THE JOURNAL cm BPXOGICAL CHEMISTRY Vol. 243, No. 3,Issue of February 10, PP. 616426, 1968

Printed in U.S.A.

Structures and Immunochemical Properties of Oligosaccharides

Isolated from Pig Submaxillary M ucins*

(Received for publication May 26, 1967)

DON 111. CARLSON~

WITH THE TECHNICAL ASSISTANCE OF CHARLES BLACKWELL

From the Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, Ohio 44106

SUMMARY

Two immunochemically distinct mucins have been isolated from pig submaxillary glands. The glands were combined according to the ability of aqueous extracts of these glands to inhibit hemagglutination of human type A erythrocytes. Mucin isolated from the glands containing blood group A activity is designated A+ pig submaxillary mucin (A+-PSM), while mucin isolated from the remaining glands is designated A- pig submaxillary mucin (A--PSM). The carbohydrate composition of both mucins is similar and comprises N- acetylgalactosamine, fucose, galactose, and N-glycolyl- neuraminic acid.

Treatment of these mucins with alkaline borohydride re- sulted in the release of a series of reduced oligosaccharides and the monosaccharide, 2-acetamido-2-deoxy-D-gala&o1 (N-acetylgalactosaminitol). Conditions are reported which give more than 90% cleavage of the sugar residues from the protein chain. The most complex oligosaccharide (desig- nated oligosaccharide I) was a pentasaccharide, 2-acetamido- 2-deoxy-oc-D-galactopyranosyl(l+3)-[cr-L-fucopyranosyl-(l --f 2)]-fi-D-galactopyranosyl-(l + 3)-[N-glycolylneuraminyl- (2 4 6)]-2-acetamido-2-deoxy-D-galactitol. In addition, the fol- lowing oligosaccharides were isolated (structures are given as related to oligosaccharide I): oligosaccharide II, I minus N-acetylgalactosamine; oligosaccharide III, a disaccharide N- glycolylneuraminyl -+N-acetylgalactosaminitol; oligosaccha - ride IV, I minus N-glycolylneuraminic acid; oligosaccharide V, II minus iV-glycolylneuraminic acid. ZV-Acetylgalac- tosaminitol (Fraction VI) was the only detectable monosac- charide. Rabbit antiserum to human type A erythrocyte stroma precipitated A+-PSM, but not A--PSM. Oligosac- charides I and IV, found only in A+-PSM, are potent in- hibitors of the anti A-A+-PSM precipitation, but oligosaccha- rides II, III, and V and a monosaccharide (Fraction VI) are completely inactive.

* This investigation was supported in part by Grants AM- 10335 and AM-08305 from the National Institutes of Health.

$ Inquiries should be addressed to the author at the Department of Pediatrics, Babies and Childrens Hospital, University Hospi- tals, Cleveland, Ohio 44106.

Detailed knowledge of the structures of glycoproteins must necessarily precede an understanding of their biosynthesis. The glycoproteins which have blood group activity are partic- ularly important. The early work of Bendich, Kabat, and Bezer (1) indicated that pig gastric mucosa contains blood group substances A, H, or AH. The carbohydrate composition and structure of substance A isolated from hog gastric mucosa are thought to be identical with those of the soluble human blood group A substances (2, 3). Bovine submaxillary mucin prepara- tions contain a highly specific blood group substance (4), and human submaxillary secretions are known to contain large amounts of blood group substances (2). Thus, studies on the structure and biosynthesis of salivary mucins may also assist in understanding the synthesis of blood group substances, since these mucins often contain similar materials.

Aminoff, Morrow, and Zarafonetis (5) found that aqueous extracts of pig submaxillary glands contained substances serolog- ically similar to the hog gastric secretions, i.e. A, H, AH, and I, which was defined as neither A nor H. These workers reported a constant ratio of fucose to hexose, and found similar sugar compositions for all serologically defined extracts. These data prompted the conclusion that the difference in immunochemical activity was not based on the absence of a particular sugar in the main oligosaccharide chain. Studies on pig serum and saliva samples (6) suggest that the interaction of alleles at the A locus with other genes is responsible for the A, 0,’ and - phenotypes, where - is defined as neither A nor 0.

Early studies of pig submaxillary mucin (7, 8) and oligosac- charides obtained from this mucin (9, 10) were carried out on pig submaxillary glands without regard for serological activity. However, recent evidence (9) suggested that a difference in pig submaxillary mucins, based on blood group activity, does exist. The present communication is a report on a detailed study of the carbohydrate moieties of two immunochemically distinct pig submaxillary mucins. Among other findings is the first proof of an oligosaccharide containing both fucose and sialic acid. The principal structural variation among the saccharides

1 Blood group 0 activity is assumed to correspond to the H activit,y reported by Aminoff et al. (5), and the phenotype “-” would be analogous to I.

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isolated from the mucins is to be seen in the number of sugar residues. The structures range from a monosaccharide, N- acetylgalactosaminito1,2 to a pentasaccharide which contains equimolar amounts of fucose, galactose, A’-acetylgalactosamine, AT-glrcolylneuraminic acid, and N-acetylgalactosaminitol. d

EXPERIMENTAL PROCEDURE

Materials

Unlees otherwise indicated, all materials used were of com- mercial origin. Sialidase was obtained from Dr. J. T. Cassidy, The L-niversity of Michigan; j%galactosidase from Dr. E. J. McGuire, The Johns Hopkins University; rabbit antihuman blood group A antiserum from Dr. G. Schiffman, Hospital of the University of Pennsylvania; crystalline N-acetyhleuraminic acid from Dr. S. Roseman, The Johns Hopkins University; Ulex europus H lectin from Dr. A. Steinberg, \STestern Reserve TJni- versity; 2,3,4-, 3,4,6-, and 2,4,6-trimethyl galactoses from Dr. P. Stoffyn, McClean Hospital, Boston; 3,4- and 4,6-dimethyl galactoses from Dr. J. K. N. Jones, Queens University, Kingston, Ontario; 2 ,Cdimethylgalactose from Dr. Betty Lewis, University of Minnesota. The author gratefully acknowledges the generous gifts of the indicated materials.

