THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 18, Issue of September 25, pp. 5704-5717, 1974

Printed in U.S.A.

Structure of the O-Glvcosidicallv Linked J J

Carbohydrate Units of Fetuin*

(Received for publication, March 11, 1974)

ROBERT G. SPIRO~ AND VISHNU D. BHOYROO

From the Departments of Biological Chemistry and Medicine, Harvard Medical School, Elliott P. Joslin Research Laboratory, and Peter Bent Brigham Hospital, Boston, Massachusetts 02215

SUMMARY

Fetuin isolated from fetal calf serum was shown to con- tam 3 carbohydrate units which are attached to serine and threonine residues on the peptide chain and are distinct from the 3 asparagine-linked heteropolysaccharides previously re- ported to occur in this protein.

The 0-glycosidically linked saccharide units were obtained as reduced oligosaccharides after alkaline borohydride treat- ment of fetuin and as glycopeptides subsequent to pronase digestion of this protein.

Gel filtration and Dowex 1 chromatography of the oligo- saccharides resulted in the isolation of a tri- and a tetrasac- charide which were made up of sialic acid, galactose, and N-acetylgalactosaminitol residues and could be converted after selective removal of the sialic acid to the same disac- charide (galactosyl-N-acetylgalactosaminitol). Studies em- ploying periodate oxidation and glycosidase digestion indi- cated that the structure of the tetrasaccharide was N-acetyl- neuraminyl- (2 -+3) - P-D - galactopyranosyl- (l -+3)[N- acetyl- neuraminyl-(2 -+6)]-N-acetylgalactosaminiitol and that the trisaccharide differed only by the absence of the sialyl residue linked to the N-acetylgalactosaminitol.

Glycopeptides containing the alkali-labile carbohydrate units were purified on Sephadex and diethylaminoethylcellu- lose columns. In this manner peptides distinguished by a high proline content and containing 1 tetrasaccharide, 1 tri- saccharide, or 2 trisaccharides were obtained. Alkaline sulfite treatment of these glycopeptides indicated that 2 serine and 1 threonine residue in each fetuin molecule is involved in the attachment of carbohydrate units. The tetrasac- charide was found to be linked only to serine while attach- ment of trisaccharide units involved a serine and a threonine residue which were located in close proximity in the peptide chain.

Smith periodate degradation of sialic acid-free glycopep- tides resulted in removal of the galactose and yielded a prod- uct from which complete release of N-acetylgalactosamine

* This work was supported by Grant AM 17325 from the Na- tional Institutes of Health and by the Adler Foundation, R.ye, New York.

3 To whom reprint requests should be addressed at the Elliott P. Joslin Research Laboratorv. 170 Pilarim Road. Boston. Massa- chusetts 02215.

”

was accomplished by digestion with a-N-acetylgalactos- aminidase although no cleavage of this sugar was effected by the action of /3- N-acetylhexosaminidase. This indicated that the alkali-labile units of fetuin are attached to the peptide chain by a-D-N-acetylgalactosaminyl-(l +J)-serine (threo- nine) bonds.

Of the total carbohydrate of fetuin 21%, including all three of the galactosamine residues, was shown to be located in the 0-glycosidically linked units with the remainder occurring in the more alkali-stable N-glycosidically bound form.

The alkaline sulfite treatment employed in this investiga- tion produced in good yield the sulfonyl derivatives of the amino acids and amino sugar involved in the glycopeptide linkage. Since the cysteic acid and Lu-amino@-hydroxy- butyric acid obtained in this manner do not separate on the amino acid analyzer, a chromatographic procedure employing Dowex 1 was developed which permitted resolution of these two components.

Fetuin, the predominant glycoprotein of fetal calf serum, has been the subject of previous structural investigations from this laboratory (l-5). These studies have indicated that fetuin con- sists of a single polypeptide chain to which three heteropolysac- charide units made up of sialic acid, galactose, N-acetylglucosa- mine, and mannose are attached through asparagine residues. While these asparagine-linked carbohydrate units account for most of the 52 sugar residues of the fetuin molecule they do not provide for the location of the N-acetylgalactosamine which is also present as part of the protein. The galactosamine of fetuin may therefore be presumed to occur in a distinct, second type of saccharide unit.

It wasthe aim of the present investigation to obtain a more complete understanding of the carbohydrate of fetuin by char- acterizing the units among which the galactosamine residues are distributed and determining the nature of the bonds by which they are linked to the peptide chain. This information was ob- tained from a study both of reduced oligosaccharides formed dur- ing alkaline borohydride treatment of fetuin and of glycopeptides isolated from pronase digests of this protein.

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EXPERIMENTAL PROCEDURE

Preparation of F&in-Fetuin was isolated from pooled fetal calf serum by low temperature ethanol fractionation, as previously described (1).

Preparation of Reduced Oligosaccharides by Alkaline Borohydride Treatment of Fetuin-Approximately 2 g of fetuin were incubated

with 0.8 M NaBH4 in 0.1 N NaOH at 37” for 68 hours. At the end of the incubation the reaction mixture was placed in an ice bath, diluted with a lo-fold volume of cold water, and brought to pH 5 by the slow addition of 4 N acetic acid to decompose the excess NaBHd. The sample was then passed through a column of Dowex 50-X2, 200 to 400 mesh (H+ form), kept at 4’, and washed with 4 column volumes of 0.01 N formic acid. The combined effluent and wash was lyophilized and the boric acid volatilized as methyl borate in a vacuum rotator by several additions of methanol.

The dried material was then dissolved in 10 ml of 0.1 M pyridine acetate buffer at pH 5.0 and fractionated on Sephadex G-25 (fine) in a column (2.1 cm in diameter) fitted with a sintered glass plate and packed to a height of 130 cm, Elution was achieved with the 0.1 M pyridine acetate buffer at a rate of 12 ml per hour. Frac- tions of 5.4 ml were collected, and aliquots of these were analyzed by the anthrone (6) and resorcinol (7) reactions. Various frac- tions were pooled and the buffer removed by lyophilization.

Chromatography of Reduced Oligosaccharides on Dowex 1 -X2- The reduced oligosaccharides obtained by the filtration on Sepha- dex G-25 were further resolved by chromatography on a column (1.0 X 82 cm) of Dowex l-X2, 299 to 400 mesh (acetate form),

which had been equilibrated with 0.002 M pyridine acetate buffer at pH 5.0. The sample dissolved in this buffer was applied to the column, after which the column was washed with 80 ml of the same buffer. Chromatography was continued with a linear concentra- tion gradient consisting of 650 ml of 0.002 M pyridine acetate, pH 5.0, in the mixing chamber and 650 ml of 0.35 M pyridine acetate, pH 5.0, in the reservoir. A flow rate of 18 ml per hour was moni- tored, and fractions of 8 ml were collected. Analysis of the frac- tions by the anthrone and resorcinol reactions were performed. Peak fractions were pooled and dried by lyophilization.

Proteolytic Digestion of Fetuin-Fetuin (25 mg per ml) was di- gested at 37” with pronase (Calbiochem) in 0.15 M Tris-acetate buffer, pH 7.8, containing 1.5 mM calcium acetate in the presence of a small amount of toluene. The enzyme was added initially in an amount equal to 1% of the weight of fetuin and again at 24 and 48 hours in amounts equal to 0.5% of the substrate weight. The reaction mixture was readjusted to pH 7.8 with 1 M Tris prior to each addition of enzyme. Incubation was carried out for a total period of 96 hours- Maximal cleavage of peptide bonds as assessed bv the ninhvdrin reaction (8) was achieved by 72 hours.

Gel Filt&ion of Pgonase Digest-T%e lyophilized pronase digest was dissolved in 0.1 M pyridine acetate buffer at pH 5.0. After removal of a small amount of insoluble material by centrifugation the digest was fractionated in this buffer on a column of Sephadex G-50 (medium) fitted with a sintered glass plate and packed to a height of 80 cm. For digests of 0.5 to 1 g of fetuin the sample was applied in a lo-ml volume to a column 2.1 cm in diameter, and elution was achieved at 8 ml per hour with a fraction size of 5.0 ml, while digests of 4 to 5 g of fetuin were applied in a 30-ml volume to a 3.4.cm diameter column which was eluted at 25 ml per hour with the collection of 12-ml fractions. Appropriate tubes from these columns were pooled and dried by lyophilization.

To achieve better separation of galactosamine-containing glyco- peptides from those containing glucosamine the lower molecular weight glycopeptides obtained from the large (3.4-cm diameter) Sephadex G-50 column were further fractionated on Sephadex G-25 (medium). For this purpose the glycopeptides dissolved in 8 ml of 0.1 M pyridine acetate-at pH 5.0 were filtered on a column 2.1 cm in diameter and nacked to a height of 130 cm. Elution was achieved with 0.1 M pyridine acetatepH 5.0 buffer at a rate of 15 ml per hour while fractions of 5.2 ml were collected.

