Presence of an O-glycosidically linked hexasaccharide in fetuin.

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.

Examination by gel filtration, thin layer and anion exchange chromatography of the 0-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetraand trisaccharides.
The structure of the hexasaccharide was determined to be NeuAca2+3Gal~1+3[NeuAca2+3Gal@l+ 4GlcNAc@1+6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal+Z-deoxygalactitol and Gabanhydromannose which were shown to be derived, respectively, from Gal+N-acetylgalactosaminitol and Gal+GlcNAc sequences. Reductive amination of the Gal+anhydromannose disaccharide with [14C]methylamine permitted identification of its linkage as 1+4. While Diplococcus pneumoniae endo-a-D-N-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra-and trisaccharides (Gal@1+3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal@l+

3[Ga1@1+4GlcNAc@1+6]GalNAc).
The number of 0-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of 0-linked N-acetylglucosamine-containing tetra-or pentasaccharides in fetuin suggests that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal@l+SGalNAc to form the NeuAcaZ+SGal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.
Fetuin, one of the first glycoproteins to be isolated and characterized (1)(2)(3)(4), has served as a model in numerous studies relating to the structure, biosynthesis, and function of glycoconjugates ( 5 ) . Such investigations have been facilitated by the ready availability of this protein and the substantial information already obtained about its N-and O-glycosidically linked carbohydrate units (6)(7)(8).
* This work was supported by Grant AM 17325 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Recipient of Capps Scholarship in Diabetes from Harvard Medical School.
To whom correspondence should be addressed Elliott P. Joslin Research Laboratory, One Joslin Place, Boston, MA 02215. The latter sugar chains which have been shown to include a trisaccharide (NeuAccu2-3Ga1/31-3GalNAc) and a tetrasaccharide (NeuAca2-3Gal/31-3[NeuAca2-6]GalNAc) (6) have now been detected in a large number of cell-surface and soluble glycoproteins (9) and are biosynthetically related, with the trisaccharide serving as the precursor of the tetrasaccharide (10). While the asparagine-linked carbohydrate units of fetuin, which are predominantly of the complex triantennary type, have also been the subject of several investigations (2-4, 7, 8, 11) recent studies have revealed the occurrence of structural variants not previously recognized (12,13).
The possibility that the 0-linked saccharides of fetuin might likewise include other less abundant species was suggested by the observation that an unexpected N-acetylglucosamine-containing saccharide fragment was formed by alkaline sulfite treatment of this glycoprotein (14). In the present report we provide evidence that such an additional 0-linked carbohydrate unit does indeed occur in fetuin in the form of a hexasaccharide with a NeuAccu2-3Galfll-3[NeuAca2-3Gal/31-4GlcNAc~l-6]GalNAc structure.

EXPERIMENTAL PROCEDURES
Preparation of Fetuin-Fetuin was isolated from pooled fetal calf serum by low temperature ethanol fractionation (1) or was purchased from GIBCO (Spiro method). Asialofetuin was prepared by mild acid hydrolysis as previously described (6).
