)+ ion at m / z 1029.

8768 Highly Branched N-Linked Pentaantennary Glycans

Gal Gall jCd4kNr

Gal-GlcNAc f. Gal+GaldGlcNAc 1321

668

SCHEME 2.

ration of all of the Gal residues of the three components in fraction HN were all p. Structural elucidation of the products derived after periodate-Smith degradation of asialo L-hyo indicated that the average number of 1.9 mol of GlcNA- cplh3Galp144 units were attached to the pentaantennary core (Fig. 5). At the same time, since no 3-0-substituted Gal but 3,4-di-O-substituted Gal was detected on methylation analysis of asialo L-hyo (see Table I), all the Gal residues of GlcNAc~l+3Gal~l+4GlcNAc~l+ units in asialo L-hyo must be substituted at the 0-4 positions. Therefore, the intact and asialo L-hyo molecules were considered to have the pentaantennary branching structure of R-.4(GlcNAcpl+3)Galpl+ 4GlcNAcpl+. The nature of the branching portion of the major asialo glycan units was elucidated by hydrazinolysis-nitrous acid deamination of mm-asialo L-hyo (the major product, Galp1+4 Galpl-.4(2,5-anhydro Man-01)) together with the information about the linkage pattern of the Fuc residue (Scheme 1). Some minor components were present, which differed in the number of the Gal residues. Further corroborative data were provided by FAB-MS analysis of the permethylated asialo glycan released by PNGase F.

FAB-MS Analysis of Asialo Glycan Units Derived from L- hyosophorin-As shown in Fig. 6, the most prominent non-reducing end fragment ion at m l z 668 corresponds to ((Gal)z(GlcNAc)l)+. In agreement with the chemical analysis which revealed heterogeneity associated with the number of Gal residues, fragment ions corresponding to ((Gal)l(GlcNAc)l)+ (mlz 464) and ((Gal)3(GlcNAc)l)+ (mlz 872) were also present. Similar heterogeneity was observed for ions at m / z 1117, 1321, 1525 (a (Gal)z(GlcNAc)l increment above m / z 464,668, and 872) which correspond, respectively, to and ,(GlcNAc),)+ with ((Gal)4(GlcNAc)z)+ (mlz 1321) being the most prominent, consistent with Gal-Gal-anhydro-mannitol being the major product derived from hydrazinolysis-nitrous acid depolymerization (see above). A further repeating unit of (GaUZ(GlcNAc), on the major components yielded the ions at mlz 1974 and 2178 corresponding to ((Gal)6(GlcNAc)3)+ and ((Gal),(GlcNAc)3)+, respectively. These results were in complete agreement with the most prevalent branching structure in asialo glycan bearing no fucose residue as depicted in Scheme 2.

A significant level of mono-fucosylation was observed for all the major antennae present, affording non-reducing end fragment ions at mlz 842 ((Fuc)l(Gal)2(GlcNAc)1)+, 1291 ((Fuc)l- (Gal)3(GlcNAc)z)+, 1495 ((Fuc)l(Gal)4(GlcNAc)z)+, 1699 ((Fucll- (Gal)5(GlcNAc)z)+, and 2148 ((Fu~)~(Gal)~(GlcNAc)~)+. In addition, minor di-fucosylated structures were also found on the major branched antennae, yielding ions at mlz 1669 ((Fuc)z(Gal)4(GlcNAc)Z)+ and 2322 ((Fuc)z(Gal)6(GlcNAc)3)+.

The Nature and Location of Sialic Acid Residues in L-hyosophorin-In the previous study, we isolated two types of biantennary sialylated free glycans from the eggs of Dibolodon hakonensis and determined their structures: the free glycans U3’-1 and U3‘-3 contain 2 mol each of the antenna, Neu5Acol2-.3(Gal~l+4)Gal~l+4GlcNAc~l-t and Neu5Ac- a2+3Galpl+4GlcNAcpl+, respectively (29). When these two free glycans were digested with the a2+3-linkage-specific sialidase (NDV-Sialidase) for 30- min, the amount of free Neu5Ac liberated from U3’-3 was seven times more than that from U3’-1, indicating that the unbranched terminal Neu5Ac residue was more susceptible to NDV-Sialidase than the branched

