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Alkaline borohydride degradation of blood group H substance
ARCHIVES
OF
Alkaline
BIOCHEMISTRY
AND
BIOPHYSICS
Borohydride
Degradation
R. N. IYER Departments
oj Biochemistry
101-105 (1971)
142,
AND
of Blood
H Substance’
DON iM. CARLSON2v3
and Pediatrics, School of Medicine, Cleveland, Ohio 44106
Received
Group
June 22, 1970; accepted
October
Case Western Reserve Cniversity, 15, 1970
Treatment of blood group H substance at a concentration of 1 mg/ml with NaOH and 1 M NaBHa at 50” results in the release of oligosaccharide side chains minimum of degradation. A procedure for the partial fractionat,ion as well chemical and immunochemical analvses of these reduced oligosaccharides scribed.
Studies on t.he carbohydrate structures of glycoproteins are facilitated by a release of the sugar chains with a minimum of degradation. The ‘Lmucin-type” of glycoprotein is particularly advantageous to such studies because the linkage of carbohydrate to prot,ein is alkali-labile. Presently, the assumption is that the linkage of carbohydrate to protein in all mucins and water-soluble blood group substances involves predominantly N-acetylgalactosamine linked glycosidically to the hydroxyl groups of serine or threonine, or both. Cleavage of this linkage occurs by an alkali-catalyzed peliminabion reaction, which results in the release of t,he sugar moiety (1) according to the following reaction: Hz-C-0-glycosyl
H-&-NH......
OH-
(-J& 1 From the Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, Ohio. This investigation was supported in part by Grants AM-03305 and AM-10335, National Institutes of Health, and by research support from the Greater Cleveland Health Fund. * Research Career Development awardee of the U. S. Public Health Service Grant Number AM11379. 3 Requests for reprints should be addressed to Don M. Calson, Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44100. 101
0.05 N with a as the is de-
O-Glycosyl-serylHz--C II C-NH..-.-
+ Glycosyl-O-
+A Dehydroalanyl-
The reducing group of the oligosaccharide chain is then susceptible to the classical alkali-catalyzed epimerizations and degradations (2, 3). However, reduction of the aldehyde group to the corresponding sugar alcohol will eliminate most, if not all, of the degradation. This undoubtedly prompted Schiffman, Rabat, and Thompson (4) to degrade the blood group substances with alkaline borohydride. Subsequent studies by Rabat et al. (3) have provided unequivocal proof for the chemical structures of the serological determinants of several blood group substances. However, reaction conditions used by these workers (0.26 M NaBH4 in 0.2 N NaOH, 25”) cause considerable degradation of the saccharide chains by alkali-catalyzed “peeling” reactions (3) as well as cleavage of model disaccharides (5). In contrast, alkaline borohydride reduction of pig submaxillary mucins using different reaction conditions (1.0 M NaBH4 in 0.05 N NaOH, 45’) results in essentially no degradation of the sugar chains (6). Thus, in alkaline borohydride the rate of degradation of oligosaccharides competes with the rate of reduction (7).