Alkaline Borohydride Treatment of Fetuin and Isolation of Reduced Oligosaccharides-Fetuin (50 mg/ml) was incubated with 1 M NaBH, in 0.1 N NaOH for 72 h at 37 "C to prepare unlabeled oligosaccharides (6), while the radiolabeled components were obtained by treating 10 mg of fetuin in 100 pl of 0.1 N NaOH containing 1 M NaB[3H]4 (Du Pont-New England Nuclear, adjusted to 81 mCi/mmol specific activity) under the same conditions. After acidification with 1 N acetic acid, passage through Dowex 50-X2,200-400 mesh (H+ form), and volatilization of the boric acid with methanol, the samples were applied to a column of Dowex 1-X2,200-400 mesh (acetate form) from which the acidic oligosaccharides were eluted with 1 N formic acid while any neutral components were removed in the effluent and water wash. The unlabeled acidic oligosaccharides were then fractionated on a column (1.8 X 130 cm) of Bio-Gel P-4 (400 mesh) equilibrated in 0.1 M pyridine acetate, pH 5.0, at a flow rate of 8 ml/h while 4-ml fractions were collected and analyzed for sialic acid. Resolution of the radiolabeled acidic saccharides was achieved by preparative thin layer chromatography on cellulose-coated plates in Solvent System A. After elution, the individual isolated oligosaccharides were further examined by chromatography on a column (0.7 X 10 cm) of Dowex In the case of the neuraminidase-treated samples the Dowex 1 column was eluted with 1 N formic acid to recover any remaining acidic components and this fraction was combined prior to chromatography with the unabsorbed material from the Dowex 50 and Dowex 1 columns. Native and asialofetuin (0.5 mg) were incubated with 4 milliunits of Diplococcus pneumoniae endo-a-N-acetylgalactosaminidase (Genzyme, 0-glycanase) in 50 pl of 10 mM sodium citrate/phosphate buffer, pH 6.0, containing 1 mM calcium acetate and 10 mM galactonol,4-lactone for 16 h at 37 "C. At the end of this period the samples and undigested controls were freeze-dried and deproteinized by extraction with 80% (v/v) ethanol. The extracts after evaporation of the solvent were then reduced with 1 mCi of NaB[3H]4 (Du Pont-New England Nuclear, 8.1 Ci/mmol) in 100 ~1 of 0.2 M sodium borate buffer, pH 9.0, for 4 h at room temperature. After decomposition of the borohydride with acetic acid the samples were applied to a 1.0 X 0.5-cm charcoal-Celite (Darco G-60-Celite 535,1:1, by weight) column (15) from which the enzyme-released reduced saccharides were eluted with 8 ml of 25% (v/v) ethanol after an 8-ml water wash.
Periodate Oxidation-The NaB[3H]4-reducedoligosaccharides (3 X lo4 dpm) were oxidized with sodium metaperiodate and then reduced with NaBH4 as previously described (6). The acidic products were absorbed on Dowex 1-X8 (acetate form) from which they were eluted with 1 N formic acid; they were then examined directly by thin layer chromatography or first subjected to mild acid (0.1 N HCl, 80 "C, 1 h) or strong acid (1 N HCl, 100 "C, 6 h) hydrolysis followed by passage through coupled columns of Dowex 50 (H+ form) and Dowex 1 (acetate form). The released aminopolyol, after elution from the Dowex 50 with HC1 was reacetylated with acetic anhydride (16) prior to chromatographic identification.
Threosaminitol and G a l p l -3 T h r N A~H~~ standards were prepared, respectively, by strong acid and mild acid hydrolysis of the NaBH4reduced periodate oxidation product of the 3H-labeled trisaccharide (NeuAccu2-3Galpl-3GalNAcH~) unit of fetuin (6).
Methylation-Permethylation of unlabeled oligosaccharide (20 nmol) was accomplished by the procedure of Hakomori (17) and was followed by hydrolysis, reduction, and acetylation as described (18) except that the neutral sugar fraction was obtained by passing the hydrolysate through coupled columns of Dowex 50-X4 (H+ form) and Dowex 1-X8 (acetate form) equilibrated and eluted with 50% (v/v) methanol. The methylated neutral sugars were analyzed on a Perkin-Elmer gas-liquid chromatography apparatus (Model 900) with a glass column (2 mm X 1.83 m) packed with 3% (w/w) ECNSS-M Gaschrom Q, 100-200 mesh (Applied Sciences) fitted with a flame ionization detector and a Hewlett-Packard Model 3390A computing integrator. The sample was injected into a column which was held at 140 "C for 2 min and this was followed by an increase in temperature to 210 'C at a program rate of 2 "C/min. Peaks were identified by comparison of their retention times to the reduced standard trimethyl ether derivatives of galactose which were gifts from Drs. R. W. Jeanloz and P. J. Stoffyn (Massachusetts General Hospital).
HydrazinelNitr0u.s Acid Treatment-After removal of sialic acid by neuraminidase, purified alkaline/NaBH4 released oligosaccharides (20 nmol) were subjected to hydrazinolysis followed by nitrous acid degradation under the conditions previously described (19). The deaminated saccharides, following passage through columns of Dowex 50-X4 (H+ form), were reduced with NaB[3H]4, in the manner already specified for the endo-a-N-acetylgalactosaminidase products, prior to examination by thin layer chromatography. Radiolabeled dGalH2 was prepared by NaB[3H]4 reduction of the 2-deoxyaldose obtained from Sigma, while standard 3H-labeled Galp1-4AnManHa and AnManHz were prepared from keratan sulfate and glucosamine, respectively, as previously described (19).