N--1cetylchondrosinitol was prepared by N acetylation of chondro.sine and reduction with sodium borohydride (see “Prep aration of N-Acetylchondrosinitol,” below).

Methods

The following substances were determined by the indicated methods: bound sialic acid by the resorcinol procedure (11) and, following sialidase treatment, by the thiobarbituric acid method (12) ; fucose by the method of Dische and Shettles (13) ; galactose by the orrinol-sulfuric acid procedure (14), corrected for fucose (15), and also by the galactose oxidase mebhod (16); protein by the procedure of Lowry et al. (17); hexosamine by the Boas modification of the Elson-Morgan determination (18); N- acetylhesoaamine by a modified Morgan-Elson procedure (19); N-acetylgalactosaminitol by hydrolysis and N-acetylation with ‘%-acetic anhydride (20); reducing substances by the Park- Johnson method (21) ; hexoses by the anthrone procedure (22) ; uranic acid by the carbazole procedure (23); and nitrogen by the ninhydrin method as described by Schiffman (24).

Quantitative studies of periodate consumption were made by iodometric titrations (25). Formaldehyde was measured by the chromotropic acid technique (26), and formic acid by a microti- tration method (3).

N-Glyrolylneuraminic acid was liberated from its glycosides with sialidase according to the procedure of Cassidy, Jourdian, and Roseman (27). Treatment with P-galactosidase was carried out as outlined by Hughes and Jeanloz (28). Enaymic treatment of the indicated oligosaccharides with N-acetylgalactosaminidase was performed by Dr. E. J. McGuire. The conditions reported by Kuhn, Baer, and Gauhe (29) for the hydrolysis of fucosyl- galacto*e and fucosyl-talose (1 x HzS04 for 180 min at 70”) were

ZThe abbreviations used are: N-acetylgalactosaminitol, 2- acetamido-2-deoxy-D-galactitol; N-acetylchondrosinitol, P-D- glucopyranosyluronic acid-(l-+3)-2-amino-2-deoxy-n-galactitol; PSM, pig submaxillary mucin; A+-PSM and A-PSM, as described in the text under “Preparation of Mucin”; N-GN and N-AN, N- glycolyl- and N-acetylneuraminic acids.

modified slightly, since incubation for 90 min at 65” was sufficient to liberate more than 80% of the fucose from some oligosac- charides. After removal of sulfate from the hydrolysis mixture with barium hydroxide, the clear supernatant fluid was N acetylated (30) to replace N-acetyl groups removed during the hydrolysis. This modified procedure will be referred to as “mild acid hydrolysis.” Optical rotations were determined in a Zeiss polarimeter at 25” with water as solvent and a 589 rnp light source. Radioactivity on paper chromatograms and electrophoretograms was detected with a Packard gas flow chromatogram scanner. A Packard Tri-Carb spectrophotometer was used for liquid scintillation counting and counting systems recommended by the manufacturer were used: a toluene system for counting paper strips, and a dioxane system (“DAM Cock- tail 611”) for aqueous solutions.

Descending paper chromatography was performed with What- man No. 1 and with Schleicher and Schuell No. 589 Green Ribbon papers for characterization and isolation, respectively, of sugars. The following solvent systems were used (all in volume for volume): A, 1-butanol-pyridine-water (6:4:3); B, ethyl acetate- pyridine-water (2 : 1: 2) ; C, ethanol-water-38% ammonium hydroxide (80:20:1); D, 1-butanol-acetic acid-water (4:1:5); E, l-butanol-1-propanol-0.1 x HCl (1:2:1); F, ethyl acetate- pyridine-acet.ic acid-water (5 : 5 : 1: 5) ; G, l-butanol-ethanol- water (4: 1:5); H, benzene-ethanol (4: 1); I, acetone-water-38% ammonium hydroxide (250 : 3 : 1.5) ; J, I-butanol-ethanol-water (10 : 1:2). Sugars and amino sugars on paper chromatograms were detected with periodate-benzidine (31), aniline-phosphoric acid (32), or ninhgdrin (32).

High voltage electrophoresis was conducted for 45 min at 50 volts per cm on Whatman No. 3MM paper with a Gilson high voltage Electrophorator. A 1 7O solution of sodium tetraborate was the buffering system. Materials susceptible to periodate oxidation were detected with the periodate-benzidine technique. To preserve the blue background resulting from this procedure, it was necessary to allow the periodate to react for a maximum of 3 min and to dip the papers in the benzidine reagent while still damp. Borate was removed from electrophoretograms, if necessary, by spraying with a mixture of acetic acid-methanol (33).

Thin layer chromatography, used mainly for the separation of

methylated sugars, was carried out on Silica Gel G. The plates were prepared as suggested by Brinkmaml Instruments, Inc., Westbury, New York. Completely methylated sugars and other nonreducing materials were detected by spraying the plates with 55% sulfuric acid containing 0.6% potassium dichromate and charring for 30 min at 110” (34).

Charcoal-Celite column chromatography was performed with Darco G-60-Celite mixtures (1 :l) prepared as described by Whistler and Durso (35). Sugars were eluted batchwise with aqueous ethanol. A Biogel P-2 column was used to desalt the oligosaccharide preparations.

The hexosamine and neutral sugar fractions of PSM were isolated as described by Spiro (36). Quantitative microprecipi- tin assays were performed as described by Schiffman (24).

Preparation of N-Acetylchondrosinitol-‘4C-N-Acetylchondro- sine was prepared by N acetylation of chondrosine (100 mg) with ‘%-acetic anhydride under the conditions previously described (30). After acetylation was complete, one-half of the reaction mixture was removed, acidified by the addition of

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618 Studies on Pig Submaxillary Mucins Vol. 243, No. 3

,O.,. 20.6.

z ; 0.4.

0;

0.2.

IO 20 30 T::E IO 20 30 7 NUMBER

FIG. 1. Chromatography of I%-N-acetylchondrosine and laC- N-acetylchondrosaminitol. I%-N-acetylchondrosine (A) and its reduced product (B), prepared as described in the text, were each applied to a column, 1 cm X 15 cm, of Dowex l-X8 (Cl-, 200 to 400 mesh) and eluted with a linear gradient of NaCl, starting with 200 ml of water in the mixing flask and 200 ml of 0.2 M NaCl in the reservoir. Fractions of 3.7 ml were collected and aliquots were assayed for N-acetylhexosamine (O), uranic acid (A), and radioactivity ( l ) .

