Oligosaccharides produced by acetolysis of blood group active(A + H)sulfated glycoproteins from hog gastric mucin.

Abstract Blood group (A + H) sulfated glycoproteins containing oligosaccharides of 14 sugar residues have been purified from hog stomach mucosa, and the structure of the carbohydrate chains was investigated. Penta-, tetra-, tri-, and disaccharides were isolated from the products of acetolysis of this material, and their structures were studied by chemical, enzymatic, and immunological methods. Periodate oxidation of a reduced sulfated disaccharide composed of N-acetylglucosamine gave predominantly erythritol, indicating 1 → 4 linkage between the two glucosamine residues. Galactose and N-acetylglucosamine were obtained from the treatment of trisaccharide with β-galactosidase. Methylation analysis of the trisaccharide revealed equal amounts of acetates of 2,3,4,6-tetra-O-methylgalactitol and 2,4,6-tri-O-methylgalactitol in addition to 3,6-di-O-methyl-N-methylglucosaminitol. This is consistent with the structure Gal (β1-3)/→ Gal (β1-4)/→ GlcNAc. Blood group H-active monofucosyl penta- and tetrasaccharides were resistant to β-galactosidase, but were cleaved to the monosaccharides by a mixture of α1,2-l-fucosidase, β-galactosidase, and β-N-acetylglucosaminidase. Analysis of the methylated pentasaccharide revealed the presence of acetates of 2,3,4-, 2,3,6-, and 3,4,6-tri-O-methylgalactitol in approximately equal amounts and, in addition, 2,3,4-tri-O-methylfucitol and 3,6-di-O-methyl-N-methylglucosaminitol. These data are consistent with the structure Fuc (α1-2)/→ Gal (β1-4)/→ GlcNAc (β1-4)/→ Gal (β1-6)/→ Gal. Methylation analyses of one of the two tetrasaccharides showed acetates of 2,3,4-tri-O-methylfucitol, 3,4,6-, 2,3,6-tri-O-methylgalactitol, and 3,6-di-O-methyl-N-methylglucosaminitol, and for the other acetates of 2,3,4-tri-O-methylfucitol, 3,4,6-, 2,3,6-tri-O-methylgalactitol, and 4,6-di-O-methyl-N-methylglucosaminitol. Those data are consistent with the structures Fuc (α1-2)/→ Gal (β1-4)/→ GlcNAc (β1-4)/→ Gal and Fuc (α1-2)/→ Gal (β1-3)/→ GlcNAc (β1-4)/→ Gal. The suggested structure of blood group (A + H) -sulfated glycoprotein presented below is further substantiated. [see PDF for sequence]

From the Department of Chemistry, Belfer Graduate School of Science, Yeshiva University, New York, New York 10033 SUMMARY Blood group (A + H) sulfated glycoproteins containing oligosaccharides of 14 sugar residues have been purified from hog stomach mucosa, and the structure of the carbohydrate chains was investigated.
Penta-, tetra-, tri-, and disaccharides were isolated from the products of acetolysis of this material, and their structures were studied by chemical, enzymatic, and immunological methods. Periodate oxidation of a reduced sulfated disaccharide composed of N-acetylglucosamine gave predominantly erythritol, indicating 1 --f 4 linkage between the two glucosamine residues. Galactose and N-acetylglucosamine were obtained from the treatment of trisaccharide with P-galactosidase.
Analysis of the methylated pentasaccharide revealed the These compounds are characterized by the presence of fucose, presence of acetates of 2,3,4-, 2,3,6-, and 3,4,6-tri-galactose, glucosamine, galactosamine, sialic acid, and sulfate 0-methylgalactitol in approximately equal amounts and, in and differ from the acidic glycoproteins of mesodermal origin (except that of keratan sulfate) primarily by the absence of hog (8) gastric mucosa were shown to possess blood group activity compatible with the blood type of the host. The structure of the carbohydrate portion of (A + H) blood group-active sulfated glycoproteins from hog stomach mucosa has been suggested recently (8).