IYER AND CARLSON
102
The conversion of hexosamines to hexosby aminitols in several glycoproteins alkaline borohydride under different conditions has been studied by Weber and Winzler (8). This communication reports a met)hod for the isolation of oligosaccharides from blood group H substance after treatment with alkaline borohydride. React,ion conditions necessary to cause p-elimination with subsequent reduction of the released saccharide are used. Partial fractionation and chemical and immunochemical analyses on the reduced oligosaccharides are given. MATERIALS
AND METHODS
Unless otherwise stated, all materials were purchased commercially. H-lectin was prepared from Ulex europus seeds obtained from F. W. Schumacher, Sandwich, Mass. Hexene tetrol (truns-3-hexene-threo-1,2,5,6tetrol) was a gift from Dr. R. S. Tipson, Natioual Bureau of Standards. Samples of blood group A and B substances were kindly provided by Dr. K. 0. Lloyd, Columbia University. Blood group H subsance was isolated from human ovarian cyst fluid by phenol extraction (9). Phenol was removed by dialysis and the contents of the dialysis bag were lyophilized yielding a white, fluffy product. The product, not completely soluble in water, was solubilised by incubating with Pronase (50 mg H-substance plus 50 rg of Pronase in 5.0 ml of 0.001 M CaCl*), for 1 hr at 37”. This treatment was necessary to dissolve the glycoprotein for analytical studies. Since the amount of protein contributed by the Pronase was negligible, the soluble material was assayed without further purification. Alternatively, cyst fluid treated first with Pronase followed by phenol extraction gave a watersoluble product which analyzed essentially the same as the product described above. The analytical methods, except for those listed, have been described previously (6). Galactose was determined by the procedure of Dische and Danilchenko (10). AT-Acetylgalactosaminitol, after deacetylation, was assayed by N-acetylation with “C-acetic anhydride (11). Sugars were identified by paper chromatography and paper electrophoresis (6). A solvent system of butanone, acetic acid, and water-saturated boric acid (9: 1: 1, v/v) was used for separating galactose and galactitol (12). High-voltage electrophoresis was performed with a Gilson High Voltage Electrophorator. Radioactive areas on paper were detected with a Packard strip scanner and quantitated in a Pack-
ard Tri-Carb liquid scintillation counter using a toluene counting system. Hexosamines were determined with the amino acid analyzer after hydrolysis as described by Boas (13). Charcoal-celite columns were prepared as described by Whistler and Durso (14). A batch elution technique was used with increasing concentrations of ethanol in water. Hemagglutination-inhibition assays were performed by microtritration (4). Isolation of oligosaccharides. Blood group H substance (450 mg) was treated with 450 ml of 1 M NaBHa in 0.05 N NaOH at 50” for 16 hr (6). The excess borohydride was destroyed by careful addition of acetic acid to pH 5, and the resulting solution was passed through a column, 4 X 30 cm, of Dowex 50-X2 (H+, 50-100 mesh). The fraction eluted with water (F-l) contained more than 95% of the carbohydrate as determined by fucose and galactose analyses. Boric acid was removed from the water eluate as methyl borate by repeated evaporation with methanol and this fraction was again passed through a fresh column of Dowex 50 H+. Remaining traces of boric acid were removed from the fraction eluted with water (F-2). Material absorbed to both Dowex 50 columns was eluted with 2 N HCl (0.5 bed volume followed by an equal volume of water). The acid eluate was neutralized immediately and desalted by passage through a column of Biogel P-2. This cationic fraction is indicated as A-2. Preparation F-2 was further fractionated on charcoal-celite by elution with aqueous ethanol solutions: 3%, lo%, 25%, and finally 50% ethanol which contained 0.5% ammonium hydroxide (v/v). Elution with each alcohol solution was continued until the eluate was negative to the anthrone reagent. RESULTS
The only carbohydrates detected in the H-substance were galactose, fucose, Nacetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Since the Hsubstance contained less than 3% N-acetylneuraminic acid, this sugar was not assayed during fractionation. The carbohydrate compositions of the H-substance and of the isolated oligosaccharide fractions are given in Table I. Almost quantitative recoveries of galactose and fucose were found in the sum of fractions F-2 and A-2. No corrections were made for sampling losses. A loss of SO85% of the N-acetylgalactosamine, with a parallel increase in N-acetylgalactosaminitol, accounted for the decrease in hexosamine contents of F-2.
STUDIES
ON BLOOD GROUP SUBSTAXCES TABLE
RECOVERIES
SUG.\R
Fraction
AND
I-
10%
25% 50%
I
SEROLOGKXL ACTIVITIES OF OLIGOSACCHARIDES ISOUTED BOROHYDRIDE-TREATED BLOOD GROUP H SUBSTANCE
Gala&se
Hexosamine
pmoles/
H-substance F-2 A-2 Charcoal-Celite fractionation of F-2 3%
103
HexosaminitoP
% ___ I 00 76 21
0.99 0.75 0.26
100 76 26
1.33 0.78 0.28
100 59 21
ALK:ILINE
Minimum amount giving inhibition (pmoles fucose/ ml)
urn&s/
H-%bstance
1.26 0.96 0.27
FROM
H%b stance I-
0
0.36 -
0
27 -
0.005
0.4
0.018
1.8
0.008
0.6
0.05
3.8
0.033 0.62 0.23
2.6 49.2 18.0
0.025 0.49 0.15
2.5 49.0 15.0
0.026 0.56 0.23
1.9 42.0 17.0
0.06 0.25~ 0.04
4.5 18.8 3.0
1.27 1.28 -
1.32 1.26 1.33
0.04b 0.8 0.2
No inhibitiond 0.58 0.3
~1Recovery calculated from the original hexosamine value of H-substance. 0 H-substance obtained from Dr. K. 0. Lloyd inhibited at a concentration of 4 fig/ml. c Other samples of blood group H substance gave values ranging from 0.1 to 0.2 pmoles galactosaminitol/mg H-substance. d No inhibition at 5.0 pmolelml. 6 An oligosaccharide isolated by Lloyd et al. (16) from II-substance inhibited at a concentration of 0.30.4 pmole fucose per ml.