Neu5Ac residue. By utilizing this nature of NDV-Sialidase, we obtained a partially desialylated L-hyo (designated NDV-L- hyo) when the intact L-hyo was digested with NDV-Sialidase as described under “Experimental Procedures.” About 44% of the Neu5Ac residues present in the original L-hyo remained unhy- drolyzed, and as judged from the disappearance of the H-3,, resonance at 1.78 ppm in the ‘H NMR spectrum of the partially desialylated L-hyo, it was concluded that almost all of the unbranched Neu5Ac residues in L-hyo were removed under such conditions of digestion. Since L-hyo was shown to contain approximately equimolar amounts of the branching and un- branching Neu5Ac residues on methylation analysis, the results indicated that about 10% of the branching Neu5Ac residues were hydrolyzed. The structural relationship between the Neu5Ac and Fuc substituents on individual antennae was further elucidated by FAB-MS analyses of permethylated glycans released by PNGase F from intact and NDV-L-hyo.

FAB-MS Analysis of the Glycans Derived from Intact L- and NDV-L-hyosophorin-As shown in Fig. 7A, although a small proportion of the intact glycan sample gave non-fucosylated, non-sialylated terminal sequence ions, viz. ((Gal)z(GlcNAc)l)+ (mlz 668) and ((Gal)3(GlcNAc)2)+ (mlz 1117), the majority of the non-reducing ends are monosialylated. Thus, the major fragment ions observed are ((Neu5Ac)1(Gal)z(GlcNAc)l)+ (mlz 1029) and ((Neu5Ac)l(Gal)4(GlcNAc)z)+ (m/z 1682). Consistent with the data obtained on the desialylated glycans (see above), ((Neu5Ac)l(Gal)l(GlcNAc)l)+ (m/z 825), ((Ne~5Ac)~(Gal),- (GlcNAc)l)+ (mlz 1233), and ((Neu5Ac)l(Gal),(GlcNAc)z)+ (mlz 1886) are also present, reflecting the (Gal),-Gal-GlcNAc- heterogeneity where n is mainly 1 but can also be 0 or 2. As before, a portion of the major terminal sequence carries a single fucose, affording ions at mlz 1203 and 1856 corresponding to ((Fuc)l(Neu5Ac)l(Gal)z(GlcNAc)l)+ and ((FucIl (Neu5Adl- (Gal)4(GlcNAc)z)+, respectively. Interestingly, non-sialylated, fucosylated antennae are not observed which may simply re- flect the low level of fucosylation (averaging 1.9 moVmol pentaantennary glycan) coupled with the low level of non-sialylated antennae (averaging 4.6 mol of Neu5Admol pentaantennary glycan). Di- or tri-sialylated fragment ions were also not detected.

After treatment with NDV-sialidase of the intact L-hyo, the product of which was deduced by NMR data to have almost completely removed Neu5Ac residues attached to terminal Gal (see above), most fragment ions attributed to monosialylated antennae, e.g. mlz 1029, 1203, 1233, 1682, 1856, and 1886 were still present in the FAB-spectrum (Fig. 7B), indicating that these structures contain NDV-sialidase-resistant Neu5Ac residues attached to internal Gal. On the other hand, the en- hanced intensity of the ion at m / z 668 ((Gal)z(GlcNAc)l)+ relative to mlz 1029 ((Neu5Ac)l(Gal)z(GlcNAc)l)+, as well as the appearance of signals at m / z 842 ((Fuc)l(Gal)z(GlcNAc)l)+, 872 ((Gal)3(GlcNAc)l)+, and 1321 ((G~~),(G~CNAC)~)+, 1495 ((Fucll- (Gal)4(GlcNAc)z)+, and 1525 ((Gal),(GlcNAc)z)+ which are ab- sent in the spectrum of permethylated intact L-hyo glycan (Fig. 7A), suggests that NDV-sialidase-susceptible Neu5Ac residues have been removed from the sialylated parents of these structures. Taken together, the data are consistent with the presence in the intact L-hyo of both types of monosialylated terminal sequences.

When one combined these results with the information for the branching structures in asialo L-hyo, the structures shown below were proposed for the major peripheral forms of the intact L-hyo (Schemes 3 and 4):

CONCLUDING REMARKS

From the data presented above, the following overall structure is proposed for the pentaantennary glycan chains of the 0.

Highly Branched N-Linked Pentaantennary Glycans

1682

8769

50

40

30

20

0 , I , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , . , , , , , , , , , , , , , , , . 13'50 14100 ld50 Id00 1150 1 d O O 16150 17100 1350 1 d O O 18150 1400 1450 M / k

FIG. 7. FAB-spectra of permethylated intact (A) and NDV sialidase-treated ( B ) glycans released from L-hyo. Ions at 14 mass units below those assigned correspond to their undermethylated counterparts. Loss of a MeOH moiety (32 units) was observed for the major A-type ions. Peaks marked with x in B correspond to "detergent-like" impurities. The additional signals clustered around m l z 1500 are characteristic of fragment ions resulting from multiple cleavages with n l z 1481 corresponding to ((HO)l(Fuc),(Gal),(GlcNAc)Z)+.

