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Alkali-labile oligosaccharides of brain glycoproteins
Biochimica et Biophysica Acta, 304 (1973) 421--429 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 27095
ALKALI-LABILE OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
RENI~E K. MARGOLIS and R I C H A R D U. MARGOLIS
Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, N.Y. 11203 and Department of Pharmacology, New York University School of Medicine, New York, N.Y. 10016 (U.S.A.) (Received November 13th, 1972)
SUMMARY
Treatment of glycopeptides prepared from rabbit brain glycoproteins with NaOH or NaOH-NaBH4 released approximately 10 7o of the carbohydrate as low molecular weight oligosaccharides. After treatment with alkali alone, a disaccharide and a trisaccharide were isolated by gel filtration and preparative paper chromatography, and have been identified as N-acetylneuraminyl-(2--,6)-O-2-acetamido-2deoxy-D-galactose (Component B-I) and di-N-acetylneuraminyl-D-galactose (Component B-III). The N-acetylgalactosamine in Component B-I was obtained as preformed Morgan-Elson chromogen, and Components B-I and B-III were not obtained when the glycopeptides were treated with alkaline NaBH4 under conditions which prevent peeling. These results indicate that the trisaccharide (Component B-III) was originally present as a substituent at C-3 of the N-acetylgalactosamine involved in the carbohydrate-peptide linkage, and was eliminated as a result of alkali treatment. A disaccharide (Component M) was isolated after alkaline NaBH4 treatment of the desialylated glycopeptides, and yielded equimolar amounts of galactose and galactosaminitol after hydrolysis. Smith degradation of Component M gave galactose and threosaminitol, indicating that the disaccharide was galactopyranosyl-(1 --, 3)-0-2aeetamido-2-deoxy-D-galactitol. From the isolation of Components B-I, B-III and M after NaOH and N a O H NaBH4 treatment of the glycopeptides, it is concluded that the major alkali-labile oligosaccharide of brain glycoproteins is the pentasaccharide di-N-acetylneuraminylD- galactopyranosyl- (1 --, 3) [N- acetylneuraminyl- (2 ~ 6) ]- O-2-acetamido-2-deoxy-Dgalactose.
INTRODUCTION"
Treatment of glycopeptides prepared from glycoproteins of rat and rabbit brain with NaOH-NaBH4 leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, ~-aminobutyric acid and galactosaminitol ~. These results indicate that N-acetylgalaetosamine at the reducing end of oligosaccharide chains in brain glycoproteins is
422
R.K. MARGOLIS, R. U. MARGOLIS
linked O-glycosidically to the hydroxyl groups of serine and threonine residues. Although most of the carbohydrate in brain glycoproteins appears to be linked by N-acetylglucosaminylasparagine linkages, it was found that approximately 10 "~, of the sialic acid, hexose and hexosamine in rat and rabbit brain glycoproteins is present in alkali-labile oligosaccharides. This report describes the partial structure of a pentasaccharide which accounts for the major portion of the alkali-labile carbohydrate in rabbit brain glycoproteins. MATERIALS AND METIffODS Materials
Rabbit brains were carefully cleaned of blood, superficial blood vessels, and meninges, and glycopeptides were prepared from brain glycoproteins as described previouslyL 2. The procedure consists of digestion of the lipid-free protein residue of brain with pronase, and precipitation of nucleic acids and acid mucopolysaccharides with cetylpyridinium chloride. Excess cetylpyridinium chloride is precipitated with KSCN, and the glycopeptides are recovered from the dialyzed supernatant by lyophilization. Glucosaminitol was a gift of Dr P. Weber, and galactosaminitol was prepared by NaBH 4 reduction of N-acetylgalactosamine 3 followed by acid hydrolysis (3 M HCI, 3 h, 100 °C). Chondrosine was obtained from Miles Laboratories, Kankakee, Ill., and N-acetylchondrosine was prepared as described by Bray et aL 4. N-acetylchondrosinitol was obtained by reduction of N-acetylchondrosine with N a B H , , and threosaminitol was prepared by Smith degradation (see below) of N-acetylchondrosinitol. Clostridium perfringens neuraminidase was from Worthington Biochemical Corp., Freehold, N. J., and Eseheriehia coli fl-galactosidase was from Boehringer Mannheim Corp., New York, N.Y. Methods
Free and bound sialic acid were determined differentially either by the thiobarbituric acid method 5 or by the periodate-resorcinol method of Jourdian et al. 6. For the release of bound sialic acid, samples were hydrolyzed for 1 h in 0.05 M H2SO, at 80 °C, and the values obtained were corrected for destruction during hydrolysis. Sulfate was measured by the barium chloranilate method of Spencer 7. Methods for the determination of total neutral sugar, galactose (using D-galactose dehydrogenase), fucose and uronic acid have been described previously 2. Glucose was measured spectrophotometrically in glycopeptide hydrolysates (1 M HCI, 3 h, 100 °C) by means of yeast hexokinase and glucose-6-phosphate dehydrogenase a, and mannose was determined enzymatically 9 after adsorption of hexosamines on a short column of Dowex