Reductive Amination with f4C]Methylamine-As an alternative to NaB['HI4 reduction the hydrazine/nitrous acid-treated saccharides were heated in conical vials (Reacti-Vials, Pierce Chemical Co.) at The abbreviations used are: ThrNAc, N-acetylthreosamine; AnMan, 2,5-anhydromannose; dGal,2-deoxygalactose; SDS, sodium dodecyl sulfate; RLacH2, chromatographic mobility relative to lactitol; Me, methyl; Et, ethyl. All sugars are presumed to be in the pyranose form and the D-COnfiguratiOn; reduced sugars are indicated by the designation H2 following the symbol for the monosaccharide. 105 "c for 4 h with 50 p1 of a 10 mM aqueous solution of ["C] methylamine (Du Pont-New England Nuclear, adjusted to 12 mCi/ mmol specific activity) containing 0.3 M sodium cyanoborohydride (Aldrich). After cooling the reaction was terminated by the addition Of 4 N formic acid (100 pl), borate was volatilized with methanol, and the samples were applied to columns containing 2 g of Dowex 50-X4 (H+ form). Following a water wash (20 ml) the glycamine products were eluted from the resin with 1 M NH4OH (15 ml) and subsequent to lyophilization subjected to thin layer chromatography. Reductive amination with ['4C]methylamine of hydrazine/nitrous acid-treated Galpl-3GlcNAcp-0-Me and Galpl-4GlcNAcp-0-Et (purchased from Sockerbolaget) was employed to prepare the standard glycamine derivatives of Galpl-3AnMan and Galpl-4AnMan, respectively.
Thin Layer Chromatography-Chromatography was carried out on plastic sheets precoated with cellulose (0.1-mm thickness; Merck) in pyridine/ethyl acetate/water/acetic acid, 5:5:3:1 (System A) or in ethyl acetate/pyridine/tetrahydrofuran/water/acetic acid, 5022: 15154 (System B). A wick of Whatman No. 3 paper was clamped to the top of the plate during the chromatography; components were located by fluorography and quantitated by scintillation counting after elution with water. For preparative separation the radioactive saccharides were solubilized with water and the eluates were extracted with peroxide-free ether to remove scintillants.
Preparative Polyacrylamide Gel Electrophoresis-Electrophoresis of fetuin was performed in SDS on polyacrylamide slab gels utilizing the buffer system of Laemmli (20) with a linear 5-15% acrylamide gradient and a 3% stacking gel. Protein bands were localized on guide lanes with Coomassie Blue and after excision were extracted by shaking for 18 h in 0.1% SDS containing 0.1% acetic acid; the SDS was removed by dialysis against Dowex 1 (acetate form) (21).
Chemical Amlysis-After acid hydrolysis (1 N HCl, 6 h, 100 "C) neutral sugars were determined as previously described by borate anion exchange chromatography (22) while amino sugars and their alcohols were separated on an amino acid analyzer (Technicon NC-2) using a borate buffer system (23). Sialic acid was determined by the thiobarbituric acid assay (24). Radiolabeled neutral sugar and re-N-acetylated hexosaminitols were separated by thin layer chromatography in Solvent System A and were quantitated after elution by scintillation counting.
Rodioactiue Measurements-Radiolabeled components on thin layer chromatography were located by fluorography at -70 "C after spraying with a scintillation mixture (25) or ENHANCE (Du Pont-New England Nuclear) using X-Omat AR film (Eastman-Kodak). Liquid scintillation counting was carried out in Ultrafluor (National Diagnostics) with a Beckman LS 7500 instrument.