Elution from the Sephadex columns was monitored by perform- ing the anthrone, resorcinol, and ninhydrin reactions on aliquots of the collected fractions.

Chromatography of Glycopeptides on DEAE-cellulose-The galac- tosamine-containing glycopeptide fractions obtained by filtration on Sephadex G-25 were lyophilized and further chromatographed on a column (1.25 X 80 cm) of DE52 microgranular cellulose fitted with a sintered glass plate and equilibrated with 0.002 M pyridine

acetate buffer at pH 5.0. After application of the sample an addi- tional 128 ml of the same buffer were passed through the column. Elution was then achieved by the application of two successive linear concentration gradients. The first gradient consisted of 420 ml of the 0.002 M pyridine acetate buffer, pH 5.0, in the mixing chamber and an equal volume of 0.11 M pyridine acetate, pH 5.0, in the reservoir while the second gradient was made up of 450 ml of the 0.11 M pyridine acetate buffer at pH 5.0 in the mixing cham- ber and an equal volume of 0.30 M pyridine acetate, pH 5.0, in the reservoir. A flow rate of 15 ml per hour was maintained, and fractions of 8.5 ml were collected, which were analyzed by the anthrone and resorcinol reactions. Fractions constituting the glycopeptide peaks were pooled and dried by lyophilization.-

Electrophoresis of Glucopeptides-The electronhoresis was carried out in a water-cooled plate apparatus for 5 hours at 19 volts per cm on Whatman No. 1 paper. The buffer used was pyridine-acetic acid-water (25:1:225), pH 6.4, and the glycopep- tides were detected by staining with ninhydrin (2).

Paper Chromatography-Chromatography was carried out on Whatman No. 1 paper by the descending technique. The follow- ing solvent systems were employed: pyridine-ethyl acetate-water- acetic acid (5:5:3:1) with pyridine-ethyl acetate-water (11:40:6) in the bottom of the chromatography chamber (9) (System A) for neutral sugars, hexosamines, and oligosaccharides; l-butanol- ethanol-water (10:1:2) (System B) for neutral sugars, polyols, and oligosaccharides; 1-butanol-acetic acid-water (4:1:5), upper phase (System C), for amino polyols and amino acids; l-butyl- acetate-acetic acid-water (3:2:1) (System D) for sialic acids and oligosaccharides; and I-butanol-pyridine-water (6:4:3) (System E) for oligosaccharides. Neutral sugars, hexosamines, polyols, and oligosaccharides were located by the silver nitrate staining procedure (10, 11) while amino polyols and amino acids were detected with a ninhydrin reagent (2).

Analyses of Sugar Components-For the determination of the neutral sugars the glycopeptides and oligosaccharides were hydro- lyzed in 1 N HCl for 4 hours at 100” in sealed tubes, and the hy- drolysates were passed through coupled columns of Dowex 50 and Dowex 1 (6). The galactose and mannose content of the neutral sugar fraction was determined by means of automated borate complex anion exchange chromatography (Technicon system) at a column temperature of 45” with an elution gradient previously described (12).

Amino sugars and amino sugar alcohols were determined after hydrolysis in 4 N HCl at 100” for 6 hours and adsorption and elu- tion from a Dowex 50 column (6). Analysis for total hexosamines was performed by the Elson-Morgan reaction (13), while the separate determination of hexosamines and hexosaminitols was performed on the amino acid analyzer (Technicon) employing a citrate-borate buffer, pH 5.28 (0.2 M in sodium ions and 0.35 M in boric acid), at a column temperature of 60”. Resolution of galac- tosaminitol, glucosaminitol, glucosamine, and galactosamine was achieved by this type of chromatography (12).-

Total sialic acid was measured with the resorcinol reagent (7) while the free sugar was estimated by the thiobarbituhc acid reaction (14).

Sialic Acid Removal-Sialic acid-free fetuin, sialic acid-free glycopeptides, and sialic acid-free oligosaccharides were prepared by hydrolysis in 0.05 N sulfuric acid at 80” for 1 hour. The re- leased sialic acid was removed from the fetuin by dialysis and from the glycopeptides and oligosaccharides by adsorption on a column of Dowex 1 (formate form) (6). For the purpose of identi- fication the sialic acid was eluted from the Dowex 1 column with 0.3 N formic acid.

N-Acetylation of Glycopeptides-Glycopeptides were N-acetyl- ated with acetic anhydride in sodium acetate as previously de- scribed (15).

Periodate Oxidation-Oxidation of glycopeptides and oligo- saccharides, both native and sialic acid-free, was carried out for 24 hours at 4” under the conditions previously described (16) with the use of a lo-fold molar excess of sodium metaperiodate to total sugar residues. The reaction was terminated by the addition of ethylene glycol in a lo-fold excess over the periodate employed. The oxidized samples were then reduced with sodium borohydride at pH 8.0 as previously reported (16) employing a 35-fold excess of sodium borohydride over the periodate used. After acidifica- tion the reduced samples were passed through coupled columns of Dowex 50-X2,200 to 400 mesh (H+ form), over Dowex l-X8,200 to

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400 mesh (formate form), to remove sodium and iodate ions. from Dr. B. Weissmann, University of Illinois College of Medicine. The effluent and wash from these columns were lyophilized, and The enzyme concentration during these digestions was 0.9 unit per the boric acid was then volatilized as methyl borate. Any glyco- ml. nentides adsorbed on the Dowex 50 column were eluted with 1.5 N Glvconentides obtained after Smith periodate degradation NHbOH at 4’ and lyophilized. Sialic acid-containing glycopep- tides or oligosaccharides were eluted from the Dowex 1 resin (with- out any displacement of iodate) by means of 2 N formic acid and then lyophilized. Controls were treated in the same manner except that the sample was added to the periodate after the ethylene glycol. The lyophilized effluent and wash and the column eluates of each sample were combined and analyzed for their sugar and sugar alcohol content after acid hydrolysis.

The amino polyols obtained after acid hydrolysis of the reduced periodate oxidation products of the oligosaccharides released by alkaline borohydride treatment were measured and identified on the Technicon amino acid analyzer, with the use of a conventional gradient and employing alanine as an internal standard (12).

4 hours) while n-galactosaminitol was prepared by reduction and deacetvlation of N-acetvl-n-salactosamine. L-Threosaminitol

Standard amino polyols were obtained in the following manner. n-Arabinosaminitol was prepared from n-arabinosamine (a gift of Dr. D. Horton, Ohio State University) by N-acetylation, sodium borohvdride reduction, and deacetylation (4 N HCl, loo”,

weresimilarly incubated with p-N-acetylhexosaminidase-prepared from jack bean meal by DEAE-Sephadex A-50 and Cm-Sephadex C-50 chromatography (21). This enzyme was present at a con- centration of 4.0 units per ml.

At the end of the galactosidase and hexosaminidase digestions the released monosaccharides were desalted and separated from enzyme by passage through coupled columns of Dowex 50-H+ and Dowex 1 (formate) and a small column of charcoal-CeZite (6). After identification of the liberated sugars by paper chroma- tography the free galactose was determined by borate complex anion exchange chromatography while the released N-acetyl- hexosamines were quantitated by the Morgan-Elson reaction (22).

All of the enzymatic digestions were accompanied by controls containing the enzyme or the substrate separately. Small amounts of toluene were added to each incubation to prevent bacterial growth.

Oligosaccharides obtained after alkaline borohydride treatment (0.8 M NaBHI-0.1 N NaOH. 37”. 72 hours) of fetuin and fetuin

Separation of Reduced Oligosaccharides on Sugar Analyzer-

was prepared by periodatk ox%ation of N-acetylchondrosinitol followed by sodium borohydride reduction of the product, acid hydrolysis, and separation on Dowex 50. (The N-acetylchon- drosinitol used was obtained by N-acetylation and sodium boro- hydride reduction of chondrosine.) Serinol was obtained from Nutritional Biochemicals. N-Acetylthreosaminitol was pre- pared by acetylation (15) of the threosaminitol.

Formaldehyde was determined by the chromotropic acid reac- tion (17) after oxidation of samples with sodium metaperiodate at pH 4.5, room temperature, for 1 hour.

Smith Periodate Degradation-Sialic acid-free fetuin was sub- mitted to Smith degradations in a manner previously reported (5). Each degradation consisted of periodate oxidation, sodium

borohydride reduction, and mild acid hydrolysis (0.05 N sulfuric acid, SO”, 1 hour). Dialysis was performed after the reduction and hydrolysis steps, and the protein was then lyophilized and a sample analyzed.

For Smith degradation of glycopeptides and oligosaccharides the periodate oxidation and sodium borohydride reduction were performed as described in the preceding section of this paper, and the mild acid hydrolysis was then performed. The hydrolysates of the glycopeptides were passed-through coupled columns of Dowex 50-X2 (H+) and Dowex l-X8 (formate) while those of the oligosaccharides were passed only t‘hrough Dowex 1. The de- graded glycopeptides were recovered from the Dowex 50 by elution with 1 M pyridine acetate, pH 5.0, followed by lyophilization while the oligosaccharides were obtained in the effluent and wash from the Dowex 1 column. The Dowex 1 columns were eluted with 0.3 N formic acid when the content of sialic acid in the oxidized glyco- peptides was to be assessed.