TABLE I

Relative frequency of blood group active substances in pig submaxillary glands

Blood group activity was determined on aqueous extracts of

glands from individual animals. Hemagglutination inhibition

under standardized conditions (26) was st.udied macroscopically and microscopically. Experiment 1 shows the results when ex- tracts were assayed only for ability to inhibit the A-anti A hemag- glutination system. A and H indicate inhibition of the human

A-anti A and O-anti H (Ulex europzts) hemagglutination systems; I designates the absence of A and H activity.

Experiment

1 2 Total

Blood group activity

A 1 AH / H / I

No. of animals

8 25

13 j 2 G / 17 23 48

Dowex 50-X8 (H+, 200 to 400 mesh), and passed through a small column of Dowex 50. The filtrate was evaporated to dryness to remove the acetic acid. The remainder of the acetylation mixture was reduced by slowly adding 200 mg of sodium boro- hydride with stirring in an ice bath over a period of 1 hour. Reduction was allowed to continue for an additional 2 hours, and the remaining sodium borohydride was destroyed by the careful addition of Dowex 50. The mixture was then treated as de- scribed above and boric acid was removed by evaporation with methanol. Both N-acetylchondrosine and the reduced product, isolated by column chromatography (Fig. l), were passed through Biogel P-2 to remove NaCI. The N-acetylchondrosinitol fraction was completely resistant to hydrolysis in 0.05 M KOH at 37” for 1 hour, whereas N-acetylchondrosine was quantitatively cleaved to yield glucuronic acid and two other periodate-suscepti- ble materials, neither of which was N-acetylgalactosamine, as determined by electrophoresis in borate buffer followed by the periodate-benzidine procedure. The ratio of glucuronic acid to N-acetylgalactosaminitol in the reduced product was 1.00 :0.97.

Methylations-Methylation of oligosaciharides was carried out by a microadaptation of the method of Kuhn and Trischmann (37). A solution containing 5 to 20 pmoles of oligosaccharide was dried in a screw cap tube and the following reagents were added: 0.5 ml of dimethylformamide, 0.5 ml of dimethyl sulfoxide, 250 mg of barium oxide, 100 mg of barium hydroxide, and 0.25 ml of methyl iodide. Glass beads were added, and the tubes were capped (with the use of Teflon liners) and were shaken vigorously for 24 hours at room temperature. Barium hydroxide and methyl iodide were again added and shaking was continued for an additional 24 hours. The methylated sugars were ex- tracted into chloroform and the chloroform was removed on a rotary evaporator. Further purification of the methylated sugars was obtained by Biogel P-2 column chromatography; the anthrone reagent was used to detect the methylated derivatives. Hydrolysis of the methylated oligosaccharides in 2 N HzS04 for 3 hours gave the constituent methylated monosaccharides. The hydrolysates were neutralized with Dowex 1 carbonate, and amino sugars were removed with Dowex 50-H+. The resulting solutions were evaporated to dryness at room temperature. Complete methylation of oligosaccharides was assumed if the methylated products were chromatographically homogeneous on thin layer chromatography (Solvent H) and if the expected products were obtained following hydrolysis. Trehalose and lactose gave products showing no OH band in their infrared analysis spectra.

Preparation of Mu&n--The pig submaxillary mucin used in previous studies (7-10) was obtained from a pooled sample of glands not selected for blood group activity. The glands used for mucin isolation as described here were pooled according to their ability to inhibit human A-anti A hemagglutination (A+- PSM) ; glands which did not contain blood group A substance and consequently did not inhibit hemagglutination were desig- nated A--PSM.

Pig submaxillary glands, obtained from a local slaughter house, were chilled in ice and excess connective tissue was removed. A small amount of tissue excised from each pair of glands was homogenized in 4 volumes of cold distilled water. The crude supernatant fluid, obtained by centrifugation for 20 min at 32,000 x g, was heated at 100” for 10 min, cooled, and again centrifuged. The clear supernatant fluid was assayed for inhibition of the human A-anti A hemagglutination system (26), and the glands were selected accordingly. The frequency of oc- currence of the A and H substances was determined for a limited number of glands (Table I). The H activity was identified by hemagglutination inhibition of human type 0 cells and Ulez-H antiserum. The mucins (A+-PSM and A--PSM) were isolated essentially by the procedure of Hashimoto, Hashimoto, and Pigman (7) as modified by de Salegui and Pigman (38).3

Isolation of Reduced Oligosaccharides-The mucins were treated with alkaline borohydride and the reduced oligosaccharides were isolated essentially as described previously (9). Approximately 1 g of mucin was incubated in 0.05 M KOH and 1.0 M sodium borohydride for 15 hours at 45” in a final volume of 400 ml. The excess borohydride was destroyed by careful addition of 4 M

acetic acid to pH 5, and the mixture was passed through a column, 4 cm x 40 cm, of Dowex 50-X8 (H+, 200 to 400 mesh). The eluate was evaporated to dryness on a rotary evaporator and

3 The modified procedure for purifying PSM was made available through the courtesy of Drs. M. de Salegui and W. Pigman, New York Medical College.