The ratio of galactose to fucose was constant in all preparat,ions indicating that there was no select’ive destruction of galactose as would occur during alkaline-degradation. None of the fractions contained detectable amounts of unsaturation as determined by pot’assium permanganat’e and bromine water. The procedures used would have detected 3-5 Fg of hexene tetrol or other unsaturated compounds produced as a result of galactose degradation. 3 % and 10 % Ethanol fractions. Generally less than 5% of the original carbohydrat,e material was found in t,hese fractions. Since disaccharides, and possibly some trisaccharides, are eluted with 10% ethanol, these data suggest that few short-chain saccharides are present. Hexosamine analysis of the 3% et’hanol fraction with the amino acid analyzer showed the presence of an unidentified amino sugar derivative apparently the same as found in earlier studies employing the alkaline borohydride treatment of oligosaccharides (5) and keratosulfate (15). Assuming identical extinction coefficients
for this “unidentified” compound and glucosamine, the ratios of this material to glucosamine in the 3 % and 10% ethanol fractions were 0.81 and 0.01, respectively. Acid hydrolysis, followed by paper chromatography in butanone-acetic acid-water saturated with boric acid, indicated that galactitol was absent from all fractions. Free N-acetylgalactosaminitol was present only in the 3% ethanol fraction. 25% Ethanol fraction,. The 25% ethanol fraction contained about 50% of the carbohydrate initially present in the H-subst,ance (Table I). The results of gel filtration on Sephadex G-25 suggested that the major portion of this material \vas in the molecular weight range of 1000-2000. The molar ratios of galactose : fucose : glucosamine : galactosaminitol were 1.00: 0.79: 0.90: 0.40, respect,ively. 14C-N-Acetylgalactosaminitol was isolated from bot’h the F-2 and the 25% ethanol fractions after acid hydrolysis and N-acetylation wit,h ‘4C-acetic anhydride, and was ident’ified by the following procedures: (1) the isolated compounds migrated during paper chromatography and paper electro-
IYER AND CARLSON
104
phoresis with standard N-acetylgalactosaminitol in systems where it was distinguishable from N-acetylglucosaminitol; and (2) periodate oxidation and sodium borohydride reduction yielded compounds which were chromatographically identical with N-acetylserinol. 50 % Ethanol fraction. Analyses on the 50 % ethanol fraction showed that this fraction contained molar ratios of galactose:fucose: hexosamine (1.00: 0.65: 1.00) similar to the 25 % ethanol fraction. However, the amount of galactosaminitol was considerably lower (0.17). The difference probably indicates t’he presence of higher molecular weight polysaccharides. Immunochemical activity of oligosuccharides. The 25 % and 50% ethanol fractions from the charcoal-celite column were potent inhibitors of the hemagglutination of 0 cells by H-lectin (Table I). No inhibition was detected with the A and B hemagglutinating systems. The H-subst’ance and oligosaccharides studied in this report had antigenic activities similar to t#hosereported by Lloyd et al. (16). DISCUSSION When blood group H substance was treated wit,h alkaline borohydride (1.0 M NaBH4 in 0.05 N NaOH at 50”) oligosaccharides containing N-acetylgalactosaminitol were isolated in good yield. The major oligosaccharide fraction was eluted from charcoal-celite with 25 % ethanol. The results of preliminary studies on this fraction with gel filtration indicated a predominance of material in the 1000-2000 molecular weight range. The extent of hemagglutination inhibition by the 25% ethanol fraction was essentially the same as that reported by Lloyd et al. (16) for oligosaccharides isolated from H-substance. Only small amounts of saccharide materials were found in the 3 % and 10% ethanol fractions. The “unidentified” compound detected with the amino acid analyzer was present, mainly in the 3% ethanol fract)ion from the charcoal-celite column. This compound has been reported in previous studies after alkaline borohydride treatment of N-acetylchondrosine (peak 3) (15) and