Digestion with Glvcosidases-Incubations were performed at 37” with various glycosidases under the conditions specified.

Neuraminidase from Vibrio cho2erae (General Biochemicals) at a concentration of 2.5 X 1OP’ unit per ml (1 unit releases 1 rmole of N-acetylneuraminic acid per min) was incubated with glycopep- tides and oligosaccharides (0.30 Mmole per ml) in 0.1 M sodium acetate buffer at pH 5.5 in the presence of 0.005 M CaCb. The released sialic acid was determined by the thiobarbituric acid reaction.

Oligosaccharides and glycopeptides were incubated with 01- and p-galactosidases at substrate concentrations of 1.5 pmoles per ml. Incubation with &galactosidase from Escherichia coli (Worthing- ton) was nerformed in 0.15 M sodium nhosphate buffer at pH 7.0

glycopeptides-were desalted by ‘passage through Dowex 50 and freed of boric acid by volatilization as methyl borate. For the purpose of quantitative estimation they were resolved before and after sialic acid removal as well as after Smith periodate degrada- tion by automated borate complex anion exchange chromatog- raphy (Technicon system) at a column temperature of 60” with the extended elution gradient previously described (23) and rham- nose as an internal standard.

Alkali and Alkaline SulJite Treatment oj Glycopeptides-The samples were treated with 0.1 N NaOH or 0.1 N NaOH containing 0.5 M Na$Os at 37” for varying periods of time (24 to 96 hours), after which they were acidified, taken to dryness, and hydrolyzed in constant boiling HCl under nitrogen for 28 hours at 105”.

Separation of Sulfonyl Derivatives Obtained by Alkaline Sul$te Treatment-An aliquot of the hydrolyzed alkaline sulfite-treated glycopeptides was placed on the amino acid analyzer (Technicon) to determine the decrease in serine and threonine as well as to quantitate the amount of sulfonylhexosamine (24) formed by the reaction. Since cysteic acid and a-amino-p-sulfonylbutyric acid do not separate on the amino acid analyzer, both appearing in the void volume, these components were resolved by chromatography of a portion of the sample on Dowex 1 at pH 3.8 in the manner already described (12) employing the Technicon ‘analytical system and using 0-phosphoserine as an internal standard.

For the purpose of paper chromatographic identification the hvdrolvzed alkaline sulfite-treated glvconeptides were desalted by passage through small coupled columns of Dowex 50-X4,200 to 400 mesh (H+ form) and Dowex l-X8, 200 to 400 mesh (formate form). The effluent and wash from these columns were lyophi- lized. Amino acids adsorbed on Dowex 50 were eluted at 4’ with 1.5 N NHkOH while those remaining on Dowex 1 were recovered by elution with 4 N formic acid. After separate lyophilizations the column eluates and effluent plus wash were combined and applied to paper for chromatography.

Standard ol-amino-p-sulfonylbutyric acid was prepared from N-benzyloxycarbonyl-L-alanyl-L-threonine amide (Cycle) by 0-acetylation, p elimination, sulfite addition, and acid hydrolysis. The acetylation was performed by adding equal volumes of dry pyridine and acetic anhydride to the dry compound and heating in a sealed tube at lC!@ at a concentration of 25 rmoles per ml for 8 hours. After removal of the excess reagent under anhydrous conditions the acetylated sample was shaken at room temperature with triethvlamine-methanol (4:96, v/v) for 4 hours at a concen-

.

in the presence of 0.02 M MgCL and 80 units per ml of the enzyme. tration of i2 rmoles per ml. ‘Subsequently the solvent was re- b-Galactosidase prepared from jack bean by DEAE-Sephadex moved in a vacuum rotator at 40”, and 0.5 M NanSO in 0.1 N A-50 chromatography (18) was digested in 0.15 M sodium citrate Na&Oz was added to permit sulfite addition to take place by buffer at pH 4.5 at a concentration of 0.8 unit per ml, while or-ga- shaking the sample (I5 pmoles per ml) at 37” for 24 hours. At lactosidase prepared from green coffee beans (Santos) (19) was the end of this incubation the sample was acidified with HCI, incubated at a concentration of 0.07 unit per ml in 0.2 M sodium dried in a vacuum rotator at 40”, and then hydrolyzed in constant acetate buffer at nH 5.0. boiling HCl in a sealed tube under nitrogen for 24 hours at 105”.

Fetuin (16 mg per ml) and glycopeptides (1.2 pmoles per ml) ob- Following removal of the acid in vacui the hydrolyzed sample tained after Smith neriodate degradation were incubated in 0.1 was dissolved in water. titrated to DH 4.0 with 0.1 N NaOH, and M sodium citrate bu’ffer at pH 4.5 with a-N-acetylgalactosamini- placed on a column of Dowex l-X8, 200 to 400 mesh (formate dase from pig liver (20) which was obtained as a generous gift form) previously equilibrated with 5 mM pyridine formate buffer

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at pH 4.0. After the sample had passed through the column it was washed with 8 volumes of the 5 rnM buffer, and the a-amino- &sulfonylbutyric acid was then eluted with 4 N formic acid. Lyophilization of this eluate gave in about 20% yield a salt-free product which upon paper chromatography in Solvent System C migrated as a single component an RA,, of 0.55 (RA., of cysteic acid = 0.42). While the compound eluted together with cysteic acid on the amino acid analyzer, it was well separated from the latter compound by Dowex l-X8 chromatography at pH 3.8 (12).

Sulfonylhexosamine (24) was prepared by alkaline sulfite treat- ment of IV-acetylchondrosine under the same conditions as em- ployed for the glycopeptides. The compound emerged just after the cysteic acid on the amino acid analyzer and migrated with an RA., of 0.87 upon paper chromatography in Solvent System C.

Amino Acid Analyses-Glycopeptides were hydrolyzed for 24 hours in glass-distilled constant boiling HCl in sealed tubes under nitrogen at 105”, and their amino acid composition was deter- mined with the Technicon amino acid analyzer.

NHz-terminal Analyses-The NH*-terminal residues of the glycopeptides were determined by the subtractive Edman pro- cedure as described by Dopheide et al. (25) with the exception that the coupling step was performed in a pyridine-triethylamine- water (60:3:37) buffer (26) for a period of 2% hours.

RESULTS

Isolation and Characterization of Reduced Oligosacchorides-Fil- t&ion of the Dowex 50 effluent and wash of the alkaline boro- hydride-treated fetuin on Sephadex G-25 resulted in the separa- tion of four distinct carbohydrate-containing peaks (Fig. 1). The material excluded from the gel (Peak A) constituted the major peak and contained glycopeptides with a sugar composi- tion consisting of sialic acid, galactose, mannose, and glucosamine. Peaks B and C were devoid of peptide material and contained as their principal sugar components sialic acid, galactose, and galac- tosaminitol. Small amounts of mannose and glucosamine were found in Peak B but not C. Peale D contained only sialic acid which on the basis of its equal reactivity in the resorcinol reaction and the thiobarbituric acid assay without prior hydrolysis, as well as its migration on paper chromatography, was shown to be in the free form (probably representing this sugar cleaved from saccharide units during passage through Dowex 50 (H+)).

On the basis of these analyses it was presumed that Peak A contained the asparagine-linked carbohydrate units of fetuin (2,

TUBE NUMBER (54mt)

FIG. 1. Gel filtration on Sephadex G-25 of alkaline borohydride- treated fetuin. The sample, representing the Dowex 50 effluent and wash from 1.8 g of fetuin, was placed on a column (2.1 X 130 cm) equilibrated with 0.1 M pyridine acetate buffer, pH 5.0. Elu- tion was achieved with this buffer, and the diagram shows sialic acid analyses by the resorcinol procedure and hexose determina- tions by the anthrone method. Lettered areas designate tubes which were pooled for further study.

3,5) while the material in Peaks B and C was made up of reduced oligosaccharides resulting from the release of alkali-labile sac- charide units of this protein.

To achieve further purification of these oligosaccharides the material in Sephadex G-25 Peaks B and C was chromatographed on Dowex l-X2 to yield 2 distinct oligosaccharides (Fig. 2). Peak C yielded a single component which was eluted with a buffer concentration of 0.12 M while the major saccharide peak resulting from chromatography of Peak B emerged when the gradient had reached a concentration of 0.29 M.