8770 Highly Branched N-Linked Pentauntennary Glycans

latipes hyosophorin (Fig. 8). The glycan structure established and depicted in Fig. 8 is novel and has a number of crucial features: ( a ) the presence of highly branched poly-N-acetyllactosamino pentaantennary structures; ( b ) the presence of a P-galactosylated Lewis X antigenic epitope, GalP1+4GalPl-. 4(Fucal-3)GlcNAcPl+; ( c ) the presence of a P-galactosylated sialyl Lewis X structure, Galp1-.4(NeuSAca2+3)Gal~l-. 4(Fucal-3)GlcNAcf31+; ( d ) the presence of Galpl-4Galpl-t and Galpl-t4GalPl+4Gal/314 as the major and minor groupings, respectively; ( e ) the presence of the branched Gal residues, ~4GlcNAc~l-.3(Gal~l+4)Gal~l~.

L-hyosophorin molecules from 0. melastigma (22) and 0. latipes are similar in having poly-N-acetyllactosaminyl core glycan structures but differ in the structure of their peripheral portions which are mostly digalactosylated in 0. Zatipes but trigalactosylated in 0. melastigma. Moreover, a portion of 0. latipes hyosophorin is fucosylated and sialylated while 0. melastigma is not fucosylated. I t should be emphasized that each family of L-hyosophorin molecules having N-linked large glycan units is homogeneous with respect to core branching and contains only tetra- or pentaantennary chains, depending on animal species, although extensive microheterogeneity occurs in their outer chain moieties (0. melastigma hyosophorin (22), tetraantennary; Paralichthys olivaceus (38) and 0. latipes hyosophorin, pentaantennary). Thus, hyosophorin molecules are uniquely N-giycosylated in vivo with no heterogeneity in antenna while most of the animal asparagine-linked glycopro-

*NeuSAca2-.3Gal~l\, 4

3 4

3

Gal61

kNeuSAca2 ir GlcNAcBl-,

=Fucal SCHEME 3.

*NeuSAca2+3Gal~l *NeuSAca2-.3Galfll\ 4 4

Galp l , , 3 4 3 \4

GalBl

*NeuSAcdfl GlcNAcf31 GlcNAc@l-.

*Fucalf 2Fucal ir 3 3

SCHEME 4.

+NeuAcfl2+3Ga1[31\ *Fucf l I~ 4 3

teins are known to show heterogeneity with respect to the antenna structures (bi-, tri-, and tetraantennary) of their glycan chains. P-Linked peripheral Gal clusters which are unusu- ally branched at galactose residues have been recently identified in hyosophorin (39) isolated from Bufojaponicus (Japanese toad), indicating that such glycan structures are not confined to a limited number of fish species but are likely to be more widespread in the animal kingdom.

Expression of diverse types of epitopes on the bulky multiantennary structures which are ubiquitous in hyosophorin molecules suggests that they may possibly play important roles in regulating the cell-cell interactions of blastomeres at certain developing stages of embryos by either inhibiting or mediating through the interactions of developing cells and L-hyosophorin molecules. A portion of the L-hyosophorin molecules was found to undergo developmental stage-specific de-N-glycosylation by the action of peptide.N-glycanase to form free glycan and post- translationally remodified (Asn + Asp) peptide (40, 41). Our finding of this developmental stage-specific change of L-hyosophorin during early embryogenesis suggests that the presence of L-hyosophorin molecules is essential for the normal development of early embryos. Identification and detailed structures of the putative receptor molecules on the developing cells during early embryogenesis and their roles in determining normal development remain to be investigated.