The aims of the present investigation were to isolate and characterize the oligosaccharides produced by acetolgsis of the sulfated glycoprotrins from hog stomach and to substantiate the suggested structure.

Preparation of Sulfated
Glycoproteins-Sulfated glycoproteins were prepared from hog gastric mucin powder (Wilson Laboratories, batch ~440) by digestion of mucin homogenate with trypsin, repeated alcohol precipitation and fractionation of the precipitate with cetylpyridinium chloride (8). The removal of nucleic acids, digestion with testicular hyaluronidase, and reprecipitation with cetylpyridinium chloride was accomplished as described by Pamer et al. (5).
Chromatography on DEAfi-Sephadex A-50-The crude sulfated glycoproteins dissolved in 0.2 N NaCl were applied in two portions to DEAE-Sephadex (Pharmacia) column (3.0 x 80 cm) equilibrated with 0.2 N NaCl.
Initial elution with 0.2 s NaCl (800 ml) was followed by elution with a linear concentration gradient consisting of 1800 ml of 0.2 N NaCl in the mixing chamber and au equal volume of 1.0 N NaCl in the reservoir.
Fractions (10 ml) were collected and the optical density at 231 nm, and the hesose content of each was determined.
Pooled fractions were dialyzed and lyophilized.
Gel Filtration-The major sulfated glycoprotein fraction from DEAE-Sephadex was dissolved in 0.5 N NaCl and applied to the column (3.6 x 220 cm) packed with Bio-Gel A50m, 50 to 100 mesli (Bio-Rad Laboratories), equilibrated with 0.5 N NaCl. Fractions (15 ml) were collected, and the elution was monitored as described above. Pooled fractions were concentrated to one-third of their volumes, precipitated with 3 volumes of ethanol, dialyzed, and lyophilized.

Final
Fractionation-The final fractionation of the sulfated glycoproteiris obtained from l&Gel column was performed on DEAESephadex A-50 equilibrated with 0.4 N NaCl. Sulfated glycoprotcins (367 mg) dissolved in 0.4 N NaCl were applied to the column (3.0 x 80 cm), and the column was washed with 1000 ml of 0.4 N NaCl.
The elution of the sulfated glycoprotein fraction was accomplished with a linear concentration gradient consisting of 1500 ml of 0.4 N NaCl in the mixing chamber and an equal volume of 0.8 N NaCl in the reservoir. Fractions (10 ml) were collected and monitored for optical density and hesose content as above. Pooled fractions were precipitated with 3 volumes of ethanol, dialyzed, and lyophilized.
The size of the major sulfated glycoprotein fraction (262.8 mg) eluted from DEAE-Sephadex was investigated on a column (2.15 x 195 cm) packed with Bio-Gel A-1.5m and on a column (1.7 x 115 cm) packed with Bio-Gel A-5m, both equilibrated with 0.5 N NaCl.
The homogeneity of the isolated sulfated glycoprotein fraction was determined by zone electrophoresis on cellulose acetate strips as previously described (8).
The mixture was stirred at room temperature for 48 hours, and the acetylated product was recovered by dialysis.
Acetolysis-The acetylated sulfated glycoprotein fraction was subjected to acetolyais with 30 ml of the mixture of acetic anhydride-acetic acid-sulfuric acid (10 : 10 : 1, by volume) (10) at 40" for 5 hours. The reaction was terminated by addition of pyridine to the cooled solution.
The mixture was poured on ice water, and the products were recovered by extraction with chloroform.
The optimum conditions for the oligosaccharide production by acetolysis was determined with a 10.mg sample incubated at 40" for 1, 3, 5, 8, and 16 hours followed by thin layer chromatography (10).
Deacylation-Deacylation was carried out at room temperature by dissolving a sample in methanol and addition of one-third of the volume of 0.5% sodium methoxide in methanol.