RESULTS
Oligosaccharides Released by Alkaline Borohydride Treatment of Fetuin-Examination by thin layer chromatography of the acidic saccharides released from two fetuin preparations by alkaline NaB[3H]4 treatment revealed three radiolabeled components (A, B, and C) which migrated in Solvent System A with an RLecH2 of 0.10, 0.20, and 0.78, respectively (Fig. 1). Components B and C represented the 0-linked tetrasaccharide and trisaccharide units of fetuin (61, while component A appeared to be a previously unrecognized oligosaccharide. A similar oligosaccharide pattern was observed in the commercial fetuin preparation and in fetuin which had undergone additional purification by preparative polyacrylamide gel electrophoresis (data not shown).
Examination by thin layer chromatography of the neutral fraction obtained after alkaline NaB[3H], treatment revealed no radiolabeled components even when severalfold larger amounts than required for visualization of component A (Fig.  1) were applied to the plate (not shown).
Analysis of the acidic oligosaccharides after preparative separation by thin layer chromatography indicated that component A like components B a n d C contained its radiolabel solely in galactosaminitol. The radioactive saccharides which showed that it contained N-linked oligosaccharides of fetuin as well as glycopeptides containing these carbohydrate units. Analysis of peak A indicated that it was a hexasaccharide derived by 8-elimination of an 0-linked unit consisting of sialic acid, galactose, glucosamine, and galactosaminitol in molar ratios of 2.1:1.9:0.9:1.0. Structural studies were undertaken to characterize this carbohydrate unit.
Determination of Sequence of Sugar Residues in Herasacchuride by Glycosidase Digestions-Upon extended neuraminidase treatment the radiolabeled hexasaccharide was converted to a neutral component ( R L~~H~ = 0.49) which was distinct from the Gal/31-3GalNAcH2 product (Rh+ = 1.60) derived from sialic acid removal of the fetuin tetra-and trisaccharides (6) (Fig. 4). Limited neuraminidase digestion (30 min) of the hexasaccharide produced an acidic radiolabeled species (Rh+ = 0.22, data not shown) which appeared to be a pentasaccharide intermediate in a stepwise removal of the 2 sialic acid residues.
When this tetrasaccharide product of neuraminidase treatment was digested with jack bean @-galactosidase a new component was produced with a chromatographic migration (RbeH2 = 0.90) consistent with a trisaccharide (Fig. 5, lane 3); removal of galactose from the hexasaccharide only took place if preceded by incubation with neuraminidase. Digestion of the galactosidase product with jack bean P-N-acetylglucosaminidase yielded a disaccharide which comigrated with Gal@1-3GalNAcH2 (Fig. 5, lane 4 ) ; this component was found to be resistant to the action of the ,&galactosidase from jack bean as well as E. coli.
Linkage Determinations by Methylation and Periodate Oxidation-After methylation the hexasaccharide yielded upon acid hydrolysis a single neutral sugar component which was identified by gas-liquid chromatography to be 2,4,6-tri-0methylgalactose (data not shown). This indicated that the 2 galactose residues of the hexasaccharide were substituted exclusively a t C-3.
The substitution of the N-acetylgalactosaminitol residue was evaluated by periodate oxidation of the "H-labeled hexasaccharide. Examination by thin layer chromatography in Solvent System A of the NaBH4-reduced product of periodate treatment obtained after strong acid hydrolysis revealed a single radiolabeled component (data not shown) which was identified after re-N-acetylation as N-acetylthreosaminitol (RbCHZ = 3.30) by comparison with the product from the fetuin trisaccharide (6). Direct chromatography of the radiolabeled reduced periodate oxidation product from the hexasaccharide demonstrated an acidic component which migrated (RbcHz = 0.95) (Fig. 6) to the same position as the sialylgalactosyl-N-acetylthreosaminitol fragment obtained by similar treatment of the fetuin tetrasaccharide unit in which the sialic acid presumably has been truncated to a seven-carbon derivative (27). Subsequent to mild acid hydrolysis a neutral component was obtained which had the same chromatographic mobility as a Galpl-3ThrNAc standard (RbeHn = 2.02). These observations suggested a NeuAca2-3Gal@l-3GalNAcH2 se- quence with the remaining sugar residues of the hexasaccharide (NeuAc, Gal, and GlcNAc) being present on a branch attached to C-6 of the N-acetylgalactosaminitol residue.