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boric acid was removed as methyl borate. The clear syrup which resulted was dissolved in 250 ml of water and the sialic acid-containing oligosaccharides were adsorbed on a column, 2 cm X 30 cm, of Dowex l-X2 (Cl-, 50 to 100 mesh). The neutral oligosaccharides, removed by washing the column with water until the eluate was negative to the anthrone reagent, were fractionated on a column, 3 cm x 4 cm, of charcoal-Celite. N- Acetylgalactosaminitol and oligosaccharide V were isolated by batch elution with aqueous ethanol (100 ml of 5% ethanol followed by 100 ml of 15% ethanol). The sialic acid-containing oligosaccharides were eluted from the Dowex l-chloride column with a linear gradient of NaCl, starting with 900 ml of water in the mixing flask and 900 ml of 0.1 M NaCl in the reservoir. A flow rate of 1 ml per min was maintained, and 5-ml fractions were collected. Aliquots from each tube were assayed by the resor- cinol and anthrone procedures. The following fractions were pooled: (a) tubes 46 to 105, containing sialic acid and hexose; (b) 106 to 140, containing sialic acid and hexose (tube 140 con- tained no hexose), (c) 141 to 230, containing sialic acid. Each combined fraction from the column was evaporated to dryness, dissolved in water, and desalted by passing through Biogel P-2. Whereas the neutral oligosaccharides from A--PSM could be completely separated by charcoal-Celite column chromatography, paper chromatography (Fig. 2) was needed to separate the sialic acid-containing oligosaccharides and for the final purification of the neutral oligosaccharides from A+-PSM. The desalted fractions from Biogel P-2 were streaked on S and S Green Ribbon paper sheets and irrigated with Solvent A (150 to 200 ml per sheet) in descending fashion. The areas which contained oligosaccharides were detected on test strips by the periodate- benzidine technique, and the corresponding areas of the chromat- ograms were eluted with water. Final purification of these materials was achieved by passage through Dowex 50-X8

I

0 0

0

0

ORIGIN

25 Cm

FIG. 2. Chromatography of oligosaccharides on S and S No. 589 Green Ribbon paper (Solvent A).

TABLE II Carbohydrate composition of pig submaxillary mucins

The carbohydrate compositions of A--PSM, isolated from combined H and I glands (Table I), and A+-PSM, in comparison with reported values, are given. The analyses on PSM isolated in the present study are based on dry weight obtained by drying an aliquot of mucin solution to a constant weight at 100”; no corrections were made for ash.

component 1 H

glands I

! A--PSIW / PsMb /p

I I

GalNAc 18.5 Fucose 6.7 Galactose 9.0 A-GN.. 15.7

Total carbohy- drate......... 49.9

I II

g/l00 g nmcin 19.2 1 23.4 23.8 18.0 24.5 G.0 8.5 6.9 6.2 5.0 8.1 11.5 12.6 9.4 19.6

16.1 14.6 19.8 11.8 15.0

49.4 58.0 63.1 45.4 64.1

Q Mucins used in the present study. b Hashimoto, Hashimoto, and Pigman (7). c Katzman and Eylar (8).

(H+, 200 to 400 mesh), neutralization of the eluate with dilute NaOH, and passage through Biogel P-2. The isolated oligosac- charides were precipitated from concentrated solutions by the addition of acetone. Although some oligosaccharides were obtained initially as syrups, repeated addition of acetone with trituration gave white, flocculent precipitates. These materials were dried in a vacuum and stored dry at 4”. Yields of the oligosaccharide fractions are given in Table IV, below.

RESULTS AND DISCUSSION

Two immunochemically distinct mucins (A+-PSM and A-- PSM) have been isolated from pig submaxillary gland extracts by methods previously reported (7, 38). The extracts were first treated at pH 4.5 and the highly charged polyanionic mucins were then precipitated as an insoluble complex with cetylpyridinium chloride. The resulting clot-like material, dis- solved by adding 0.9% NaCl solution, was further purified by ethanol fractionation. Although the isolated mucins were monodisperse in the ultracentrifuge, no claim can be made for “homogeneity” or “purity” of the mucin samples. The carbohy- dra.t,e compositions of much usd in d~i.s study (Table II) are approximately the same as reported for PSM by Hashimoto, Hashimoto, and Pigman (7). Although purified A+-PSM con- tains more GalNAc than A--PSM, it would be indeed difficult, if not impossible, to determine blood group activity of an indi- vidual gland based on its gross sugar composition. Results based on analyses of this type undoubtedly prompted Aminoff et al. (5) to conclude that differences in the sugar chains did not exist among the serologically different mucins.

Studies of the carbohydrate structures of glycoproteins are facilitated by quantitative release of the sugar chains with a minimum of degradation. Other workers, using alkali concen- trations similar to that reported here (see “Methods”) and incubating for 5 to 7 days at room temperature to release the sugar residues from mucins (10, 39) and blood group substances (3), have experienced low yields and reported degradation of the

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TABLE III

Molar ratios and sugar recoveries from alkaline borohydride-treated A--PSM

See “Preparation of oligosaccharides” for details.

N-GP I FUCOSe~ I GalactoseC

Fraction I I Sugar : recovered REXXWt-Iye Sugar

recovered Recoverye

A--PSM. ~ 400 100 260 Dowex 50-H+ eluate. 375 94 235 Dowex l-Cl- water eluate.. 100 Dowex l-Cl- NaCl eluate 250 62 87

pmoles/g mu& %

100 90 39 33

/moles/g mucin %

320 1 100 225 70

98 31

104 32 -

GalNA& I

Protein

/moles/g w/g ?M ucin % mucin 70

840 100 610 100 0 <2 <80 <5

1 I / a Molar ratio for sugar in this preparation, 0.96. * Molar ratio for sugar in this preparation, 0.63. c Molar ratio for sugar in this preparation, 0.77. d Molar ratio for sugar in this preparation, 2.00. e Percentage recovery of constituents, based on calorimetric analysis.

P 1 I L 2 4 6 6 IO 12 14

HYDROLYSIS TIME (HOURS)

FIG. 3. Studies on alkaline borohydride conditions for cleavage of the carbohydrate to protein linkage. A--PSM was incubated at 25” (0, 0, A) and at 45” (0, H, A) at concentrations described in the text. The alkaline conditions were 0.05 N KOH (A, A), 1 M sodium borohydride (0, 0 ), and 0.05 N KOH plus 1 M sodium borohydride (0, n ). The incubation mixtures were neutralized at the indicated time intervals and dialyzed. The contents of the dialysis bags were assayed for N-GN by the resorcinol procedure. Controls neutralized at zero time were included.

oligosaccharides. However, the saccharide moieties of PSM are almost quantitatively released with the conditions described under “Methods,” as shown in Table III. The results of a study of the effects of alkali concentration and temperature on oligosaccharide release are shown in Fig. 3. While only the results for nondialyzable N-GN are shown, similar values were found for galactose and fucose. Incubation for 15 hours at 45” in the presence of 1 M NaBH4 and 0.05 M KOH gave greater than 90% release of the sugar moieties. Similar results were ob- tained with 1 M NaBH4. At 25” a slow rate of release was found, which appeared to be independent of the alkali concentrations used.

