Structures and Immunochemical Properties of Oligosaccharides Isolated from Pig Submaxillary Mucins

Structures and Immunochemical Properties of Oligosaccharides Isolated from Pig Submaxillary MucinsTHE JOURNAL cm BPXOGICAL CHEMISTRY Vol. 243, No. 3,Issue of February 10, PP. 616426, 1968
Printed in U.S.A.
DON 111. CARLSON~
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
618 Studies on Pig Submaxillary Mucins Vol. 243, No. 3
,O.,. 20.6.
z ; 0.4.
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
No. of animals
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.
Issue of February 10, 1968 D. M. Carlson 619
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
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
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)…