similar disaccharides (5). Presumably this unidentified amino sugar derivative arises from the action of sodium borohydride on the alkali degradation product (Morgan-Elson chromogen) of N-acet,ylgalactosamine. The amount of t’his material (peak 3) formed alkaline borohydride treatment during should then be a measure of saccharide degradation. Calculat’ions based on t#his proposal gave less than 5% degradation of the N-acetylgalactosamine released. Oligosaccharides not released from the peptide chain were present in fraction A-2 and possibly to a very small ext,ent in the 50% ethanol fraction from t)he charcoalcelite column. These oligosaccharides are probably linked t,o N-t,erminal serine or threonine residues since the amino group must be substituted to achieve elimination from the peptide chain. However, the presence of the N-acylglycosylamine linkage involving the amide-nitrogen of asparagine (1) remains a possibility. Calculated LLaverage” molecular weights of oligosaccharides. Theoretically, the reduced oligosaccharides should contain 1 mole of N-acetylgalactosaminitol per oligosaccharide moiety. By comparing the sugar ratios of the 25 % ethanol fraction and the 50% ethanol fraction it is possible to calculate an oligosaccharide average molecular weight. Sugar ratios in the 25 % ethanol fraction are : N-acet’ylgalactosaminitol, 1.00; galactose, 2.5; fucose, 2.0; glucosamine, 2.2; t’he calcuated average molecular weight is 1350. Average values for sugar residues in t,he 50 % ethanol fraction are: N-acet(ylgalactosaminitol, 1.00; galactose, 5.8; fucose, 3.8; glucosamine, 5.8; the calculated average molecular weight is 2880. A small error in N-acetylgalactosaminitol analyses on the 50% ethanol fraction could result in a high calculated average molecular weight. “Calculated” molecular weight,s for the composite structure of blood group H substance proposed by Lloyd, Rabat, and Licerio (3) could range from a simple straight chain of 7 sugar residues (mol wt, 1240) to a complex branched chain of about 14 sugar residues (mol wt approximately 2400).
STUDIES
ON BLOOD GROUP SUBSTANCES
REFERENCES 1. NEUBERGER, A., GOTTSCWBLK, A., AND MARSHALL, R. D., in “Glycoproteins-Their Composition, Structure and Function” (A. Gottschalk, ed.), p. 273, Elsevier, New York (1966). 2. B.4~~017, C. E., Advan. Carbohyd. Chem. 9. 91 (1954). 3. LLOYD, K. O., KABAT, E. A., AND LICERIO, E., Biochemistry 7, 2976 (1968). 4. SCHIFFMAN, G., KABAT, E. A., AND THOMPSON, W., Biochemistry 3, 113 (1964). 5. LLOYD, K. O., AND KABAT, E. A., Carbohyd. Res. 9, 41 (1969). 6. CARLSON, D. M., J. Biol. Chem., 243, 616 (1968). 7; MAYO, J. W., AND CARLSON, D. M., in press, Carbohyd.
Res.
105
8. WEBER, P., AND WINZLER, R. J., Arch. Biothem. Biophys. 129, 534 (1969). 9. MORGAN, W. T. J., AND KING, H. K., Biochem. J. 37, 640 (1943). 10. DISCHE, Z., AND DANIU~HENKO, A., Anal. Biochem. 21, 119 (1967). 11. C.IRLSON, D. M., Anal. Biochem., 20, 195 (1967). 12. REM, W. R., AND REYNOLDS, T., Nature London 181, 767 (1958). 13. BOAS, N. F., J. Biol. Chem. 204, 553 (1953). 14. WHISTLER, R. L., AND DURSO, D. F., J. Amer. Chem. Sot. 72, 677 (1959). 16. BRAY, B. A., LIEBERMAN, R., AND MEYER, K. J. Biol. Chem. 242, 3373 (1967). 16. LLOYD, K. O., KABAT, E. A., L.QYUG, E. J., AND GRUEZO, F., Biochemistry& 1489 (1966)
OF
Alkaline
BIOCHEMISTRY
AND
BIOPHYSICS
Borohydride
Degradation
R. N. IYER Departments
oj Biochemistry
101-105 (1971)
142,
AND
of Blood
H Substance’
DON iM. CARLSON2v3
and Pediatrics, School of Medicine, Cleveland, Ohio 44106
Received
Group
June 22, 1970; accepted
October
Case Western Reserve Cniversity, 15, 1970
Treatment of blood group H substance at a concentration of 1 mg/ml with NaOH and 1 M NaBHa at 50” results in the release of oligosaccharide side chains minimum of degradation. A procedure for the partial fractionat,ion as well chemical and immunochemical analvses of these reduced oligosaccharides scribed.