The reaction was terminated after 25 min by the addition of ethyl acetate, and the solvents removed by evaporation.
Gel Filtration of Acetolysis Products-The fractionation of the deacylated acetolysis products as well as their desalting was performed on a Bio-Gel P-2, 50 to 100 mesh (Bio-Rad) column (1.7 X 215 cm) developed with 10% aqueous ethanol.
Fractions (3 ml) were collected, and the optical density at 231 nm and the hexose content of each was determined.
The pooled fractions were concentrated to a small volume and rechromatographed individually on the same column. The material excluded from the Bio-Gel P-2 was subjected to gel filtration on a column (1.7 X 220 cm) packed with Bio-Gel P-100 and the column developed with 0.5 N NaCl.
Fractions (5 ml) were collected and monitored as above.
The material excluded from this column was dialyzed, lyophilized, acetylated, and acetolyzed again under the conditions described above. The fraction retarded on Bio-Gel P-100 was desalted on Bio-Gel P-2 column and lyophilized.
Paper Chromatography-Descending paper chromatography (Systems A, B, and C) on Whatman No. 3 paprr (previously washed with the solvent system used) was applied as a final step in the oligosaccharide purification. Whatman No. 1 was used for monosaccharide identification.
Enzymatic Assays-Sulfated glycoprotcin fraction and the oligosaccharides derived from it by acetolysis were incubated in the presence of toluene with fi-galactosidase (EC 3.2.1. 23,Worthington) under the conditions described previously (8). The galactose of the dialyzable fraction released from the sulfated glycoproteins wasquantitized by gas-liquid chromatography. Sugars released from the oligosaccharides were determined by paper chromatography (Systems A and B) Treatment of the sulfated glycoprotein fraction and the fucose containing oligosaccharides with otl ,2-r-fucosidase (kindly provided by Dr. Bahl, State University of New York, Buffalo, N. Y.) was performed according to Bahl (12). The sugars released from the sulfated glycoproteins fraction were quantitized on a dialyzable fraction, and those released from the oligosaccharides were identified by paper chromatography (Systems A and B). Controls consisting only of substrates or enzymes accompanied each experiment.
Metlaylation-The oligosaccharides were methylated by the method of Hakomori (13). The samples dissolved in dimethyl sulfoxide were stirred at room temperature with methylsulfinyl carbanion under nitrogen.
After 6 hours methyl iodide was added and stirring continued for another 18 hours. This was followed by addition of dimethyl sulfoxide, methylsulfinyl carbanion, stirring for 6 hours, addition of methyl iodide, and stirring for another 24 hours. Cpon termination of the reaction with 4.0 JI iSaC the methylated material was extracted into chloroform.
The permethylatcd oligosaccharide products were subjected to formolysis and hydrolysis (14). The neutral sugars were separated from the methylated products of hexosamine on an SG 5OWX8 (H+) column (14).
Periodate Oxidation of Reduced Sulfated Disaccharide-The sulfated disaccharide obtained from the sulfated glycoprotein fraction by acetolysiu, deacylation of the aqueous portion of the acetolysis products, gel filtration on Bio-Gel P-2 column, and paper chromatography (System D) was reduced with sodium borohydride (16 hours at room temperature). Excess borohydride was destroyed with acetic acid and borate removed by evaporation with methanol.
The residue was oxidized with excess 0.15 M sodium metaperiodate at room temperature for 50 hours. The periodate was destroyed with et.hylene glycol and the residue reduced with sodium borohydride.
The residue obtained after destruction of excess borohydride and removal of borate wad hydrolyzed in 1.0 K sulfuric acid (16 hours at 100'). The hydrolysate was neutralized with barium hydroside, tentrifugrd, aiid the supernatant filtered through a mixed bed resin column.
The eluate was concentrated to dryness and the residues derivatized with silylating reagents (15).