HydrazinelNitrous Acid Degradation of the Hexasaccharide-To obtain further information about the C-6 branch of the hexasaccharide, the asialohexasaccharide was fragmented by nitrous acid treatment following deacetylation with hydrazine. After NaB['H], reduction two radiolabeled products were observed on thin layer chromatography in Solvent System B (Fig. 7). The slower moving compound (Di-I RbeH, = 1.73; molar yield, 33%) which was also produced from the fetuin asialotetrasaccharide unit gave rise upon acid hydrolysis to dGalH2 as anticipated from the reported action of nitrous acid on hexosaminitols (28). Since @-galactosidase was observed to cleave this component (Fig. 8) with the release of radiolabeled dGalH, its identity as Galpl-3dGalH2 was established and this indicated that the Gall-3GalNAcH2 sequence of the hexasaccharide is indeed in a 8-anomeric configuration.
The second product (Di-2) of the hydrazine/nitrous acidtreated asialohexasaccharide migrated to the position of AnManH,! after @galactosidase digestion (Fig. 8) as well as after Smith periodate degradation (data not shown). This disaccharide, which was not formed from the fetuin tetrasaccharide unit (Fig. 7), is presumed to be derived from a GalP-GlcNAc substitution on C-6 of the N-acetylgalactosaminitol residue of the hexasaccharide.
Reductive Amination of Disaccharide from Nitrous Acid Degradation-Since GalB1-3GlcNAc and GalS1-4GlcNAc sequences yield the identical Galp-AnManH, disaccharide due to the symmetry of the reduced anhydromannose residue, a charide product from the hexasaccharide unit identified it as the 1-4 isomer (Fig. 9).
Effect of Endo-a-N-Acetylgalactosaminidase on 0-Linked Units of Fetuin-When asialofetuin was digested with the D.
pneumoniae endo-a-N-acetylgalactosaminidase effective distinction between a C-3 and a C-4 substituted N-acetylglucleavage of the GalP1-3GalNAc disaccharide was brought cosamine could not be made. The introduction Of a ['"I about as revealed by thin layer chromatography of the methylamine group a t c-1 by reductive amination of the NaB['HI4-reduced products (data not shown). However, no nitrous acid product permitted its asymmetry to be preserved released tetrasaccharide (Gal~l-3(Gal~1-4GlcNAc~l-6)Galand made possible a chromatographic discrimination between NAc) could be detected even when 30 times the amount the C-3-and C-4-substituted GalP-AnMan isomers (Fig. 9). required for visibility was applied to the chromatograph. This The mobility of the 1-aminomethyl derivative of the disac-resistance of the asialohexasaccharide unit to cleavage by the endoglycosidase, in contrast to the Galbl-3GalNAc originating from the fetuin tetra-and trisaccharides, is consistent with the restricted specificity of this enzyme (29).

DISCUSSION
The widespread use of fetuin as a model glycoprotein in biological and biochemical investigations makes a thorough understanding of the structure of its N-and 0-linked saccharide units a matter of considerable importance. The present study contributes to our knowledge of this protein by demonstrating the occurrence of a previously undetected 0glycosidically linked hexasaccharide. After release by alkaline borohydride treatment this distinctive carbohydrate unit could be resolved from the 0-linked tetra-and trisaccharides (6) by Bio-Gel P-4 filtration as well as by thin layer chromatography.
Our investigations have shown that the hexasaccharide (Fig. 10) is structurally related to the fetuin trisaccharide (NeuAca2-3Gal~l-3GalNAcH,) and tetrasaccharide (Neu-Ac(u2-3Gal~l-3[NeuAca2-6]GalNAcH2) units, differing from the latter by the occurrence of a sialyl-N-acetyllactosamine branch on C-6 of the N-acetylgalactosaminitol instead of a sialic acid residue. Indeed the structure of the hexasaccharide indicates that the NeuAca2-3Gal~l-4GlcNAc sequence is not restricted to the N-linked carbohydrate units of fetuin (3,4).