Sugar Recoveries

IMolar ratios and recoveries of the constituent carbohydrate moieties of X--PS?II are given in Table III. High recoveries of sialic acid and fucose with a complete loss of hexosamine were

obtained, following alkaline borohydride treatment and Dowex 50-H+ column chromatography. Hydrolysis of A+-PSM gave the same results, except that the recoveries of GalNAc varied from 30 to 40yG. Galactose recoveries ranged from 70 to 85%, but there were no detectable galactitol or unsaturated compounds as in oligosaccharides from blood group substances (3) and from PSM as reported by Katzman and Eylar (39). Chromatography on Dowex l-Cl- resulted in a 20 to 25% loss of sialic acid-con- taining oligosaccharides and a 10 to 20 y0 loss of neutral oligosac- charides. Chromatography on charcoal-Celite or Biogel P-2 gave better than 90% recovery of sugars.

Characterization of Saccharides

All saccharide fractions were homogeneous in five chromato- graphic solvent systems and on electrophoresis in borate buffer. Oligosaccharides II, III, V, and VI were isolated from A--PSM; this preparation did not contain oligosaccharides I and IV. A+-PSM contained the oligosaccharides found in A--PSM and, in addition, oligosaccharides I and IV. The proposed structures are shown in Fig. 4. Oligosaccharide I, a pentasaccharide, contains equimolar quantities of GalNAc, galactose, fucose, N-GN, and N-acetylgalactosaminitol. The remaining oligosac- charides lack one or more of these sugar residues, but all contain N-acetylgalactosaniinitol.

Configurations of the component sugars, except sialic acid, were shown to correspond to L-fucose, D-galactose, and D-galactos- amine. The monosaccharides were isolated from A--PSM; thus the galactosamine fraction should reflect the configuration of the amino sugar attached to the protein (see Fig. 4), since this is the only hexosamine moiety present. Fucose and galactose were the only detectable neutral sugars. The fucose, isolated as a syrup, had a specific rotation of -73” (reported value, -76” (40)). A preparation of the crystalline fucose phenylhydrazone (40) melted at 169-172”; the reported value is 170-172”. A mixture of this material with authentic fucose phenylhydrazone had a melting point of 169-171”. Galactose and galactosamine both occur in the D configuration, as shown by their rates of reaction with galactose oxidase as compared with standard compounds. The galactosamine and galactose isolated from PSM gave 73 y0 and 98 ‘j& respectively, of the color obtained with standard galactose (16).

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Neutral Fractions

Fraction VI-This fraction, not bound by Dowex l-Cl-, was eluted from charcoal-Celite with 5 ‘% ethanol. The material was identical with N-acetylgalactosaminitol in Solvents A, B, and F and in borate electrophoresis. After crystallization from ethanol the material melted at 174-175” (uncorrected). Crystalline N- acetylgalactosaminitol, prepared by the reduction of GalNAc with sodium borohydride (41), melted at 174176”. The melting point of a mixture of Fraction VI with N-acetylgalactosaminitol was 174-176”. Treatment with periodate, followed by reduction with borohydride, converted both Fraction VI and N-acetyl- galactosaminitol into products the RF values of which were identical with that of N-acetylserinol in three solvent systems (A, B, and D). The N-acetyl content of Fraction VI was 12.1% (theoretical, 11.6Q.4

Oligosaccharide V-Mild acid hydrolysis liberated 89% of the fucose from oligosaccharide V, as followed by the increase in reducing substances (Fig. 5), with no detectable release of galactose or N-acetylgalactosaminitol. Fucose was separated from the galactosyl-+N-acetylgalactosaminitol moiety by char- coal-Celite chromatography. The disaccharide had a specific rotation of -48” (Table IV), and was cleaved by&galactosidase. The high negative rotation of the trisaccharicle ( -114.7”, Table IV) suggests that fucose is bound by an o( linkage (42). The specific rotation of oligosaccharide V is close to those of similar oligosaccharides isolated from blood group H substance (-111’ and-ll60 (3)).

The galactose of oligosaccharide V is substituted at carbon atom 2, as shown by the facts that (a) the trisaccharicle is active with galactose oxidase (16), and (5) methylation gave 3,4,6- trimethyl galactose (Table VI, below).

Methylation and acid hydrolysis of the disaccharide, galac- tosyl+N-acetylgalactosaminitol, gave 2,3,4,6-tetramethyl galactose (Table VI, below) and a product that was removed by Dowex 50-H+. Elution of this cationic material and treatment with periodate resulted in the formation of reducing substances, which suggests that galactose had been linked to C-3 of N- acetylgalactosaminitol. Treatment of both the disaccharide galactosyl+N-acetylgalactosaminitol and N-acetylchondrosinitol with periodate, followed in succession by borohydride reduction, acid hydrolysis, and N-acetylation with W-acetic anhydride, gave identical i4C-products (Fig. 6). N-Acetylserinol, the prod- uct arising from N-acetylgalactosaminitol, does not migrate with the above conditions and is not detectable by the periodate- benzidine technique. The absence of radioactivity at the origin eliminates the presence of this 3-carbon fragment. These data suggest that galactose is linked to C-3 of N-acetylgalactosaminitol and not to C-4 as suggested by Katzman and Eylar (10). The results of periodate oxidation experiments (Table V) provide additional evidence for the proposed structure of oligosaccharide V.

Oligosaccharide IV-Mild acid hydrolysis (180 min) released more than 80% of the fucose from oligosaccharide IV with concomitant formation of a trisaccharide which contained GalNAc, galactose, and N-acetylgalactosaminitol (Table IV). The galactose residues of both the parent oligosaccharide and the resulting trisaccharide were resistant to periodate oxidation, indicating that the galactose was substituted at C-3. Methyla-

4The N-acetyl analyses were performed by Crobaugh Labora- tories, Cleveland.