50 (H +). Preformed Morgan-Elson chromogen t° was determined by a modification of the method of Reissig et aL 11 in which the heating step after the addition of potassium tetraborate was omitted 4. Standards of N-acetylgalactosamine were carried through the test as described by Reissig et al. l l Hexosamines and amino sugar alcohols were separated on a 56-cm column of Beckman UR-30 resin using a Beckman Model 120C amino acid analyzer. The columns were eluted either with a 0.35 M sodium citrate buffer (pH 5.06) containing 0.3 M
O L I G O S A C C H A R I D E S OF BRAIN GLYCOPROTEINS
423
boric acid 12, or with the pH 5.29 citrate-borate buffer described by Weber and Winzler 13. For the quantitation of amino sugars, samples were hydrolyzed for 8 h at 100 °C in 4 M HCI, evaporated to dryness in a rotary evaporator, and redissolved in 0.02 M HC1 for application to the amino acid analyzer. These hydrolysis conditions gave greater than 95 9/0 recovery of hexosamine and hexosaminitol standards. When NaBH4 reduction was used to determine the nature of the reducing end in oligosaccharides, treatment was carried out for 6 h at 4 °C with 0.5 M NaBH4, after which the solution was adjusted to pH 6 with acetic acid and passed through a small column of Dowex 50 (H ÷). The eluate was concentrated to dryness under reduced pressure and boric acid was removed as trimethylborate by repeated evaporation with HCl-methanol (1 : 1000, v/v). For periodate oxidation or Smith degradation, glycopeptides or oligosaccharides, at a concentration of less than 3 mg/ml, were treated with 0.03 M sodium metaperiodate in 0.1 M sodium acetate buffer, pH 4.0, at 4 °C in the dark. After 48 h excess periodate was destroyed with ethyleneglycol. To measure the extent of destruction of hexosamines, samples were evaporated to dryness under vacuum, hydrolyzed, and analyzed for glucosamine and galactosamine using the amino acid analyzer. For Smith degradations, periodate-treated samples (1-10 mg/ml) were reduced with 1 M NaBH4 (24 h at 4 °C). Excess NaBH4 was destroyed with glacial acetic acid, and the solution was desalted by gel filtration on a 1 cm × 50 cm column of Sephadex G-10. After concentration in a rotary evaporator, the residue was hydrolyzed for 45 rain in 4 M HC1 at 100 °C and neutralized with NaOH. Paper chromatography was performed both on Whatman No. 1 and on prewashed Whatman No. 3 MM paper (for preparative separations) using the following solvent systems: I, butanol-acetic acid-water (4 : 1 : 5, by vol., upper phase); II, ethyl acetate-acetic acid-water ( 6 : 3 : 2 , by vol.); III, propanol--ethyl acetate-water (7 : 1 : 2, by vol.); IV, ethyl acetate-pyridine-butanol-butyric acid-water (10 : 10 : 5 : 1 : 5, by vol.); V, pyridine-acetic acid-butanol-water (20 : 6 : 30 : 24, by vol.); VI, ethanol-pyridine-water-ammonia (30 : 3 0 : 4 0 : 2 . 5 , by vol.); and VII, glycerol-butanolacetone-water-ammonia (5 : 35 : 30 : 30 : 2.5, by vol.). Oligosaccharides were hydrolyzed for 2 h in 2 M trifluoroacetic acid at 120 °C, and sugars on paper chromatograms were detected with alkaline AgNO 3. RESULTS
The composition of the rabbit brain glycopeptides used in this study is given in Table I. When these glycopeptides were treated with 0.2 M NaBH 4 in 0.2 M NaOH for 48 h at 25 °C and analyzed for amino acids and hexosamines 1, a peak was observed on the amino acid analyzer at the same elution volume as Peak 3 described by Bray et aL 4, which they tentatively identified as a reduction product of the unsaturated chromogen I postulated by Kuhn and Kriiger I o. However, when the glycopeptides were treated at 45 °C with 0.4 M NaBH4 in 0.02 M NaOH, Peak 3 was much reduced in size or absent. Similar alkaline NaBH4 treatment conditions have been shown to favor reduction of N-acetylgalactosamine residues at the reducing ends of oligosaccharides released by fl-elimination, in preference to alkali-mediated "peeling" of substituents from C-3 (refs 14, 15). It therefore appears likely that the difference between the galactosamine destroyed (7 pmoles/100 mg) and the galactosaminitol produced
424
R. K. M A R G O L I S , R. U. M A R G O L I S
TABLE I COMPOSITION
OF GLYCOPEPTIDFS
Component
RABBIT BRAIN
GLYCOPROTEINS
of moisture-free weight*
N-Acetylglucosamine** N-Acetylgalactosamine** Galactose Mannose Fucose Glucose N-Acetylneuraminic acid A m i n o acids Sulfate*** Phosphorus Ash
19.5 3. I 14.6 8.4 4.2 0.2 20.6 21.4 I. I 0.3 1.5
Total
94.9
* ** during ***
FROM
W a t e r content was 7.0 ~0, as d e t e r m i n e d by drying to c o n s t a n t weight in vacuo at 60 °C. D e t e r m i n e d using an a u t o m a t e d a m i n o acid analyzer. Values are corrected for destruction hydrolysis a n d expressed as acetylated sugars. Uronic acid was n o t present in these glycopeptide preparations (cf. ref. 2t.