Studies on t.he carbohydrate structures of glycoproteins are facilitated by a release of the sugar chains with a minimum of degradation. The ‘Lmucin-type” of glycoprotein is particularly advantageous to such studies because the linkage of carbohydrate to prot,ein is alkali-labile. Presently, the assumption is that the linkage of carbohydrate to protein in all mucins and water-soluble blood group substances involves predominantly N-acetylgalactosamine linked glycosidically to the hydroxyl groups of serine or threonine, or both. Cleavage of this linkage occurs by an alkali-catalyzed peliminabion reaction, which results in the release of t,he sugar moiety (1) according to the following reaction: Hz-C-0-glycosyl
H-&-NH......
OH-
(-J& 1 From the Departments of Biochemistry and Pediatrics, Case Western Reserve University, Cleveland, Ohio. This investigation was supported in part by Grants AM-03305 and AM-10335, National Institutes of Health, and by research support from the Greater Cleveland Health Fund. * Research Career Development awardee of the U. S. Public Health Service Grant Number AM11379. 3 Requests for reprints should be addressed to Don M. Calson, Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44100. 101
0.05 N with a as the is de-
O-Glycosyl-serylHz--C II C-NH..-.-
+ Glycosyl-O-
+A Dehydroalanyl-
The reducing group of the oligosaccharide chain is then susceptible to the classical alkali-catalyzed epimerizations and degradations (2, 3). However, reduction of the aldehyde group to the corresponding sugar alcohol will eliminate most, if not all, of the degradation. This undoubtedly prompted Schiffman, Rabat, and Thompson (4) to degrade the blood group substances with alkaline borohydride. Subsequent studies by Rabat et al. (3) have provided unequivocal proof for the chemical structures of the serological determinants of several blood group substances. However, reaction conditions used by these workers (0.26 M NaBH4 in 0.2 N NaOH, 25”) cause considerable degradation of the saccharide chains by alkali-catalyzed “peeling” reactions (3) as well as cleavage of model disaccharides (5). In contrast, alkaline borohydride reduction of pig submaxillary mucins using different reaction conditions (1.0 M NaBH4 in 0.05 N NaOH, 45’) results in essentially no degradation of the sugar chains (6). Thus, in alkaline borohydride the rate of degradation of oligosaccharides competes with the rate of reduction (7).