Although previous studies a t a submicro level using radio- isotope-labeled materials have implied the presence of bulky multiantennary glycan structures, this and the previous re- ports (22, 38) represent the first comprehensive elucidation of such glycan structures by employing chemical (periodate- Smith degradation and hydrazine-nitrous acid depolymerization), biochemical (endo-P-galactosidase digestion of chemical degradation products), and instrumental methods (FAB-MS and 1D and 2D ‘H NMR). It should be noted that digestion with endo-P-galactosidase, which is conventionally carried out in the structural elucidation of poly-N-acetyllactosaminyl glycans, was not useful for the intact L-hyosophorin of both 0. latipes and 0. melastigma because they showed complete resistance to digestion although they had poly-N-acetyllactosamino skeletal structures. It should again be pointed out that Smith degradation and hydrazinolysishitrous acid deamination methods have been proved to be essential and effective to establish pre-

tNeuAca2+3Gal~1-.4GalPl (-4GlcNAc~1+3Gal~l),, +4GlcNAc(3I 3 3

+NeuAcaZ” tFucal’

+NeuAca2+3GaI[31\ *Fucfllr 4 3 \ 6 Asp I

rNeuAca2+3Galf31+4GaIPl (+4GlcNAc~1-.3Galpl),, +4GlcNAcP1&4 Manal 3 3

Ala I

Ala +NeuAca2+3Gal~lL +Fucalb I

6 I I

iNeuAca2’ 2Fucal’ / \ 4 +NeuAca2-.3Gal~1~4GaIpl (+4GlcNAc~1-.3Gal~l), -4GlcNAcPl

3 Ser

3 M a n ~ 1 - . 4 G l c N A c p l ~ 4 G I c N A c ~ ~ l ~ ~ ~ ~ 3 +NeuAca2’ 2Fucfll’’

+ N e u A c a 2 + 3 G a l ~ 1 ~ 4 G a l ~ l (-4GlcNAcP1+3GalPl), -.4GlcNAcP1

eNeuAcay 2FucaI’ 3 3

Manal -NeuAcf12-.3Gal()lL

4 zNeuAca2+3Galfil+4Galpl (+4GlcNAcpl+3Gal~l), -4GlcNAcPl

3 3 +NeuAcflZ? +Fucal/”

Gln I

Thr I

Val I

Scr

p, q, r, s, t t 0, lhe mean value of (ptqtrts+t) = 1.9.

FIG. 8. The overall structure of the 0. latipes L-hyo. The average number of sialic acid residues was about 4.6 moL’molecule of L-hyo.


cisely the structures of large glycan chains of the type described Hakomori, S., and Paulson, J. C. (1990) Science 250, 1130-1132 herein. 19. Walz, G., Aruffo, A,, Kolanus, W., Bevilacqua, M., and Seed, B. (1990) Science

The proposed structures as depicted in Fig. 8 are biosyntheti- 20. Sawada, R., Lowe, J . B., and Fukuda, M. (1993) J. Biol. Chem. 268, 12675- 250, 1132-1135

cally consistent with what is understood about the biosynthesis 12681 of N-linked multiantennary glycan chains (42). Thus, although 21. Inoue, Y. (1993) in Polysialic Acid: From Microbes to Man (Roth, J., Rut-

N-glycans with the trisubstituted a-Man structure 22. Taguchi, T., Seko, A,, Kitajima, K., Inoue, s., Iwamatsu, T., Khoo, K.-H., Mor-

GlcNAc81~6(GlcNAc~l-4)(GlcNAc~l-~2)Mana have to date 23, Aminoff, D, (1961) Biochem, J , 81, 384-392

it is more likely that GlcNAc-transferase VI activity may be 1433 more widespread than has been presently recognized.

ishauser, U., and Troy, F. A., eds) pp. 171-181, Birkhauser Verlag, Basel

r is , H. R., Dell, A., and Inoue, Y. (1993) J. Biol. Chem. 268, 2353-2362

been found Only in hen ovomucoid beside flounder hyosophorin, 24. Uchida, Y., Tsukada, Y., and Sugimori, T. (1977) J. Biochem. (Tokyo) 82,1425-

25. Svennerholm, L. (1963) Methods Enzymol. 6, 4 5 9 4 6 2 26. Dubois. M.. Gilles, K. A.. Hamilton. J . K., Rebers. P. A,. and Smith, F. (1956)

1.

2.

3.

4. 5.

6.

7.

8.

10. 9.

11. 12.

13.

14.

15. 16.

17. 18.

REFERENCES Fukuda, M., Spooncer, E., Oates, J. E., Dell, A., and Klock, J . C. (1984) J. Biol.

Spooncer, E., Fukuda, M., Klock, J. C., Oates, J. E., and Dell, A. (1984) J. Biol.

Fukuda, M., Dell, A,, Oates, J . E., and Fukuda, M. N. (1984) J. Bid. Chem.