Compositional analyses of these 2 oligosaccharides indicated that the less acidic component, C (64 to 69), was a reduced tri- saccharide consisting of equimolar amounts of sialic acid, galac- tose, and N-acetylgalactosaminitol while the more acidic com- pound, 13 (142 to 148), was a tetrasaccharide in which sialic acid, galactose, and N-acetylgalactosaminitol occurred in a molar ratio of 2 : 1: 1 (Table I). The difference in size between these 2 oligo- saccharides resides in a single sialic acid residue and accounts for their distinct elution during the Sephadex G-25 filtration (Fig. 1). Paper chromatographic examination of the sialic acid released by mild acid hydrolysis of both oligosaccharides revealed only the presence of N-acetylneuraminic acid and indicated that the small amount of N-glycolylneuraminic acid which is present in fetuin (1) does not reside in these saccharide units.

Upon paper chromatography in Solvent System A the tetra- saccharide and trisaccharide each migrated as a single component with an RGal of 0.23 for the former and an RGal of 0.51 for the latter. In Solvent System D the R oai of the tetrasaccharide was 0.13 while that of the trisaccharide was 0.38.

The molar ratio of trisaccharide to tetrasaccharide calculated from the yield obtained by Dowex 1 chromatography (Table I) was 3.64, indicating that 22% of the oligosaccharides were present in the form of the tetrasaccharide.

Structural Studies on Reduced Oligosaccharides-After removal of the sialic acid from either the tetrasaccharide or the trisac- charide a disaccharide consisting of galactose and galactosamini- to1 was formed which migrated with an RGal of 0.57 upon paper chromatography in Solvent System B (Fig. 3), 0.86 in Solvent

FIG. 2. Chromatography on Dowex l-X2 of oligosaccharide fractions obtained by gel filtration of alkaline borohydride-treated fetuin. The samples, representing Sephadex G-25 Peak C (upper) and Peak B (lower), were placed on a column (1.0 X 82 cm) equili- brated with 0.602 M pyridine acetate buffer, pH 5.0. After elution with this buffer a linear gradient was started as described in text from 0.092 M (tube 11) to 0.30 M (tube 152). The tubes making up the principal peak from each separation were pooled for further study and designated C (64 to 69) and B (142 to 148), respectively.

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TABLE I Composition of oligosaccharides obtained from alkaline

borohydride-treated fetuin by Dowex l-X& chromatography of Sephadex G-.&5 peaks

OligosaccharidtJ Component

C (64-69) B (142-148)

&t?nolcs/1oo n&b

N-Acetylneuraminic acid. _ _ _ . . . 150 (1.03)” 207 (2.05) Galactose........................ 146 (1 .OO) 107 (l-06) N-Acetylgalactosaminitold. _ . . 146 (1.00) 101 (1.00) Galactosyl-N-acetylgalactos-

aminito18. . . . . 168 (1.15) 117 (1.16)

0 Letters B and C in oligosaccharide designations refer to the Sephadex G-25 peaks which were chromatographed on Dowex 1 to yield peak eluting in the tubes indicated by the hyphenated numbers (Fig. 2).

b Analysis expressed per weight of oligosaccharide which was determined from the sum of the saccharide residues in each peak. The yield of oligosaccharide B (64 to 69) was 31.4 mg while that of C (142 to 148) was 10.9 mg.

c Numbers in parentheses represent molar ratios relative to N-acetylgalactosaminitol.

d Meaeured as the deacetylated sugar. c Meaeured directly by borate complex anion exchange chro-

matography after selective removal of the sialic acid residues (see Fig. 4).

FIG. 3. Paper chromatography of tetrasaccharide (2) and tri- saccharide (3) after removal of sialic acid. The oligosaccharides were obtained as Dowex 1 Peaks B (142 to 148) and C (64 to 69) (Fig. 2), and the sialic acid was released by mild acid hydrolysis. Standards, 1 and 4. Chromatography was performed for 7 days in Solvent System B, and the paper was stained with the silver reagent. Lac, lactose; Cal-GQ~NAc-H,, galactoeyl-N-acetylga- lactosaminitol; Gal, galactose.

System A, and 0.84 in Solvent System E. The component formed by removal of sialic acid eluted during borate complex anion exchange chromatography at a considerably earlier time than either the trisaccharide or tetrasaccharide from which it was

derived (Fii. 4) and was obtained in essential quantitative yield (Table I).

Incubation of the oligosaccharides with 77. ch0Zera.e neura minidase resulted in almost complete release of sialic acid in 48 hours (93% cleavage from the trisaccharide and 91% from the tetrasaccharide). No galaetose was released when the tri- and tetrasaccharides were digested with either E. eoZi 8-galactosidase or coffee bean o-galactosidase for 96 hours. However, when either of these oligosaccharides, after removal of their sialic acid, were incubated with j3-galactosidase for that period of time com- plete cleavage took place, and equimolar amounts of galactose and N-acetylgalactosaminitol were formed. In contrast, treat- ment of the sialic acid-free oligosaccbarides with cr-galactosidase failed to effect any scission of galactose.

Periodate oxidation of the tetrasaccharide as well as the tri- saccharide resulted in complete destruction of the sialic acid res- dues, as determined by paper chromatography of the acidic components released by the mild acid hydrolysis (Table II). Such chromatographic examination was essential as oxidation products of sialic acid react with the various calorimetric reagents used in the determination of this sugar (5). No loss of galactose was evident upon oxidation of either the tetra- or trisaccharide although the galactosaminitol was lost and quantitatively con- verted to threosaminitol (Table II). This amino sugar alcohol was identified by its time of elution from the amino acid analyzer on which it was well separated from galactosaminitol as well as arabinosaminitol and serinol which are the other possible reduced products of galactosaminitol oxidation (Fig. 5). Upon paper chromatography in Solvent System C the amino polyol product moved to the same position as standard threosaminitol (RG~w-IS,, = 1.35) while arabinosaminitol migrated 1.18 and serinol 1.67 relative to the N-acetylgalactosaminitol.

When the reduced product of periodate oxidation of either the tetrasaccharide or trisaccharide was submitted to mild acid hy- drolysis (Smith degradation), a substance was formed which mi- grated as a single component on paper chromatogr&phy more rapidly than galactosyl-N-acetylgalactosaminitol (Fig. 6) and which yielded equimolar amounts of gala&se and threosaminitol on more vigorous acid hydrolysis. Treatment of this compound with jack bean &galactosidase resulted in its complete cleavage into equivalent amounts of galactose and N-acetylthreosaminitol. The latter sugar was identified by its migration to the same posi- tion as the standard N-acetylthreosaminitol (RG~IN = 1.75, RG~INA~ = 1.35) in Solvent System A. When the neutral re- duced products of Smith degradation of the tetra- and trisac- charides were chromatographed on the sugar analyzer the disaccharide (galactosyl-N-acetylthreosaminitol) emerged about 3 hours prior to the galactosyl-N-acetylgalactosaminitol (Fig. 7) and was found to be present in amounts approximately equal to the original oliiosaccharides.

Periodate oxidation of the disaccharide (galactosyl-N-acetyl- galactosaminitol) formed after removal of the sialic acid from either the tetra- or trisaccharide resulted in total destruction of the gala&se and the galactosaminitol with the formation after reduction of threosaminitol (Table II).

The results of the periodate oxidation indicate that in the tri- saccharide the sialic acid is linked to C-3 of the gala&se while the galactose in turn is attached to C-3 of the N-acetylgalactos- aminitol. In the tetrasaccharide the same sequence and linkages appear to prevail, and in addition an extra sialic acid residue is linked to this sialyl-galactosyl-N-acetylgalactosaminitl chain.

The formation of threosaminitol and close to 2 moles of formal- dehyde after periodate treatment of the tetrasaccharide indicates

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FIG. 4. Separation of tetrasaccharide (NAN-Gal-(NAN)- GalNAc-Hz), trisaccharide (NAN-Gal-GalNAc-He), and disac- charide (Gal-GalNAc-Hz) by borate complex anion exchange chro- matography on sugar analyzer (Technicon). The tetrasaccharide (Peak B (142 to 148) and trisaccharide (Peak C (64 to 69)) were

TABLE II Effect of periodate oxidation on oligosaccharides obtained

from alkaline borohydride-treated jetuin

Trisaccharideb DisaccharideC /

I moles/naolc oligosaccharide’

N-Acetylneuraminic acid.. 2.05 0 1.03 0 Galactose................. 1.06 1.07 1.00 0.98 1.04 0 N-Acetylgalactosaminitolf 1.00 0 1.09 0 1.00 0 N-Aeetylthreosaminitolf. 0.96 1.12 0 Formaldehydeh. 2.38 2.06 1.13 GaIactosyl-N-acetylthreos-

aminitol” 1.14 1.03 I I I I

a Obtained as Dowex 1 peak (142 to 148) by chromatography of Sephadex G-25 Peak B (Fig. 2).

6 Obtained as Dowex 1 peak (64 to 69) by chromatography of Sephadex G-25 Peak C (Fig. 2).

c Obtained after selective removal of sialic acid from trisac- charide; same compound produced after removal of sialic acid from tetrasaecharide (see Fig. 3).

d Periodate oxidation carried out for 24 hours at 4” followed by sodium borohydride reduction as described in text.