IYER AND CARLSON
102
The conversion of hexosamines to hexosby aminitols in several glycoproteins alkaline borohydride under different conditions has been studied by Weber and Winzler (8). This communication reports a met)hod for the isolation of oligosaccharides from blood group H substance after treatment with alkaline borohydride. React,ion conditions necessary to cause p-elimination with subsequent reduction of the released saccharide are used. Partial fractionation and chemical and immunochemical analyses on the reduced oligosaccharides are given. MATERIALS
AND METHODS
Unless otherwise stated, all materials were purchased commercially. H-lectin was prepared from Ulex europus seeds obtained from F. W. Schumacher, Sandwich, Mass. Hexene tetrol (truns-3-hexene-threo-1,2,5,6tetrol) was a gift from Dr. R. S. Tipson, Natioual Bureau of Standards. Samples of blood group A and B substances were kindly provided by Dr. K. 0. Lloyd, Columbia University. Blood group H subsance was isolated from human ovarian cyst fluid by phenol extraction (9). Phenol was removed by dialysis and the contents of the dialysis bag were lyophilized yielding a white, fluffy product. The product, not completely soluble in water, was solubilised by incubating with Pronase (50 mg H-substance plus 50 rg of Pronase in 5.0 ml of 0.001 M CaCl*), for 1 hr at 37”. This treatment was necessary to dissolve the glycoprotein for analytical studies. Since the amount of protein contributed by the Pronase was negligible, the soluble material was assayed without further purification. Alternatively, cyst fluid treated first with Pronase followed by phenol extraction gave a watersoluble product which analyzed essentially the same as the product described above. The analytical methods, except for those listed, have been described previously (6). Galactose was determined by the procedure of Dische and Danilchenko (10). AT-Acetylgalactosaminitol, after deacetylation, was assayed by N-acetylation with “C-acetic anhydride (11). Sugars were identified by paper chromatography and paper electrophoresis (6). A solvent system of butanone, acetic acid, and water-saturated boric acid (9: 1: 1, v/v) was used for separating galactose and galactitol (12). High-voltage electrophoresis was performed with a Gilson High Voltage Electrophorator. Radioactive areas on paper were detected with a Packard strip scanner and quantitated in a Pack-
ard Tri-Carb liquid scintillation counter using a toluene counting system. Hexosamines were determined with the amino acid analyzer after hydrolysis as described by Boas (13). Charcoal-celite columns were prepared as described by Whistler and Durso (14). A batch elution technique was used with increasing concentrations of ethanol in water. Hemagglutination-inhibition assays were performed by microtritration (4). Isolation of oligosaccharides. Blood group H substance (450 mg) was treated with 450 ml of 1 M NaBHa in 0.05 N NaOH at 50” for 16 hr (6). The excess borohydride was destroyed by careful addition of acetic acid to pH 5, and the resulting solution was passed through a column, 4 X 30 cm, of Dowex 50-X2 (H+, 50-100 mesh). The fraction eluted with water (F-l) contained more than 95% of the carbohydrate as determined by fucose and galactose analyses. Boric acid was removed from the water eluate as methyl borate by repeated evaporation with methanol and this fraction was again passed through a fresh column of Dowex 50 H+. Remaining traces of boric acid were removed from the fraction eluted with water (F-2). Material absorbed to both Dowex 50 columns was eluted with 2 N HCl (0.5 bed volume followed by an equal volume of water). The acid eluate was neutralized immediately and desalted by passage through a column of Biogel P-2. This cationic fraction is indicated as A-2. Preparation F-2 was further fractionated on charcoal-celite by elution with aqueous ethanol solutions: 3%, lo%, 25%, and finally 50% ethanol which contained 0.5% ammonium hydroxide (v/v). Elution with each alcohol solution was continued until the eluate was negative to the anthrone reagent. RESULTS
The only carbohydrates detected in the H-substance were galactose, fucose, Nacetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid. Since the Hsubstance contained less than 3% N-acetylneuraminic acid, this sugar was not assayed during fractionation. The carbohydrate compositions of the H-substance and of the isolated oligosaccharide fractions are given in Table I. Almost quantitative recoveries of galactose and fucose were found in the sum of fractions F-2 and A-2. No corrections were made for sampling losses. A loss of SO85% of the N-acetylgalactosamine, with a parallel increase in N-acetylgalactosaminitol, accounted for the decrease in hexosamine contents of F-2.
STUDIES
ON BLOOD GROUP SUBSTAXCES TABLE
RECOVERIES
SUG.\R
Fraction
AND
I-
10%
25% 50%
I
SEROLOGKXL ACTIVITIES OF OLIGOSACCHARIDES ISOUTED BOROHYDRIDE-TREATED BLOOD GROUP H SUBSTANCE
Gala&se
Hexosamine
pmoles/
H-substance F-2 A-2 Charcoal-Celite fractionation of F-2 3%
103
HexosaminitoP
% ___ I 00 76 21
0.99 0.75 0.26
100 76 26
1.33 0.78 0.28
100 59 21
ALK:ILINE
Minimum amount giving inhibition (pmoles fucose/ ml)
urn&s/
H-%bstance
1.26 0.96 0.27
FROM
H%b stance I-
0
0.36 -
0
27 -
0.005
0.4
0.018
1.8
0.008
0.6
0.05
3.8
0.033 0.62 0.23
2.6 49.2 18.0
0.025 0.49 0.15
2.5 49.0 15.0
0.026 0.56 0.23
1.9 42.0 17.0
0.06 0.25~ 0.04
4.5 18.8 3.0
1.27 1.28 -
1.32 1.26 1.33
0.04b 0.8 0.2
No inhibitiond 0.58 0.3
~1Recovery calculated from the original hexosamine value of H-substance. 0 H-substance obtained from Dr. K. 0. Lloyd inhibited at a concentration of 4 fig/ml. c Other samples of blood group H substance gave values ranging from 0.1 to 0.2 pmoles galactosaminitol/mg H-substance. d No inhibition at 5.0 pmolelml. 6 An oligosaccharide isolated by Lloyd et al. (16) from II-substance inhibited at a concentration of 0.30.4 pmole fucose per ml.