Fukuda, M., Dell, A., and Fukuda, M. N. (1984) J. Biol. Chem. 259,47824791 Yurewicz, E. C., Sacco, A. G., and Suhramanian, M. G . (1987) J. Biol. Chem.

Muramatsu, H., Ishihara, H., Miyauchi, T., Gachelin, G., Fujisaki, T., Tejima,

Muramatsu, T., Gachelin, G., Nicolas, J. F., Condamine, H., Jakob, H., and

Maemura, K., and Fukuda, M. (1992) J. Biol. Chem. 267, 24379-24386 Fukuda, M. (1991) J. Biol. Chem. 266,21327-21330 Carlsson, S. R., Roth, J . , Piller, F., and Fukuda, M. (1988) J. Biol. Chem. 263,

Fukuda, M. (1985) Biochim. Biophys. Acta 780, 119-150 Fukuda, M., Koefler, H. P., and Minowada, J. (1981) Proc. Natl. Acad. Sci.

Kamada, Y., Arita, Y., Ogata, S., Muramatsu, H., and Muramatsu, T. (1987) U. S. A. 7 8 , 6 2 9 9 4 3 0 3

Hakomori, S., Fukuda, M., and Nudelman, E. (1982) in Teratocarcinoma and Eur J. Biochem. 163, 497-502

Embryonic Interactions IMuramatsu, T., Gachelin, G., Moscona, A. A,, and Ikawa, Y., eds) pp. 179-200, Japan Scientific Societies Press, Tokyo

Chem. 259, 10925-10935

Chem. 259,47924801

259,8260-8273

262,564-571

S., and Muramatsu, T. (1983) J. Biochem. (Tokyo) 94, 799-810

Jacob, F. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2315-2319

18911-18919

Pierce, M., and Arango, J . 11986) J. Bid. Chem. 261, 10772-10777 Youseti, S., Higgins, E., Daoling, Z., Pollex-mger, A,, Hindsgaul, O., and

Noguchi, S., and Nakano, M. (1992) Eur J. Biochem. 209, 883-894 Phillips, M. L., Nudelman, E., Gaeta, F. C. A., Perez, M., Singhal, A. K.,

Dennis, J . W. (1991) J. Biol. Chem. 266,1772-1782

27. Kitajima, K., Inoue, S., and Inoue, Y. (1989) Deu. Biol. 132, 544-553 28. Spiro, R. G. (1966) Methods Enzymol. 8, 26-52 29. Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J . Biol.

Chem. 264, 18520-18526 30. Dell, A,, Khoo, K.-H., Panico, M., McDowell, R. A., Etienne, A. T., Reason, A. J.,

31. Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. and Morris, H. R. (1994) Glycobiology, in press

32. Vliegenthart, J. F. G., Dorland, L., and van Halbeek, H. (1983)Adu. Carbohydr (1989) J. Biol. Chem. 264, 1623-1630

33. Iwasaki, M., Seko, A,, Kitajima, K., Inoue, Y., and Inoue, S. (1992) J. Biol. Chem. Biochem. 41,209-374

34. Kitagawa, H., Nakada, H., Kurosaka, A., Hiraiwa, N., Numata, Y., Fukui, S., Chem. 267,24287-24296

Funakoshi, I., Kawasaki, T., Yamashina, I., Shimada, I., and Inagaki, F. (1989) Biochemistry 28, 88914897

35. Woodward, J . R., Craik, D., Dell,A., Khoo, K.-H., Munro, S. L. A,, Clarke,A. E., and Bacic, A. (1992) Glycobiology 2, 241-250

36. Priem, B., Solokwan, J. , Wieruszeski, J.-M., Strecker, G., Nazih, H., and Mor- van, H. (1990) Glycoconjugate J . 7, 121-132

37. Nimtz, M., Martin, W., Wray, V., Kloppel, K.-D., Augustin. J., and Conradt, H. S. (1993) Eur J. Biochem. 213,39-56

38. Seko, A,, Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929

39. Shimoda, Y., Kitajima, K., Inoue, S., Wardrip, N. J . , Hedrick, J. L., and Inoue, Y. (1993) Glycoconjugate J. 10, 264

40. Seko, A,, Kitajima, K., Inoue, S., and Inoue, Y. (1991) Biochem. Biophys. Res. Commun. 180, 1165-1171

41. Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114

42. Brockhausen, I., Hull, E., Hindsgaul, O., Schachter, H., Shah, R. N., Michnick, S. W., and Carver, J . P. (1989) J. Biol. Chem. 264, 11211-11221

Anal. Chem. 28, 350-356

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