6 Expressed per mole of oligosaccharide on the basis of the galactosaminitol.

f Measured as the deacetylated sugars on the amino acid analyzer; the molar color yield of threosaminitol was 0.34 that of alanine (color factor = 2.94).

n Threosaminitol identified by paper chromatography but not quantitated on the amino acid analyzer.

h Measured by the chromotropic acid reaction after periodate oxidation at room temperature for 1 hour ‘at pH 4.5.

i Measured by borate complex anion exchange chromatography after mild acid hydrolysis (0.05 N HzS04, 80”, 60 min) of reduced product of the periodate oxidation (see Fig. 7).

that the attachment of the additional sialic residue is to C-6 of the N-acetylgalactosaminitol. Although threosaminitol could result from a C-3 substitution of the hexosaminitol alone as well as from C-3 plus C-6 substitutions of this sugar alcohol (12), in the former case an extra mole of formaldehyde beyond the two

isolated by Dowex l-X2 chromatography while the disaccharide was obtained by removal of sialic acid from either of these 2 oligo- saccharides. Rhamnose (0.25 Fmole) was used as an internal standard, and the elution scheme employed has been described 06).

FIG. 5. Identification on the amino acid analyzer (Technicon) of amino polyol resulting from periodate oxidation of reduced oligosaccharides. Upper, hydrolysate of sodium metaperiodate- oxidized, sodium borohydride-reduced trisaccharide (C (64 to 69)). Lower, standards. Alanine was used as internal standard. GalN- Hz, galactosaminitol; AraN-Hz, arabinosaminitol; ThrN-Hz, threosaminitol; Ser-Hz, serinol.

contributed by the sialic acid moieties would have been formed. The single mole of formaldehyde yielded by the disaccharide and the 2 moles obtained from the trisaccharide (Table II) are con- sistent with an unsubstituted C-6 on the N-acetylgalactosaminitol in both these compounds.

The possibility that a (2-t@ sialyl-sialyl linkage occurs in the tetrasaccharide as has been described in certain gangliosides (27) and colominic acid (28) is ruled out by the fact that such a bond would have provided protection from destruction by periodic acid to 1 mole of the sialic acid.

Isolation and Characterization of Glycopeptides-Filtration of the pronase digest of fetuin on Sephadex G-50 resulted in the sep- aration of the glycopeptides from the large amount of peptide material (Fig. 8). A major glycopeptide peak (Fraction I) was obtained which showed two distinct shoulders (Fractions II and III) of lower molecular weight glycopeptide components. The elution curves indicated that the ratio of sialic acid to hexose was higher in the later fractions than in the main peak and suggested that a partial resolution of glycopeptides with different carbohy- drate units may have been achieved. Analyses of the three glycopeptide fractions did indeed show a remarkable difference in

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FIG. 6. Paper chromatography of Smith periodate-degraded trisaccharide (C (64 to 69)). The sample (Z), obtained after periodate oxidation, borohydride reduction, and mild acid hydroly- sis of the trisaccharide, is compared to the disaccharide formed by mild acid hydrolysis alone of this trisaccharide (3). Standards, 1 and 4. The chromatograph waz run for 80 hours in Solvent Sys- tem B, and the components were visualized with the silver reagent Gal-ThrNAc-Hz, galactosyl-N-acetylthreosaminitol; Gal-GalNAc- HZ, galactosyl-N-acetylgalactosaminitol.

FIG. 7. Borate complex anion exchange chromatography of tri- saccharide after Smith periodate degradation (upper) compared to disaccharide formed after mild acid hydrolysis (lower) as de- scribed in legend to Fig. 6. Chromatography was performed on the Technicon sugar analyzer with rhamnose az internal standard.

the ratios of galactosamine to glucosamine in these fractions which ranged from 0.02 in Fraction I to 94.0 in Fraction III and can be compared to a ratio of 0.20 which was found to occur in the undigested protein (3.1 moles of galactosamine and 15.7 moles of glucosamine per mole of fetuin).

For the purpose of isolating on a large scale the glycopeptides containiig galactosamine, the lower molecular weight glycopep- tides obtained by gel filtration on a large Sephadex G-59 column, equivalent to tubes 31 to 4’7 from the small column (Fig. 8), were further filtered on Sephadex G-25 to yield three fractions (Fig. 9).

Analyses of the higher molecular weight glycopeptides from the pronase digest as represented by the combined Fraction A from the Sephadex G-25 column and tubes .% to 30 from Sepha- dex G-50 indicated the occurrence of the sugar components sialic

u , L I I I I I I 10 20 25 30 35 40 TUBE NUMBER Wnl;

50 55 60

FIG. 8. Gel filtration on Sephadex G-59 of pronase digest of 5.59 mg of fetuin. A column (2.1 X 80 cm) was equilibrated and eluted with 0.1 M pyridine acetate buffer, pH 5.0. Elution dia- gram shows sialic acid by the resorcinol procedure, hexose by anthrone, and peptide by the ninhydrin method. Numbered seas designate tubes which were combined for further study, and the molar ratio of galactosamine to glucosamine is given for these pools.

- Y -

TUBE NUMBER (5.2ml)

FIG. 9. Hefiltration on a Sephadex G-25 column (2.1 X 139 cm) of lower molecular weight glycopeptides obtained from Sephsdex G-50 filtration of pronase digest from 4.5 g of fetuin. The sample, containing 146 mg of hexose, was obtained from a Sephadex G-50 column (3.4 X 80 cm) and was equivalent to a pool of tubes 31 to 47 from the smaller column shown in Fig. 8. Elution was per- formed with 0.1 M pyridine acetate buffer, pH 5.0. Lettered areas designate the tubes which were combined for further study.

acid, galactose, glucosamine, and mannose in ratios characteristic of the asparagine-linked carbohydrate units of fetuin, while only very small amounts of galactosamine were present (Table III). In contrast the glycopeptides which penetrated more deeply into the Sephadex G-25 gel (Fractions B and C) contained primarily galactosamine, galactose, and sialic acid. Mannose and glucos- amine were absent from Fraction C and occurred only as minor constituents of Fraction B (Table III). In terms of ammo acids, a high prolme content distinguished the galactosamine-containing glycopeptides most notably from the glycopeptides in whieh glucosamine predominated (Table III).

When the glycopeptides in Fraction B from Sephadex G-25 were chromatographed on DEAE-cellulose, resolution into a num- ber of components was accomplished (Fig. 10) which accounted for 92% of the hexose and 91% of the sialic acid placed on the column. All of the major peaks contained glycopeptides in which

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TABLE III Composition of glycopeptide fractions obtained by gel

filtration of pronase digest of fetuin

Component

Sialic acids............. 98.9 102.4 Galactose................ 88.4 N-acetylgalactosamined...

76.1 4.0 69.8

N-acetylglucosamined..... 131.1 9.2 Mannose.................. 76.7 4.0

Aspartic acid ............ 44.1 45.4 Threonine ................ 5.6 23.8 Serine ................... 34.6 58.6 Glutamic acid ............ 6.4 20.0 Proline .................. 15.1 135.6 Glycine .................. 16.8 33.1 Alanine .................. 17.2 96.4 Valine ................... 2.4 37.0 Half-cystine ............. 15.6 2.7 Isoleucine ............... 0.3 Leucine .................. 0.6 Tyrosine ................. 0.3 Phenylalanine ............ 0.7 Lysine ................... 1.5 Histidine ................ 2.1 Arginine ................. 8.5

-

Fraction

Aa I I

Bb Cb

clmoles/lOO

58.9 57.5 53.3

e

117.2 40.1 66.2 38.9

171.9 59.6 37.5 25.1 54.9 12.4

6.8

4.6 1.1 3.8

a Analyses of fraction A from Sephadex G-25 (Fig. 9) combined with glycopeptides from large Sephadex G-50 column (equivalent to tubes 25-30, Fig. 8; see text) which were not filtered on Sephadex G-25.

,C

b Analyses of fraction B and C from Sephadex G-25 column (Fig. 9).

' Analyses expressed per weight of glycopeptides which was determined from the sum of the saccharide and amino acid residues in each fraction. The yields were as follows: A, 934 mg; B, 184 mg; C, 75 mg.

d Determined as the deacetylated sugars.

e Blank entries indicate components were below the range of detection.

the sugar components were exclusively sialic acid, galactose, and galactosamine (Table IV). In Pealcs %’ and 6 mannose and glu- cosamine were found in addition to sialic acid and galactose indicating the presence of glycopeptides with the asparagine- linked carbohydrate unit.

Chromatography on DEAE-cellulose of Fraction C from the Sephadex G-25 column yielded one glycopeptide peak which eluted in a position between Peaks 1 and d of Fraction B (Fig. 10, inset) and contained only sialic acid, galactose, and galactosamine as its saccharide constituents (Table IV).