OLIGOSACCHARIDE I

yH20H

OLIGOSACCHARIDE II

NHAc

OLIGOSACCHARIDE III

FIG. 4. Proposed structures for oligosaccharides isolated from PSM. Gly, glycolyl. Oligosaccharides IV, V, and VI are identical with I, II, and III, respectively, with the sialic acid moieties removed.

z30 L-_THE_ORE_TEAL _______ ---------__

w l - d25 - 5

15 30 45 90 105 120 TIME

6(hW;~S)

FIG. 5. Mild acid hydrolysis of oligosaccharide V. A mixture containing 29.2 bmoles of oligosaccharide V in 4 ml of 1 N H&SO, was incubated at 65”; 5-~1 aliquots were removed at the indicated time intervals and placed in 1 ml of water, and reducing values were determined (21). n-Fucose was used as a standard.

tion and acid hydrolysis of oligosaccharide IV gave a dimethyl galactose that chromatographed with 4,6-&methyl galactose (Table VI). Treatment of oligosaccharide IV with N-acetyl- galactosaminidase released 85 y0 of the GalNAc. Since oligosac- charide IV gave a r4C-product identical with oligosaccharide V

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622 Studies on Pig Submaxillary Mucins Vol. 243, No. 3

TABLE IV

Yields, analyses, and specijic rotations of oligosaccharide fractions

I Yield’” Molar ratios

Percentage of dry weight*

A+-PSM Galactose FUCOStY GalNAc N-GN Y-Acetyl- galactos- aminitol

1.06 0.85 1.06 0.52

1.00 1.00

1.00

1.00

1.00 1.00 1.00

0.82 1.05

0.83

1.05

0.16 <0.05

0.98 1.04

-

0.94

99.7 94.5

101.9 103.2 101 .o

91.0

t-6.9”

-10.3 +19.6

-114.7

+19.8

-48

Fraction I. 0 Fraction II. _. _. _. 43 Fraction III. _. 55

Fraction IV.. 0 Fraction V.. 105

Fraction VI.. -c Fraction I minus N-GNd. Fraction II minus N-GNd

II-a...........................

II-b. Fraction IV minus fucosee.. Fraction V minus fucosee..

0.86

0.81

0.91

0.89

0 Yields of oligosaccharides are based on dry weights of materials precipitated with acetone.

b Calculated weight of oligosaccharide (based on analytical values)

Dry weight of oligosaccharide analyzed c -, not tested.

x 100 = percentage of dry weight

d Sialidase treated to remove N-GN. 8 Fucose removed by mild acid hydrolysis.

TABLE V

Results of perioclafe oxidations

Periodate uptake and formaldehyde and formic acid formation were measured after a 24-hour incubation period.

-ANODE

l’i

CATHODE -

:A j: ORIGIN

:

Actual

Oligosaccharide I. ,I 6 .OO

Oligosaccharide III.. _. 1.00 Oligosaccharide IV. 5.00 Oligosaccharide V 5.00

I

moles/mole oligosaccharide

5.82 1.00 1.08 2.00 4.30 1.00 0.96 3.00 5.10 1.00 0.93 2.00

4.90 1.00 0.93 2.00

1.32 1.18

Mannitol standard.. 5.00 5.30 1 2.00 / 1.92 1 4.00 3.72

16 16 I4 I2 IO 6 6 4 2 0

DISTANCE (cm)

FIG. 6. Identification of periodate oxidation products of sub- stituted N-acetylgalactosaminitols. Mixtures containing A, N- acetylchondrosinitol, 1.5 pmoles, or B, galactosyl-+N-acetylgal- actosaminitol, 1.3 pmoles; sodium acetate, pH 4.5, 12 pmoles and NaIO+ 40 rmoles, in a final volume of 0.06 ml, were incubated for 12 hours at 4” in the dark. The reaction was terminated by adding 1 ml of 1 M NaBHd and the mixture was allowed to stand at room temperature for 2 hours with intermittent mixing. Excess borohydride was destroyed with 1 M acetic acid and the solution was applied to a column of Dowex 50-H+. The eluate, evaporated to dryness, was treated with methyl alcohol to remove boric acid. N-Acetylgalactosaminitol (1.1 pmole) was carried through the remaining procedure, together with the oxidized samples. Fol- lowing hydrolysis in 1.0 ml of 4 N HCl for 5 hours, all samples were evaporated to dryness and N-acetylated with’%-acetic anhydride. The W-N-acetylated derivatives and the indicated standards were separated by electrophoresis in borate buffer. Radioactive areas were detected by strip scanning; areas reacting with perio- date-benzidene are cross-hatched. The W-products from galac- tosyl+N-acetylgalactosaminitol and Kacetylchondrosinitol were also identical when chromatographed in Solvents A, D, and G. The remaining oligosaccharides (Table IV), except oligosaccha- ride III and Fraction VI, gave the same W-products when ex- posed to the above conditions.

a Oligosaccharides IV and V gave consistently low values (60

to G5’j$, of theoretical) for periodate consumption and formic acid formation after incubation in water at 4’.

(Fig. 6)) the galactosyl moiety of oligosaccharide IV also appears

to be linked to C-3 of N-acetylgalactosaminitol. A second

radioactive product, not further identified and presumably

arising from the terminal GalT\;Ac residue, was present.

The specific rotation of oligosaccharide IV (+19X’) is close to that reported ($28.7”) for a similar oligosaccharide isolated from blood group A substance (ARL 0.52) (3). N-Acetyl analy- sis of oligosaccharide IV (found, 20.1%; theoretical, 19.8%) indicated that galactosamine and galactosaminitol are present as the N-acetyl derivatives.