Gas-Liquid Chromafographic Analysis-Gas-liquid chromatography analyses were performed with a Hewlett-Packard Research chromatograph, model 5750 apparatus. Stainless steel columns (6 feet x f4 mch) packed with 3% ECNSS-M on Gaschrom Q, 100 to 120 mesh, or packed with 37, SE-30 on Gaschrom Q, 100 to 120 mesh, were used. Trimethylsilyl derivat.ives of methyl glycosides and reduced glycosides were analyzed on SE-30 columns developed for 6 min at 150" and then programmed at 2" per nun to 200". Analysis of the trimetlrylsilyl derivatives of ethylene glycol, glycerol, and erythritol was performed on the same columns but developed at. 65" for 4 min and then programmed at 6" per min to 170". The conditions for analy& of the alditol acetates on ECNSS-M columns were the same as described previously (8).
The methylated neutral sugars after reduction and acetylation were analyzed on ECNSS-M columns at 150" and the similarly prepared amino sugar derivatives were analyzed on the same columns developed at 170" for 3 min and then programmed at 2" per miii to 220".
The determination of the reducing end of oligosaccharides, their romposition, and sugar ratios was performed as described previously (8). Individual sugar components were identified also by paper chromatography (Systems 4, B, D) following hydrolysis (2 p\' HCl, 100" for 6 hours).
Infrared spectra were obtained with potassium bromide pellets in a PerkinElmer model 21 spectrophotometer with sodium chloride prism.
Hemagglutination and hemagglutination inhibition assays were performed with a Takatsy microtitrator.
The calorimetric analyses were the same as cited previously The sample (810 mg) was applied in 0.2 h' NaCl to the column (3.0 X 80 cm) and eluted with a linear NaCl gradient as indicated.

Isolation
OJ Sulfated Glycoproteins-The crude sulfated glycoproteins were obtained from hog gastric mucin powder (50 g) by tryysin digestion, alcohol precipitation, fractionation with cetylpyridinium chloride, removal of nucleic acids, and testicular hyaluronidase digestion, followed by refractionation of the nondialyzable fraction with cetylpyridinium chloride (5, 8). The fractionation of crude sulfated glycoprotcins (1.62 g) was accomplished on DEAE-Sephadex (Fig. 1). The major sulfated glycoprotein fraction, Fraction III (998 mg) from this column was separated further from the testicular hyaluronidasc-resistant mucopolysaccharides by gel filtration on Bio-Gel A-50m, Fraction IlIC (Fig. 2). The hexose-rich fraction, Fraction IIIB (367.4 mg) from this column was finally fractionated on DEAE-Sephadex (Fig. 3). The chemical composition of the purified sulfated glycoprotein fraction, Fraction IIIB2 (262.8 mg) is given in Table I. The isolated sulfated glycoproteins were devoid of uranic acid and exhibited a single band upon electrophorcsis on cellulose acetate strips (8). This material also gave a single, slightly retarded peak when chromatographed on Rio-Gel A-5m and was eluted in the void volume from the Bio-Gel A-l .5m column.
The carbohydrate components of the isolated sulfated glycoprotein fraction, identified by paper chromatography (Systems A, B, and D) and by gas-liquid chromatography as alditol acetates were fucose, galactose, glucosamine, and galactosamine. The molar ratios of these sugar components determined by gasliquid chromatography as alditol acetates (Table II) corresponded to the previously (8) characterized sulfated glycoprotein fractions composed of 14 sugar residues.
Acetolysis of Xulfated Glycoproteins-The optimum conditions for the oligosaccharide production by acetolysis of acetylated sulfated glycoproteins were determined by withdrawing aliquots of acetolysis mixture at several time intervals and examining the products on thin layer plates for the number and intensity of spots.
Short acetolysis (1 and 3 hours) resulted in a small yield of oligosaccharides in addition to monosaccharides, whereas 8 and 2 (left). Gel filtration on Bio-Gel A-50m of sulfated glycoprotein Fraction III from DEAE-Sephadex.