The ratio of the sugar residues in the galactosamine-containing glycopeptides suggested the presence in them of the trisaccharide (N - acetylneuraminyl - galactosyl -N - acetylgalactosaminyl) and tetrasaccharide (N - acetylneuraminyl - galactosyl - (N - acetylneu- raminyl)-N-acetylgalactosaminyl) units obtained by the alkaline borohydride treatment of fetuin. This supposition was con-

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firmed by the finding that alkaline borohydride treatment of glycopeptides in which the ratio of sialic acid to gala&se to N- acetylgalactosamine is close to 1: 1: 1 (Peaks B-l, B-3, B-4, and C-l) yielded the trisaccharide, while such treatment of Peak B-8 in which the ratio of sialic acid to galactose to N-acetylgalactosa- mine is 2: 1: 1 resulted in the formation of only the tetrasac- charide. When the glycopeptides of Peak B-7 were exposed to alkaline borohydride both trisaccharide and tetrasaccharide were released in molar ratios relative to the galactosamine in this peak, of 0.56 and 0.54, respectively, as determined by borate complex anion exchange chromatography on the sugar analyzer. The approximateiy equimolar occurrence of tri- and tetrasaccharide in Peak B-7 is consistent with the nonintegral ratios of sialic acid to galactose or to galactosamine (Table IV).

NHz-terminal analyses of the glycopeptides indicated that B-3 and B-4 contained 2 carbohydrate units per peptide chain while the others contained only a single saccharide unit (Table IV).

Only a limited number of amino acids were present in the glyco- peptide peaks (Table IV). Noteworthy was the occurrence in each glycopeptide of 1 residue of serine and from 2 to 4 residues of proline per terminal amino acid. Threonine u-as found in most of the glycopeptides containing the trisaccharide unit but in none in which the tetrasaccharide occurred. B-3 and B-4 are of particular interest in that they indicate that 2 trisaccharide units, 1 serine- and 1 threonine-linked, occur within a short seg- ment of the fetuin peptide chain. Since B-4 has only 7 amino acid residues and neither serine nor threonine is in NH&rminal position a maximum of 4 amino acids can separate the two units.

On electrophoresis the glycopeptides moved toward the anode with mobilities increasing in the order of their elution from the DEAE-cellulose column (Fig. 11). When the net, negative charge on the glycopeptide is calculated with the assumption that the aspartic and glutamic acids are not in the amide form a direct relationship between this value, the migration on electrophoresis, and the elution volume from DEAE-cellulose is obtained (Table IV). The single trisaccharide unit of B-l and C-l imparts one extra negative charge to these glycopeptides while B-3 and B-4 have a net negative charge of 2 due to the presence of 2 trisac- charide units. B-5 has a charge of -2 as it contains a residue of aspartic acid in addition to the trisaccharide while a negative charge of 3 is ascribed to B-7 on the basis of an approximately equal molar mixture of a glycopeptide containing a tetrasac- charide with aspartic acid and one containing a trisaccharide with aspartic plus glutamic acid. Finally, B-8 may be presumed to have a net, charge of -4 as it has a tetrasaccharide, as well as 1 residue each of aspartic and glutamic acid.

Studies on Structure of Glycopeptides-Since alkaline borohy- dride treatment of isolated glycopeptides released components with the same chromatographic properties and composition as the tetra- and trisaccharide obtained from the undigested fetuin it appeared likely that the structure of the carbohydrate portion of the glycopeptides would be the same as that of the oligosac- charides.

Periodate oxidation of several glycopeptides (B-4, B-7, B-8, and C-l) gave results which were consistent with this contention. Sialic acid was destroyed although galactose and galactosamine were spared during periodate treatment of undegraded glycopep- tides. After sialic acid removal the galactose was oxidized while the galactosamine still remained intact. However, when two Smith periodate degradations were carried out on either the sialic acid-free glycopeptides or on sialic acid-free fetuin complete de- struction of the galactosamine was accomplished.

V. ch0Zera.e neuraminidase released 94yo of the sialic acid from

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TABLE IV

Composition of glycopeptides containing galactosamine obtained by DE62 cellulose chroma

N-acetylneuraminic acid ...... Galactose .................... N-acetylgalactosamine ........

Aspartic acid ................ Threonine .................... Serine ....................... Glutamic acid ................ Proline ...................... Glycine ...................... Alanine ...................... Valine .......................

NH2-terminal amino acids (total)d .............

Number of carbohydrate units per glycopeptidee ....

Net negative charge per glycopeptidef ..... .........

Yield (mg)g

lb 3 4 5 8 lC

1.01 1.07 1.00

0.21 0.63 1.02

2.11 0.83 0.66 0.28

0.95

1

1

( 4.7)

1.07 1.03 1.00

1.05 0.97 1.00

1.28 1.10 1.00

1.03

1.03

2.28 0.14 1.93 1.07

1.61 1.03 1.00

2.16 1.00 1.00

1.02

1.06 1.04 1.00

1.00 0.49 0.45 0.50 0.43

2.04 1.10 0.96 0.46 1.04 0.82

0.92 0.44 2.34

0.94 1.02 2.18

2.09 1.10

0.58 0.95

1.97 0.99

1.72 0.84 0.34 0.34

0.52 0.51 0.92 1.05 1.18 0.97

2

2

(14.1)

2

2

1

2

(11.0)

1

3

(25.6) (27.1)

1

4

( 7.9 :

1

(22.6)

Sephadex Fractioq B

-

wq.h” Sephadex

Fraction C

(1 Analyses are expressed as molar ratios relative to galactosamine; values below 0.10 are not included in the table. * Numbers refer to peaks obtained by chromatography of Sephadex G-25 Fraction B (see Fig. 10). c Refers to peak obtained by chromatography of Sephadex G-25 Fraction C (see inset, Fig. 10). d Sum of terminal residues determined by the subtractive Edman degradation; glycine and alanine were the terminal residues in all

peaks except B-7 which contained NHz-terminal glutamic acid, proline, and alanine and B-S which had glutamic acid and alanine in terminal positions.

e Number of units determined from ratio of galactosamine to NHZ-terminal amino acids. Analyses of the products of alkaline- borohydride treatment indicated that glycopeptides B-l, 3,4, 5 and C-l contained trisaccharide units; B-S had a tetrasaccharide unit and B-7 contained both trisaccharide and tetrasaccharide in a ratio of 0.56 to 0.54 (see text).

f Calculated on the assumption that the b-carboxyl group of the aspartic acid and y-carboxyl group of the glutamic acid residues are unsubstituted (see text).

@ Calculated from the sum of the sugar and amino acid residues in each peak.

a glycopeptide containing the trisaccharide unit (B-4) and 95oj, of this sugar from a glycopeptide containing the tetrasaccharide unit (B-S) after 48 hours of incubation. Sialic acid-free glyco- peptides (B-4, B-8, and C-l) proved to be resistant to the action of coffee bean cr-galactosidase upon prolonged incubation (96 hours) as in the case of the oligosaccharides. Unexpectedly, how- ever, incubation for that period of time with P-galactosidase from E. coli or jack bean also failed to release more than 10% of the galactose. When the N-acetylated sialic acid-free glycopeptides were incubated with the jack bean P-galactosidase for 96 hours the liberation of galactose was enhanced but still remained at only 25 to 35% of the theoretical.

In order to determine the anomeric configuration of the N- acetylgalactosaminylserine (threonine) linkage the galactose of the tri- and tetrasaccharide units were removed by one Smith degradation of sialic acid-free fetuin and sialic acid-free galactos- amine-containing glycopeptides (Fraction B) . Incubation of such a sialic acid- and galactose-free fetuin preparation with pig liver cY-N-acetylgalactosaminidase resulted in the release of 5.56 pmoles of N-acetylgalactosamine per 100 mg of protein in 96 hours, while digestion of the Smith-degraded glycopeptides with this enzyme brought about complete liberation of the N-acetyl- galactosamine in that period of time.

Treatment of either the sialic acid-free fetuin or the sialic acid- free glycopeptides with the a-N-acetylgalactosaminidase without

prior Smith degradation did not accomplish any saccharide re- lease.

When the sialic acid-free Smith-degraded glycopeptides were incubated with jack bean P-N-acetylhexosaminidase for 72 hours not even a trace of N-acetylgalactosamine was liberated.

While the release of galactosaminitol-containing oligosac- charides after alkaline borohydride treatment of fetuin and fetuin glycopeptides suggested the presence of carbohydrate units at- tached to the peptide chain of this protein through glycosidic bonds involving N-acetylgalactosamine and an cr-amino-@hy- droxy acid, the identity of the ammo acid(s) remained to be established. Incubation of galactosamine-containing glycopep- tides in 0.1 N NaOH at 37” for various lengths of time resulted in a destruction of 89% of the galactosamine by 96 hours and an almost equivalent loss of serine plus threonine (Table V). The destruction of serine proceeded at a more rapid rate than the loss of threonine while all of the other amino acids in the glycopeptide fraction were unaffected by this treatment.