Sialic Acid-containing Fractions

Oligosucchuride 1l1--Oligosaccharide III is similar to the disaccharide, il’-acetylneuramlin\-l-(2~6)-N-acetylgalactosamin-

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Issue of February 10, 1968 D. M. Carlson 623

itol, isolated from sheep submaxillary mucin (43, 44), except that it contains N-GN instead of N-AN. Treatment with sialidase yielded only N-GN (95 ‘%) and N-acetylgalactosaminitol. Periodate oxidation followed by borohydride reduction gave two components: one which was absorbed by Dowex l-Cl- and which contained the sialic acid portion, and the other a neutral fragment. Hydrolysis of the neutral material in 4 N HCl for 2 hours at 100” yielded serinol, as identified by borate electrophore- sis and chromatography on borated paper in Solvents A, B, and D. Hydrolysis of the sialic acid-containing material (1 N

HzS04, 30 min, 100”) released ethylene glycol, as detected by paper chromatography in Solvents A and D. These data show that oligosaccharide III contains the same linkage as reported for similar disaccharides from other submaxillary mucins in which sialic acid is linked to C-6 of N-acetylgalactosamine (43).

O~~gosc~~charide II-The fraction designated oligosaccharide II is a mixture of two oligosaccharides (9). Attempts to separate these compounds prior to sialidase treatment were unsuccessful. Sialidase cleaved 89% of the N-GN from oligosaccharide II. Two neutral oligosaccharides, 11-a and II-b, were then isolated. Oligosaccharide II-a contained fucose, galactose, and N-acetyl- galactosaminitol (Table IV), and had the properties of oligosac- charide V. Oligosaccharide II-b contained only galactose and N-acetylgalactosaminitol (Table IV), and was identical with the disaccharide isolated from oligosaccharide V following mild acid hydrolysis. The relative amounts of oligosaccharides 11-a and 11-b varied with different preparations of PSM.

Oligosucchatide I-The most complex oligosaccharide isolated was the pentasaccharide, oligosaccharide I. Sialidase treatment

T.4BLE VI

Chromatographic identi$calion of methylated sugars

The methylated oligosaccharides were acid hydrolyzed, de- ionized, and chromatographed on thin layer chromatographic plates and paper.

Methylated derivative

Oligosaccharide IV. Oligosaccharide V

Oligosaccharide I minus M-GN

Oligosaccharide V mi- nus fucose..

2,3,4,6-Tetramethyl galactose

2,3,4-Trimethyl fucose.

Trimethyl galactoses 2,3,4-...............

2,4,6-............... 3,4,6-...............

Dimethyl galactoses 2,4-.................

3,4-................. 4,6-.................

1

Solvent system II

~TMGC

0.30 0.48

0.30

0.87

0.88

0.87

0.47

0.30

TMF

j.86 b.87

j.86

Solvent Solvent system I system G

I.53

I.84

I.84

0.90

0.67'

0.76 0.67

‘TMF

1.00

L.00

a &h-G, &etramethylplueose.

b RTMF, Rtrimethylfucose*

c 2,3,4-Trimethyl galactose does not migrate under the condi- tions used for borate electrophoresis; 3,4,6-trimethyl galactose and the indicated derivatives from oligosaccharides migrate toward the anode.

TIME WOURS) w. I

FIG. 7. Periodate consumption curve for oligosaccharide IV. A mixture containing, in a final volume of 1.0 ml, sodium acetate, pH 4.5, 200 pmoles, NaIOd, 10 pmoles, and oligosaccharide IV, 1.0 Mmole, was incubated at 4” in the dark. A control incubation lacking the oligosaccharide was included. At the indicated time intervals, 0.1 ml of each was removed, placed in 0.45 ml of a solu- tion containing 0.1 ml of saturated NaHC03 and 5pmoles of AstOs, and titrated with a standard iodine solution.

59 AMS)

\ . d 100

FIG. 8. Precipitin assays. A antiserum (0.1 ml) and the in- dicated amounts of A--PSM or A+-PSM in 0.2 ml of sterile NaCl solution were incubated at 4” overnight. The precipitates were assayed for nitrogen (24).

gave N-GN (91y0) and a product which was identical with oligosaccharide IV. Treatment of 0.08 pmole of oligosaccharide I with periodate followed by reduction with borohydride gave two compounds. The anionic fragment, removed by Dowex l-Cl-, was eluted with 0.1 x NaCl and contained 0.063 pmole of sialic acid, as determined by the resorcinol procedure. Galactose (0.077 /Imole), measured by the anthrone method and, following hydrolysis, with galactose oxidase, was quantitatively eluted from the resin with water. Acid hydrolysis of this latter material and N-acetylation with W-acetic anhydride gave two radioactive compounds which were chromatographically identical with the products prepared in the same manner from oligosaccharide IV. These data support the proposed 1+3 linkage of galactose to N-acetylgalactosaminitol and suggest that N-GN is glycosidically linked to C-6 of N-acetylgalactosaminitol, as in oligosaccharide III.

Periodate Oxidation Studies

Periodate oxidation studies, performed in buffered solutions at pH 4.5, resulted in periodate consumption and formaldehyde formation data which are in agreement with the proposed

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624 Studies on Pig

P

200 OLIGOSACCHARIDE ADDED (mp MOLES)

FIG. 9. Inhibition of the precipitin reaction by oligosaccha- rides. All incubations were carried out at 4” in 0.1 ml of A anti- serum, 5 pg of A+-PSM, and the indicated amounts of oligosac- charides, in a total volume of 0.3 ml.

Submaxillary Mu&s Vol. 243, No. 3

FIG. 10. Thin layer chromatography of methylated standards and oligosaccharides (Solvent H). ChonOHNAc, N-acetylchon- drosinitol; (2X) and (3X) indicate the number of methylation treatments for oligosaccharide IV (see “Methods”).

oligosaccharide structures. The periodate oxidation curve for oligosaccharide IV is shown in Fig. 7. Data on periodate con- sumption and formic acid and formaldehyde formation are given in Table V. Formic acid determinations were performed in water and consistently gave results of 60 to 65% of theoretical; this was in agreement with periodate consumption data obtained under the same conditions.

Quantitative Precipitin Assays

Rabbit antiserum to human type A red cell stroma precipitated A+-PSM, but not A--PSM (Fig. 8). Oligosaccharides I and IV inhibited the reaction of A antiserum with A+-PSM, whereas oligosaccharides II, III, V, and VI were inactive (Fig. 9). These

data support the proposed structures for oligosaccharides I and IV, which contain the same antigenic sugar determinant group- ings as blood group A substance (3).