The structural formulation for the hexasaccharide is based on the results of periodate oxidation, methylation, and glycosidase digestions; furthermore, our studies were facilitated by the employment of hydrazine/nitrous acid treatment to fragment the asialohexasaccharide into its constituent disaccharide units by deaminative cleavage of the bond between glucosamine and galactosaminitol (Fig. 10). Reductive amination with [l4Cjmethylamine of the Gal-AnMan product released by this treatment made it possible to distinguish between its pl-3 and Dl-4 isomers. The other disaccharide obtained from the hydrazine/nitrous acid degradation, namely Gall-3dGalHp in which the N-acetylgalactosaminitol residue of the hexasaccharide had been converted to 2-deoxygalactitol, permitted us to evaluate the anomeric configuration of the Gall-3GalNAcH2 bond since it was readily cleaved by 8galactosidase in contrast to the known (30) glycosidase resistance of Galpl-3GalNAcHp.
Although the observation that the hexasaccharide was released along with the tetra-and trisaccharide units by pelimination indicates that it is attached to a serine or threonine residue, the resistance of the asialohexasaccharide to  Expressed as mole/mol of oligosaccharide; values for the tri-and tetrasaccharides have been previously reported (6).
* Expressed as mole/mol of fetuin and calculated from the molar ratios of the units as measured by aalactosaminitol analvses on the cleavage by endo-a-N-acetylgalactosaminidase, which is consistent with the restricted specificity of this enzyme (29), precluded an assignment of the anomeric configuration of its linkage to the peptide chain.
An accounting of the 0-glycosidically linked units of fetuin indicates that the hexasaccharide, like the tetrasaccharide, is present in submolar amounts (Table I) and therefore would be present in only a minority of fetuin molecules. This tabulation moreover raises the possibility that the hexa-and tetrasaccharide could occupy the same glycosylation site; the latter oligosaccharide has been previously shown to be attached only to serine in contrast to the two trisaccharide units which are linked to a serine and threonine residue in close proximity on the peptide chain (6).
Since the hexasaccharide was found in all preparations of fetuin purified by the low temperature ethanol fractionation procedure as well as in a preparation eluted from polyacrylamide electrophoresis gel there is no reason to doubt that it is an integral saccharide unit of this glycoprotein. Indeed a comparable occurrence in fetuin of N-linked carbohydrate unit variants in substantially less than molar amounts has recently been reported (12,13).
Carbohydrate units of the 0-linked type which contain a GlcNAcDl-6GalNAc branch point as in the fetuin hexasaccharide, have been observed in a variety of secreted and membrane glycoproteins (9) and indeed a glycosyltransferase which functions to attach N-acetylglucosamine to C-6 of 0glycosidically linked N-acetylgalactosamine has been found in canine submaxillary glands (31) as well as a number of other tissues (32). The absence in fetuin of 0-linked pentaand tetrasaccharides containing the incomplete sialyl-N-acetyllactosamine sequence, such as have been detected in other glycoproteins (33)(34)(35), suggests that the attachment of the Nacetylglucosamine is the rate-limiting step in the assembly of this branch of the fetuin hexasaccharide unit. If the 0-linked hexa-and tetrasaccharides do indeed occupy the same glycosylation site, the relatively small amount of the former might reflect a competition between the a2-6-sialyltransferase and the 61-6GlcNAc transferase for a common NeuAca2-3GalD-3GalNAc substrate. If, however, in fetal calf liver the D l -6GlcNAc transferase acts specifically on 0-linked GalO1-3GalNAc, as it does in other tissues (31,32), the predominating activity of the a2-34alyltransferase in this organ (10) may make this disaccharide acceptor a very transitory intermediate. Indeed in this case the pl-6GlcNAc transferase may paradoxically be in competition with the a2-3 rather than the a2-6-sialyltransferase as the latter enzyme has been shown to act exclusively on the NeuAca2-3Galpl-3GalNAc trisaccharide in the fetal liver (10). The possibility that the sequence of assembly of the fetuin tetrasaccharide and hexasaccharide units differ in these salient respects is currently being explored as it may account for the relative proportion of the three 0-linked carbohydrate units in the fetuin molecule.
Aside from its relevance to an understanding of the biosynthesis of 0-linked saccharide chains the identification and characterization of a hexasaccharide unit in fetuin may prove to be of importance in the continued utilization of this glycoprotein as a model in biological and biochemical investigations.