(2/~moles/100 mg) by alkaline NaBH 4 treatment of brain glycopeptides under the milder reducing conditions I can be accounted for as reduced Morgan-Elson chromogen (i.e., reduced chromogen I of Kuhn and Krfiger).
Components B-I, B-H and B-Ill Rat and rabbit brain glycopeptides were also treated at a concentration of 10-30 mg/ml with 0.2 M NaOH at 25 '~C under nitrogen, and neutralized with HCI after 48 h. This alkali treatment without NaBH~ resulted in cleavage of O-glycosidic carbohydrate-peptide linkages and yielded preformed Morgan-Elson chromogen in an amount 10-20 ~ greater than that of the N'acetylgalactosamine destroyed. Since only N-acetylhexosamines which are substituted in the 3-position (and unsubstituted at C-4) react in the modified Morgan-Elson reaction used 4, it would appear that most of the N-acetylgalactosamine linked to serine and threonine, and destroyed by alkali treatment, is also 3-substituted. The results of periodate oxidation studies are consistent with this assumption, insofar as only 17 ~ and 20'3o of the N-acetylgalactosamine in glycopeptides from rat and rabbit brain, respectively, was destroyed by periodate. When glycopeptides prepared from rat or rabbit brain glycoproteins were treated with 0.2 M N a O H (withot, t NaBH4) and submitted to gel filtration on Sephadex G-25, several retarded peaks containing sialic acid were observed, and fractions were pooled as indicated in Fig. 1. The major peak of alkali-stable glycopeptides (A) was followed by two retarded peaks containing bound sialic acid (B), as well as a peak of free sialic acid (C). The free sialic acid was evidently released by the nonspecific alkaline hydrolysis of a small percentage of the total sialic acid in the glycopeptides, rather than by peeling, since retreatment of glycopeptides with 0.2 M NaOH after prior alkaline NaBH4 treatment under conditions shown to prevent peeling (0.4 M
OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
425
1.2 1.0 A
I,--- B--'l'-c--I-----
i 0.8 0.6
0 - --'1
M-EIC
-
0,4 0.2
0
I
90 102
I
I
I
I
ll4
126
138
150
I
16;) EFFLUENT VOLUME (mi)
Fig. 1. Rabbit brain glycopeptides were treated with 0.2 M NaOH for 48 h at 25 °C under nitrogen, neutralized with I-[C1, and aliquots containing 130 mg of alkali-treated glycopeptides were eluted with water from a 2 cm x 58 cm column of Sephadex G-25. Free (O-O) and bound (Q-Q) sialic acid were determined by the thiobarbituric method. Free sialic acid was also identified by its chromatographic mobility in Solvents I, II and III. Peak A, containing the alkali-stable glycopeptides, emerged mostly with the void volume as a single symmetrical peak. Preformed Morgan-Elson chromogen (M-E C) was measured as described under Methods, and appeared as two peaks (90 ~o in B and 10 ~o in D) at the positions designated by arrows. NANA -- N-acetylneuraminicacid. NaBH4, 0.02 M N a O H , 16 h, 45 °C) resulted in the release of approximately the same amount of free sialic acid. 90 % of the preformed Morgan-Elson chromogen was eluted at a position coinciding with the second retarded peak of bound sialic acid, while the remaining 10 ~ appeared in Fraction D, slightly ahead of a glucose marker. When Fraction A, obtained by 0.2 M N a O H treatment of the glycopeptides, was subsequently treated with 0.5 M NaBH4, no galactosaminitol was detected after acid hydrolysis. This finding demonstrates that no large alkali-labile oligosaccharides containing N-acetylgalactosamine at the reducing end were present in the fraction of alkali-stable glycopeptides excluded from Sephadex G-25, and implies that all of the alkali-labile carbohydrate is present in oligosaccharide chains which undergo at least some degree of peeling. The tubes comprising Fraction B were pooled and separated into three components by preparative paper chromatography in Solvent I. The most rapidly migrating component had an Rtactos e of 1.7, and was designated Component B-I. It contained only preformed Morgan-Elson chromogen and sialic acid, but no hexose or hexosamine. F r o m its composition and its elution position on G-25 between free sialic acid and the first retarded peak of bound sialic acid (which was shown to contain trisaccharides), Component B-I appeared to be a disaccharide. The molar ratio of Morgan-Elson chromogen to bound sialic acid (measured by the periodate-resorcinol method) was 1.82 : 1.00. When an aliquot of B-I was treated with neuraminidase and reapplied to Sephadex G-25, all of the Morgan-Elson chromogen was eluted in Fraction D, at an effluent volume slightly less than that of glucose. The high apparent molar ratio of Morgan-Elson chromogen to sialic acid is in agreement with previous findings that substitution at the C-3 position greatly enhances color formation in the Morgan-Elson reaction 16-19. Two other more slowly-migrating components of the pooled B fractions could also be resolved, and were designated Component B-II (Rt,c,os ~ 1.06) and Component