The ratio of galactose to fucose was constant in all preparat,ions indicating that there was no select’ive destruction of galactose as would occur during alkaline-degradation. None of the fractions contained detectable amounts of unsaturation as determined by pot’assium permanganat’e and bromine water. The procedures used would have detected 3-5 Fg of hexene tetrol or other unsaturated compounds produced as a result of galactose degradation. 3 % and 10 % Ethanol fractions. Generally less than 5% of the original carbohydrat,e material was found in t,hese fractions. Since disaccharides, and possibly some trisaccharides, are eluted with 10% ethanol, these data suggest that few short-chain saccharides are present. Hexosamine analysis of the 3% et’hanol fraction with the amino acid analyzer showed the presence of an unidentified amino sugar derivative apparently the same as found in earlier studies employing the alkaline borohydride treatment of oligosaccharides (5) and keratosulfate (15). Assuming identical extinction coefficients
for this “unidentified” compound and glucosamine, the ratios of this material to glucosamine in the 3 % and 10% ethanol fractions were 0.81 and 0.01, respectively. Acid hydrolysis, followed by paper chromatography in butanone-acetic acid-water saturated with boric acid, indicated that galactitol was absent from all fractions. Free N-acetylgalactosaminitol was present only in the 3% ethanol fraction. 25% Ethanol fraction,. The 25% ethanol fraction contained about 50% of the carbohydrate initially present in the H-subst,ance (Table I). The results of gel filtration on Sephadex G-25 suggested that the major portion of this material \vas in the molecular weight range of 1000-2000. The molar ratios of galactose : fucose : glucosamine : galactosaminitol were 1.00: 0.79: 0.90: 0.40, respect,ively. 14C-N-Acetylgalactosaminitol was isolated from bot’h the F-2 and the 25% ethanol fractions after acid hydrolysis and N-acetylation wit,h ‘4C-acetic anhydride, and was ident’ified by the following procedures: (1) the isolated compounds migrated during paper chromatography and paper electro-
IYER AND CARLSON
104
phoresis with standard N-acetylgalactosaminitol in systems where it was distinguishable from N-acetylglucosaminitol; and (2) periodate oxidation and sodium borohydride reduction yielded compounds which were chromatographically identical with N-acetylserinol. 50 % Ethanol fraction. Analyses on the 50 % ethanol fraction showed that this fraction contained molar ratios of galactose:fucose: hexosamine (1.00: 0.65: 1.00) similar to the 25 % ethanol fraction. However, the amount of galactosaminitol was considerably lower (0.17). The difference probably indicates t’he presence of higher molecular weight polysaccharides. Immunochemical activity of oligosuccharides. The 25 % and 50% ethanol fractions from the charcoal-celite column were potent inhibitors of the hemagglutination of 0 cells by H-lectin (Table I). No inhibition was detected with the A and B hemagglutinating systems. The H-subst’ance and oligosaccharides studied in this report had antigenic activities similar to t#hosereported by Lloyd et al. (16). DISCUSSION When blood group H substance was treated wit,h alkaline borohydride (1.0 M NaBH4 in 0.05 N NaOH at 50”) oligosaccharides containing N-acetylgalactosaminitol were isolated in good yield. The major oligosaccharide fraction was eluted from charcoal-celite with 25 % ethanol. The results of preliminary studies on this fraction with gel filtration indicated a predominance of material in the 1000-2000 molecular weight range. The extent of hemagglutination inhibition by the 25% ethanol fraction was essentially the same as that reported by Lloyd et al. (16) for oligosaccharides isolated from H-substance. Only small amounts of saccharide materials were found in the 3 % and 10% ethanol fractions. The “unidentified” compound detected with the amino acid analyzer was present, mainly in the 3% ethanol fract)ion from the charcoal-celite column. This compound has been reported in previous studies after alkaline borohydride treatment of N-acetylchondrosine (peak 3) (15) and