Since the loss of galactosamine, serine, and threonine was pre- sumed to be the result of scission of the glycopeptide bond by the process of /I elimination, the alkali incubation was performed in the presence of sodium sulfite in order to obtain the sulfonyl de- rivatives of the amino acids and ammo sugar by an addition reac- tion involving their unsaturated products (29). Alkaline sulfite treatment caused a somewhat more rapid destruction of the p-

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Fxa. 10. Chromatography on DE52 cellulose of glycopeptides containing galactosamine obtained by gel filtration of pronase digest of fetuin. The sample, containing 34 mg of sialic acid, was taken from Fraction B obtained by Sephadex G-25 filtration (Fig. 9) and was placed on a column (1.25 X 80 cm) in 0.002 M pyridine acetate buffer at pH 5.0. After elution with this buffer two suc- cessive linear gradients were applied as described in text, ranging from 0.002 M (tube 16) to 0.25 M (tube 193). Numbered area6 designate tubes which were pooled for study. The inset shows the position of elution of the only major peak obtained when a sample containing 6.8 mg of sialic acid, taken from Fraction C of the Sephadex G-25 column, was chromatographed on the DE52 column under the same conditions as described for Fraction B. The tubes making up this peak were pooled and designated as C-l.

) -

FIG. 11. Paper electrophoresis of glycopeptides obtained by DE52 cellulose chromatography. The glycopeptides originating from Sephadex G-25 Fraction B are further designated by the number of the peak from the DE52 column (Fig. 10). C-l refers to the glycopeptide from Sephadex G-25 Fraction C which emerges as a single peak on DE52 chromatography (Fig. 10, inset). Elec- trophoresis was performed at 19 volts per cm for 5 hours in pyri- dine acetate buffer at pH 6.4, and the paper was stained with nin- hydrin. The shaded spots were yellow in color while the clear spots appeared blue with this stain.

hydroxyamino acids and galactosamine than alkali alone, so that after 72 hours at 37” 94% of the hexosamine had been lost (Table V) .

When individual glycopeptides were exposed to alkaline sulfite,

TABLE V

Eflect of varying lengths of alkali and alkaline sulfite treatment on threonine, set&e, and galactosamine

content of fetuin glycopeptidesa

Component

Threonine. Serine . . Galactosamine

Untreated

T&l /H?Wld

42 102 122

Length of treatment (hours)

0.5 tx N&O3 in 0.1 NaOH, 37’ 0.1 N NaOH,

0

24 ( 48 1 72 1 96 24 ‘\ 72

m Fraction B from Sephadex G-25 column (Fig. 9). b Content, of entire Fraction B without treatment. c Expressed as micromoles in entire Fraction B.

a substantial portion of the galactosamine was destroyed (greater than 92% in all except C-l) along with an approximately equi- molar loss of serine or serine plus threonine (Table VI). The lost serine was converted in an essentially quantitative manner to cysteic acid while the destroyed threonine appeared as a-amino- P-sulfonylbutyric acid in an average yield of 82% (Table VI). The sulfonyl amino acids, which were absent in the untreated glycopeptides, were well resolved by Dowex 1 chromatography (Fig. 12) although they could not be resolved on the amino acid analyzer (Figs. 13 and 14).

The destruction of galactosamine was accompanied in each glycopeptide by the appearance of a sulfonylhexosamine (average conversion, 99%) which emerged on the amino acid analyzer shortly after cysteic acid at the same position as the hexosamine product of alkaline sulfite treatment of N-acetylchondrosine (Figs. 13 and 14). On the basis of the work of Weber and Winz- ler (24) this C-3 sulfonic acid derivative of hexosamine appears to arise from amino hexoses substituted at position 3 and either located in a terminal-reducing position or involved in 0-glycosidic linkage to a b-hydroxyamino acid in the peptide chain.

Paper chromatography also permitted identification of the sulfonyl derivatives obtained after alkaline sulfite treatment to be made (Figs. 15 and 16) with cysteic acid, a-amino-@-sulfonyl- butyric acid, and sulfonylhexosamine being well resolved. Gly- copeptides containing only serine-linked oligosaccharides could be clearly distinguished from those in which carbohydrate-pep- tide bonds to both serine and threonine exist by the absence in the former of a component migrating to the level of a-amino-p- sulfonylbutyric acid.

Since the data presented in Table VI accounts for the major galactosamine-containing glycopeptides it permits an assessment of the relative proportion of serine and threonine residues in- volved in the 0-glycosidic attachments of the tri- and tetrasac- charide units in fetuin. A total of 47.4 pmoles of serine and 21.8 pmoles of threonine was found to be destroyed by alkaline sulfite treatment of these glycopeptide peaks, indicating that 68% of the N-acetylgalactosaminylpeptide linkages are through the former and 32% to the latter amino acid.

DISCUSSION

It is evident from the results of the present investigation that the fetuin molecule contains, in addition to the previously de- scribed asparagine-linked heteropolysaccharides (2, 3,5), distinct

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TABLE VI Effect of alkaline sd$te treatment on glycopeptides containing galactosamine

components

Glycopeptide

B-3 Untreated.... 0 0 0 6.2 6.3 12.6 Treated=..... 5.7 4.0 10.3 0.8 0.6 0.7 A..........., +5.7 +4.0 +10.3 -5.4 -5.7 -11.9

B-4 Untreated.... 0 0 0 Treatedc..... 11.0 8.3 20.5 A..........., +11.0 +8.3 +20.5

B-5 Untreated.... Treatedc..... A............

0 7.5

+7.5

0 0 0

0 7.5

+7.5

11.8 2.1

-9.7

ed

11.5 26.6 1.8 2.0

-9.7 -24.6

1.9 0.8

-7.1

7.7 0.3

-7.4

B-7 Untreated.... 0 0 0 0 16.0 17.4 Treated=..... 15.0 0 15.4 0 0.6 0.3 A s........... t15.0 0 +15.4 0 -15.4 -17.1

B-8 Untreated.. . . 0 0 0 0 4.1 4.4 Treated=..... 4.6 0 3.8 0 0.1 0 A . . . . . . . . . . . . +4:6 0 +3.8 0 -4.0 -4.4

C-l Untreated.... 0 0 0 12.1 19.7 20.6 Treated=.. . . . 6.9 5.8 12.8 5.4 14.2 6.4 A . . . . . . . . . . . . +6.9 +5.8 +12.s -6.7 -5.5 -14.2

1.27

1.04

1.11

1.10

1.16

a Glycopeptides were obtained by DE-52 chromatography of Sephadex G-25 fraction B (see Fig. 10) and fraction C (inset, Fig. 10).

b Expressed as micromoles in each entire glycopeptide peak.

c Analyses performed after treatment of glycopeptide with alkaline sulfite of 37" for 72 hours ss described in text.

d Dash indicates that there was less than 0.1 mole of threonine per mole of glycopeptids.

FIG. 12. Separation on Dowex l-X8 of sulfonyl amino acids ob- tained after acid hydrolysis of alkaline sulfite-treated glycopep- tides. Upper, glycopeptide B-7; lower, glycopeptide C-l. Chro- matography was performed at pH 3.8 employing a gradient previ- ously described (12). The portion of the elution diagram contain- ing aspartic acid, cr-amino-&sulfonylbutyric acid (Abu-S), and cysteic acid (Q/s-A) is shown. 0-Phosphoserine (SW-OP) was used as an internal standard.

carbohydrate units which are attached by 0-glycosidic bonds to the peptide chain. These hitherto unrecognized saccharide units, which were obtained both in their free form after alkaline boro- hydride treatment of the protein as well as connected to small segments of the peptide chain after pronase digestion, were shown to be acidic components made up of sialic acid, gala&se, and

FIG. 13. Segments of elution diagrams from amino acid analyzer (Technicon) of acid hydrolysates of a glycopeptide with serine- linked carbohydrate unit before (upper) and after (lower) alkaline sulfite treatment. Glycopeptide B-5 was used for these studies. The positions of elution of cysteic acid (Cys-A) and a sulfonyl- hexosamine derivative of galactosamine (G&N-S) are shown in the lower curve.

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FIG. 14. Segments of elution diagrams from amino acid analyzer of acid hydrolysates of a glycopeptide with serine- and threonine- linked carbohydrate units before (upper) and after (lower) alka- line sulfite treatment. Glycopeptide Peak C-l was used for these studies. Cysteic acid (Cys-A) and a-amino-&sulfonylbutyric acid (Abu-8) emerge at the same position but can be resolved by Dowex 1 chromatography (Fig. 12).

FIG. 15. Paper chromatography of desalted acid hydrolysate of glycopeptide C-l before (4) and after (3) alkaline sulfite treat- ment. Standards, 1, d, 6, and 6. Chromatography was in solvent system C for 114 hours, and the components were visualized with the ninhydrin reagent. GalN-S, sulfonylhexosamine derivative of galactosamine; Abu-S, cy-amino-&sulfonylbuturic acid; Cys-+4, cysteic acid.