Methylation Studies

Standards of n-fucose, trehalose, N-acetylchondrosinitol, oligosaccharide V, and oligosaccharide V minus fucose appear to be completely methylated by the procedure described (Fig. 10). Oligosaccharide IV required three treatments to achieve es- sentially complete methylation. Chromatographic identifica- tion of the constituent methylated monosaccharides is given in Table VI.

Hydrolysis of the galactose and fucose methyl ethers with boron tribromide in dichloromethane (45) resulted in the release of galactose and fucose, respectively, as shown by paper electro- phoresis in borate buffer and paper chromatography with Solvent Systems A and J.

DISCUSSION

Studies on the carbohydrate composition of purified PSM (7-9) indicate that N-GN, GalNAc, galactose, and fucose ac- count for almost the entire carbohydrate content. Traces of N-AN, mannose, and GluNAc are found in some fractions, but these components may still represent a small contamination with plasma glycoproteins. Many problems are involved in demon- strating the purity of isolated mucins. Highly charged polymers, such as these, produce viscous solutions which do not adapt readily to the usual physical methods of determining purity and homogeneity (46).

PSM, isolated by different methods, has been shown to be monodisperse in the ultracentrifuge (7, 8)) electrophoretically homogeneous (7, 8), and homogeneous by polyacrylamide gel electrophoresis and immunoelectrophoresis (8). The present investigation shows that at least two immunochemically different glycoproteins are present in pig submaxillary glands. Thus, it is difficult to define a “homogeneous” polymer of this type.

Physical and chemical studies on glycoproteins purified from pooled pig submaxillary glands by different procedures (7,8) have some striking differences. This is particularly true for glycopro- tein II isolated by DEAE-cellulose-Celite column chromatog- raphy (8). High salt concentration (5 N NaCl) failed to elute this tightly adsorbed material. However, 5 N NaCl combined with a gradient of 0.05 N NaOH resulted in the elution of glyco- protein II. This material was found to be of relatively low molecular weight (180,000) and exhibited a low intrinsic viscosity (1.32 dl pm-l), as compared with previous values for PSM of 810,000 and 6.7 dl gm-I, respectively (7). These differences were attributed to dissociation of PSM on DEAE-cellulose under “neutral” conditions and “moderate” ionic strength (8). Fur- ther studies are required, but glycoprotein II may be the result of degradation of the mucin molecule and not a discrete glycopro- tein fraction.

The products of alkaline treatment of mucopolysaccharides, mucins, and blood group substances have been studied exten- sively (47). The mechanism whereby the sugar moiety is released is now commonly accepted as a fi elimination reaction involving glycosidic linkages to the hydroxyl groups of serine or threonine or both. While these linkages have been well defined in sheep and bovine submaxillary mucins (47), the carbohydrate to protein linkage in PSM remains to be studied. The release of

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Issue of February 10, 1968 D. M. Carlson 625

N-acetylgalactosaminitol by alkaline borohydride indicates that the linkage in PSM is the same as that found in other mucins.

We described a series of partially characterized oligosaccharides in a preliminary report (9), including the trisaccharide, fucosyl4 galactosyl+N-acetylgalactosaminitol (oligosaccharide V). Sub- sequently, Katzman and Eylar (10) reported the structure of a neutral trisaccharide isolated from PSM to be cY-L-fucopyranosyl- (l~2)-~-n-galactopyranosyl-(1~4)-N-acetyl-n-galactosaminitol. Sufficient data were presented to define the linkage of fucose to galactose, but these workers failed to provide evidence for the proposed anomeric configurations or the D- or L-sugar configura- tions. Results of the present study provide no evidence for a l-4 linkage of galactose to N-acetylgalactosaminitol in the oligosaccharides isolated from PSM. Instead the data indicate only a l-3 linkage. However, the existence of a l-4 linkage in an oligosaccharide fraction of PSM cannot be completely excluded, since the studies by Katzman and Eylar were per- formed on a trisaccharide that accounted for only 0.1 to 0.2% of the total carbohydrate in the mucin.

The structural complexity of blood group substances A, B, and H must be appreciated. Considerable degradation of the oligosaccharidc chain is encountered during alkaline borohydride treatment, with the formation of galactitol and 3-hexene-l , 2,5,6- tetrol, an unsaturated galactitol derivative (3). Although different conditions were used, neither of these compounds was detected in oligosaccharides from PSiLI. Two oligosaccharides (IV and V) isolated from PSM closely resemble oligosaccharides isolated from A and H blood group substances. They are, respectively, ARL 0.52 and HRL 0.75 (3).

Blood group A activity of oligosaccharide I increases following removal of the sialic acid residue. This result agrees with studies on the inhibition of A-anti iz precipitation by oligosac- charides isolated from blood group A substance (48), in which a second fucose residue, attached to GluNAc (possibly analogous to the N-acetylgalactosaminitol residue in oligosaccharide I), decreased the inhibitory ability. Also, the release of an ionic component may influence the relative serological activity of oligosaccharide I.

Preliminary experiments in this laboratory suggest that mucins isolated from H and I type glands contain the same oligosac- charide chains. If this is so, then the immunochemical differ- ence may be the result of variation in the relative amounts of the different oligosaccharides.

Studies are now in progress, with the precipitin technique, to attempt to show that the oligosaccharide moieties all reside on the same protein chain.

Acknowledgments-The author wishes to thank Dr. G. Schiff- man for his assistance in the microprecipitin assays, Dr. E. McGuire for treating oligosaccharide IV with N-acetylgalactos- aminidase, Dr. P. Stoffyn for suggesting Solvent I for the separa- tion of trimethylgalactose derivatives, Mr. T. Saari for assaying the pig submaxillary gland extracts for blood group activity, and Dr. Judith Ramseyer for assisting in the infrared spectral analyses.

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Don M. Carlson and With the technical assistance of Charles BlackwellPig Submaxillary Mucins

Structures and Immunochemical Properties of Oligosaccharides Isolated from

1968, 243:616-626.J. Biol. Chem. 

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