similar disaccharides (5). Presumably this unidentified amino sugar derivative arises from the action of sodium borohydride on the alkali degradation product (Morgan-Elson chromogen) of N-acet,ylgalactosamine. The amount of t’his material (peak 3) formed alkaline borohydride treatment during should then be a measure of saccharide degradation. Calculat’ions based on t#his proposal gave less than 5% degradation of the N-acetylgalactosamine released. Oligosaccharides not released from the peptide chain were present in fraction A-2 and possibly to a very small ext,ent in the 50% ethanol fraction from t)he charcoalcelite column. These oligosaccharides are probably linked t,o N-t,erminal serine or threonine residues since the amino group must be substituted to achieve elimination from the peptide chain. However, the presence of the N-acylglycosylamine linkage involving the amide-nitrogen of asparagine (1) remains a possibility. Calculated LLaverage” molecular weights of oligosaccharides. Theoretically, the reduced oligosaccharides should contain 1 mole of N-acetylgalactosaminitol per oligosaccharide moiety. By comparing the sugar ratios of the 25 % ethanol fraction and the 50% ethanol fraction it is possible to calculate an oligosaccharide average molecular weight. Sugar ratios in the 25 % ethanol fraction are : N-acet’ylgalactosaminitol, 1.00; galactose, 2.5; fucose, 2.0; glucosamine, 2.2; t’he calcuated average molecular weight is 1350. Average values for sugar residues in t,he 50 % ethanol fraction are: N-acet(ylgalactosaminitol, 1.00; galactose, 5.8; fucose, 3.8; glucosamine, 5.8; the calculated average molecular weight is 2880. A small error in N-acetylgalactosaminitol analyses on the 50% ethanol fraction could result in a high calculated average molecular weight. “Calculated” molecular weight,s for the composite structure of blood group H substance proposed by Lloyd, Rabat, and Licerio (3) could range from a simple straight chain of 7 sugar residues (mol wt, 1240) to a complex branched chain of about 14 sugar residues (mol wt approximately 2400).
STUDIES
ON BLOOD GROUP SUBSTANCES
REFERENCES 1. NEUBERGER, A., GOTTSCWBLK, A., AND MARSHALL, R. D., in “Glycoproteins-Their Composition, Structure and Function” (A. Gottschalk, ed.), p. 273, Elsevier, New York (1966). 2. B.4~~017, C. E., Advan. Carbohyd. Chem. 9. 91 (1954). 3. LLOYD, K. O., KABAT, E. A., AND LICERIO, E., Biochemistry 7, 2976 (1968). 4. SCHIFFMAN, G., KABAT, E. A., AND THOMPSON, W., Biochemistry 3, 113 (1964). 5. LLOYD, K. O., AND KABAT, E. A., Carbohyd. Res. 9, 41 (1969). 6. CARLSON, D. M., J. Biol. Chem., 243, 616 (1968). 7; MAYO, J. W., AND CARLSON, D. M., in press, Carbohyd.
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8. WEBER, P., AND WINZLER, R. J., Arch. Biothem. Biophys. 129, 534 (1969). 9. MORGAN, W. T. J., AND KING, H. K., Biochem. J. 37, 640 (1943). 10. DISCHE, Z., AND DANIU~HENKO, A., Anal. Biochem. 21, 119 (1967). 11. C.IRLSON, D. M., Anal. Biochem., 20, 195 (1967). 12. REM, W. R., AND REYNOLDS, T., Nature London 181, 767 (1958). 13. BOAS, N. F., J. Biol. Chem. 204, 553 (1953). 14. WHISTLER, R. L., AND DURSO, D. F., J. Amer. Chem. Sot. 72, 677 (1959). 16. BRAY, B. A., LIEBERMAN, R., AND MEYER, K. J. Biol. Chem. 242, 3373 (1967). 16. LLOYD, K. O., KABAT, E. A., L.QYUG, E. J., AND GRUEZO, F., Biochemistry& 1489 (1966)