The sample (998 mg) was applied in 0.5 N NaCl to the column (3.6 X 220 cm) and eluted with the same salt solution.  16 hours incubation in acetolysis medium resulted in cstensive monosaccharide production in addition to the oligosaccharides.
The 5-hour acetolysis appeared to be the most feasible for the oligosaccharide production from the acetylated sulfated glycoproteins.
Isolation of Oligosaccharides-The acetolysis products obtained by the chloroform extraction from the acetolysis medium were deacylated with sodium methoxide in methanol and fractionated on a &o-Gel P-2 column.
Four fractions were obtained (Fig. 4). The fraction, eluted from this column in the void volume, Fraction I, was chromatographed further on Bio-Gel P-100, to give two components (Fig. 5), one of which (I) was eluted in the void volume; this material was reacetylated and subjected once more to acetolysis and fractionation on Bio-Gel P-2 and P-100 columns.
The fraction (II) retarded on a Bio-Gel P-100 column was concentrated, desalted on P-2 column, and lyophilized.
The corresponding fractions obtained from the Gel filtration on Bio-Gel P-100 column (1.70 X 220 cm) of deacylated products excluded from P-2 column (Fraction I, Fig. 4). From 220 mg of the sulfated glycoproteins subjected to two consecutive acetolysis procedures 35.5 mg were obtained in glycoprotein Fraction I and 6.8 mg in Fraction II. fractionatiou of the acetolysis products on Bio-Gel P-2 column were combined, rechromatographed individually on the same column, and lyophilized.
The material eluted from P-2 column in the galactose region (Fraction IV, Fig. 4) was examined for the possible presence of components larger than monosaccharide by paper chromatography (Systems A and 1%). Since it contained only monosaccharides it was discarded.
The material remaining in the aqueous phase after chloroform extraction was deacylated and chromatographed on Bio-Gel P-2 column to give a single fraction in the region of Fraction III (Fig. 4). This fraction after rechromatography on the same column was concentrated and lyophilized to give 4.7 mg.
Paper Chromatography of Oligosaccharides-The oligosaccharide fractions obtained from fractionation of the deacylated acetolysis products and %&Gel P-2 and l-'-100 columns were purified further by preparative paper chromatography (Systems A, 13, C). This procedure resulted in the isolation of a single component (oligosaccharide I) from the oligosaccharide fraction (II) retarded on P-100 (Fig. 5). From Fraction II (Fig. 4) two major components (oligosaccharides II and III), and from Fraction III (Fig. 4) a single component (oligosaccharide IV) were obtained.
The RGal values of these oligosaccharides are given in Table  III.
tography performed on this oligosaccharide after its reduction, The silylated products identified by gas-liquid chromatography composition, and its molar ratios (Table II) to the previously (8) were erythritol, glycerol, and ethylene glycol. Erythritol derived characterized sulfated glycoprotein composed of 14 carbohydrate from the reducing end of the sulfated disaccharide is indicative units' of a 1 + 4 linkage between the two glucosamine residues.
Only The glycoprotein was resistant to the action of fl-galactosidase glycerol would result from the procedure if the glucosamine res-and in this respect was also similar to the previously (8)  a short time (25 min) galactose in addition to a spot migrating Acetolysis of the sulfated glycoproteins for 5 hours at 40" identically with N-acetyllactosamine standard was demonstrated with a mixture of acetic anhydride-acetic acid-sulfuric acid (10) by paper chromatography (Systems A and B). Prolonged in-was found optimal for the production of oligosaccharides. Short cubation (50 hours) of oligosaccharide IV with P-galactosidase incubation periods resulted in small yields of oligosaccharides.
gave only galactose and N-acetylglucosamine in the chroma-whereas longer incubation produced mainly monosaccharides. togram.
Four oligosaccharides in addition to the monosaccharides have Incubation of the native sulfated glycoprotein fraction with been purified by gel filtration and paper chromatography from the mixture' of crl , 2-n-fucosidase, fl-galactosidase, and P-N-the deacylated products of chloroform extracts of the acetolysis