FIG. 16. Comparison by paper chromatography of the sulfonyl derivatives obtained after acid hydrolysis of alkaline sulfite- treated glycopeptide C-l with serine- and threonine-linked carbo- hydrate units (8) and glycopeptide B-5 with serine-linked unit (4). Standards, 1, d, and 6. Chromatography was carried out for 120 hours in Solvent System C, and the paper was stained with nin- hydrin.

TABLE VII Carbohydrate units of fetuin

Number of units. Number of residues per unit

Sialic acid. . . Galactose............... __. N-Acetylgalactosamine. N-Acetylglucosamine.. Mannose.

Percent protein weight . Percent distribution by weight

of carbohydrate.. . .

Aspar. agine- linked

3”

3d 3 0 5 3

17.8

79

;erine. linked

2

l-28 1 1 0 0 3.3

15

Thre- onine- linked

1

1 13 13.6 1 12 12.4 1 3 3.1 0 15 15.7 0 9 8.1 1.4 22.5 22.9

6 100

~- 6

m Sum of values in the three types of units. * Actual analyses of whole protein aa previously determined

(1, 3); analyses for galactosamine and glucosamine are from the present study.

c Number of asparagine-linked units previously determined (2). d Average values for this unit given; some microheterogeneity

exits. o Serine-linked units occur as 1 trisaccharide and 1 tetrasac-

charide.

N-acetylgalactosamine residues. The latter sugar was found to be located solely in these alkali-labile carbohydrate units and to be involved in the glycopeptide bonds which link them to serine and threonine in the peptide chain.

On the basis of its galactosamine content and the composition of its alkali-labile oligosaccharides, fetuin would appear to con- tain a total of three 0-glycosidically linked carbohydrate units (Table VII) which along with an equal number of asparagine- linked units account for the total number of sugar residues in the molecule. Information obtained in the present study of the galactosamine-containing glycopeptides, moreover, indicates that, 2 serine and 1 threonine residues are involved in the aIkali-sensi tive linkages and that the tetrasaccharide unit is attached only

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H 0

AcNH H OH COOH H OH

0

CH20H H H H

FIG. 17. Proposed structure and peptide attachment of the 0-glycosidically linked tetrasaccharide unit of fetuin, N-acetyl- neuraminyl - (24 3) -0 -D -galactopyranosyl - (14 3)[N - acetylneur- aminyl-(2+6)]-a-D-N-acetylgalactosaminyl- (l-+3) -serine. This

to the serine (Table VII). While the carbohydrate of fetuin is distributed between an equal number of N- and O-glycosidi- tally linked units the considerable difference in size between the two types results in an uneven division by weight (Table VII). Out of a total of 52 sugar residues in fetuin, 42 can be attributed to the asparagine-linked heteropolysaccharides while the re- mainder are components of the serine (threonine)-linked oligo- saccharides.

Since the primary structure of the fetuin peptide chain has not yet been established the location of the 6 saccharide units on this chain remains unspecified. The results of the present study would indicate, however, that 2 trisaccharide units, one linked to serine and the other attached to threonine, are situated in close proximity and that no more than 4 amino acid residues intervene between their attachments. Noteworthy also is the high concentration of proline in the vicinity of the O-glycosidi- tally bound carbohydrate units of fetuin. Numerous residues of this amino acid have also been observed near the attachment site of the serine (threonine)-linked saccharides of rabbit IgG immunoglobulin (30) and human chorionic gonadotropin (31).

Studies employing periodate oxidation and glycosidase diges- tion have permitted the formulation of structures for the tetra- and trisaccharide units of fetuin and their peptide attachments (Fig. 17). The 2 oligosaccharide units have in common an N- acetylneuraminyl-(2--r3)-~-n-galactopyranosy1-(1~3)-N-acetyl- galactosamine sequence, while the tetrasaccharide has in addi- tion a branch in the form of an N-acetylneuraminyl residue attached by an cy-ketosidic bond to C-6 of the N-acetylgalactos- amine. Both units are linked to the hydroxyamino acid through cu-glycosidic bonds involving N-acetylgalactosamine. No al- kali-labile units without sialic acid, or more specifically, without sialic acid linked to gala&se were observed in fetuin.

If the tetrasaccharide unit is to be considered the completed form of the 0-glycosidically linked units of fetuin, and the tri- saccharide a biosynthetic variant, the occurrence of the latter in only one structural form suggests a disparity in the activity of the two sialyltransferases responsible for the assembly of the total unit, with transfer of sialic acid to galactose occurring more rapidly than that to N-acetylgalactosamine. Since the tetra- saccharide unit was found linked to serine but not to threonine, steric hindrance imposed by the latter, somewhat bulkier, hy- droxyamino acid may also play a role in determining whether sialic acid can be attached to the N-acetylgalactosamine during the process of biosynthesis.

y-y Hj;v; H HNAc

unit also occurs in a trisaccharide variant in which the N-acetyl- neuraminic acid linked to N-acetylgalactosamine is missing and in which the N-acetylgalactosaminyl bond is to either serine or threonine.

That the peptide chain may influence the action of enzymes on the 0-glycosidically linked carbohydrate units was certainly evident from the studies performed with @-galactosidases. While these glycosidases obtained from two sources were highly effec- tive in releasing galactose from the sialic acid-free oligosaccha- rides, only minimal activity was observed when these oligosac- charides were attached to the peptide chain.

The sialyl-galactosyl-N-acetylhexosamine sequence observed in the alkali-labile units of fetuin also occurs in the oligosaccha- ride branches of the asparagine-linked heteropolysaccharides of this protein (5). In the latter chains N-acetylglucosamine is substituted for the N-acetylgalactosamine, and t,he linkage of the gala&se is to C-4 instead of C-3 of the hexosamine, but in both units the attachment of sialic acid is to C-3 of the galactose. Because of the similarity of these linkages and sequences three sequential Smith periodate oxidations applied to the whole pro- tein degrade both types of units in a stepwise manner (5).

The occurrence of carbohydrate linked to serine or. threonine in the peptide chain through an 0-glycosidic bond involving N- acetylgalactosamine has been observed in a number of glyco- proteins from diverse sources. These have included submaxil- lary glyeoproteins, IgG and JgA immunoglobulins, erythrocyte membrane glycoprotein, chorionic gonadotropin, and the freez- ing point-depressing glycoproteins from Antarctic fish (32). The structure of the 0-glycosidically linked saccharide units of the porcine submaxillary glycoprotein (33) and human erythro- cyte glycoprotein (34, 35) have been investigated after their release from the protein by alkaline borohydride treatment. The porcine mucous protein yielded a reduced pentasaccharide as its most complete unit while the human red cell membrane provided a tetrasaccharide as its largest reduced saccharide product. It is of interest that the structure of this erythrocyte carbohydrate unit appears to be very similar to that found in fetuin in the present study.

The anomeric configuration of the N-acetylgalactosaminyl- serine (threonine) bond has been determined to be cy in ovine and bovine submaxillary glycoproteins with the use of the mam- malian cw-N-acetylgalactosaminidase (20) which was also effec- tive in cleaving this linkage when used with fetuin in the current investigation.

The observation that the fetuin molecule contains both N- and 0-glycosidically linked carbohydrate places it in the growing list of glycoproteins which have more than one distinct type of carbohydrate unit (32). While the difference in unit types may

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be limited to the occurrence of single and complex asparagine- linked units in the same protein, as in the case of calf thyro- globulin (36) and porcine ribonuclease (37), different glycopep- tide attachments may also coexist on the same molecule. The occurrence of both serine (threonine)-linked units and units bound to asparagine has been observed in IgA and IgG immuno- globulins, chorionic gonadotropin, cornea1 proteoglycan, erythro- cyte membrane glycoprotein, and human thyroglobulin (32), in addition to fetuin.

One of the major tasks in characterizing a glycoprotein in which alkali-labile carbohydrate units occur is to identify and quantitate the cY-amino-P-hydroxy acids to which they are attached on the peptide chain. While a useful technique for accomplishing this purpose appears to be reduction with sodium borohydride and palladium chloride of the unsaturated amino acids formed during @ elimination to yield alanine, and a-amino butyric acid (38), this method when employed on the fetuin glycopeptides produced disappointing yields of these reduced amino acids. However, when /3 elimination was carried out in the presence of sodium sulfite as first suggested by Harbon et al. (29), a satisfactory conversion of the unsaturated products of the hydroxyamino acids to their sulfonyl derivatives, namely cysteic acid and a-amino-@-sulfonylbutyric acid, took place.

Since these two sulfonyl amino acids can not be resolved on the amino acid analyzer, both appearing in the void volume, a chromatographic procedure employing Dowex 1 was developed which clearly separated these two components from an acid hydrolysate (12).

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Robert G. Spiro and Vishnu D. Bhoyroo-Glycosidically Linked Carbohydrate Units of FetuinOStructure of the

1974, 249:5704-5717.J. Biol. Chem. 

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