426
R.K. MARGOLIS, R. U. MARGOLIS
B-III (R,.c,ose 0.80). Component B-II contained sialic acid, galactose, glucosamine and galactosamine, in molar ratios of 1.2 : 0.35 : 0.6 : 1.0. After treatment with NaBH4, all of the galactosamine was reduced to galactosaminitol, while the glucosamine was unaffected. No galactitol was detected by paper chromatography of the reduced, hydrolyzed oligosaccharides in Solvents II or III, and all of the hexosamine was destroyed by periodate oxidation. Component B-Ill contained only sialic acid and galactose, in a molar ratio of 1.8 : 1 (traces of glucosamine and galactosamine present in Component B-Ill could be removed by rechromatography in Solvent I). When sialic acid was removed from Component B-Ill by mild acid hydrolysis, chromatography in Solvents I, I!. and IlI revealed only galactose and sialic acid. No mannose or fucose were found in hydrolysates of Components B-I, B-II or B-Ill when examined by paper chromatography in Solvents II, Ill, and IV, and Components B-II and B-Ill contained no preformed Morgan-Elson chromogen.
Component M When rat or rabbit brain glycopeptides were treated with N a O H - N a B H ~ under conditions which prevent peeling and submitted to gel filtration on Sephadex G-25, the carbohydrate was eluted as a single peak, with most of the material appearing in the void volume. These results were obtained whether the elution was carried out in water, 0.1 M NaCI or 1 ~,,,acetic acid, and several other chromatographic and electrophoretic methods also failed to completely separate the reduced alkali-labile oligosaccharides from the much larger amount of alkali-stable carbohydrate. In order to obtain further evidence in support of the structures suggested by the results of treatment with alkali alone, 200 mg of rabbit brain glycopeptides were desialylated by mild acid hydrolysis (0.05 M H2SO4, I h, 80 °C). The desialylated glycopeptides were separated from free sialic acid by gel filtration on a 2 cm ~: 60 cm column of Sephadex G-25, and eluted with water as a single peak which was only slightly retarded. The fractions containing the desialylated glycopeptides were pooled and lyophilized, and the glycopeptides were then treated with 0.4 M NaBH4 in 0.02 M N a O H as described above. When the alkaline NaBH4-treated, desialylated glycopeptides were again submitted to gel filtration on Sephadex G-25, a retarded peak of carbohydrate was detected as well as the major peak of desialylated alkali-stable glycopeptides. The retarded carbohydrate-containing fractions were pooled and examined by paper E
La
o
v
o ¢n I
I
I
I
I
I
I
I
I
BO
100
t20
140
160
180
200
220
240
minutes
Fig. 2. Elution of amino sugars and amino sugar alcohols using Beckman UR-30 resin and pH 5.29 citrate-borate buffer (see Materials and Methods). Lower tracing: 1, galactosaminitol; 2, glucosamine; 3, galactosamine; 4, threosaminitol; and 5, serinol. Upper tracing: amino sugar alcohol obtained by Smith degradation of Component M.
OLIGOSACCHA.RIDESOF BRAIN GLYCOPROTEINS
427
chromatography. In most of the solvent systems tested there was a major, rapidly migrating component, as well as a minor component of lower mobility or which remained at the origin. The major component (M) had an Rtacto~e of 1.44 in Solvent I, and was isolated in approximately 8 ~ yield (based on the hexose and hexosamine content of the glycopeptides) by preparative paper chromatography using this solvent system. Component M contained equimolar amounts of galactose and galactosaminitol. Threosaminitol was obtained as the amino sugar fraction after Smith degradation (Fig. 2). These results support the proposed structure of Component M as galactopyranosyl-(1--+3)O-2-acetamido-2-deoxy-D-galactitol. The disaccharide was not cleaved by a fl-galactosidase from E. coli, suggesting a possible ~ configuration of the glycosidic bond.
Sulfate and fucose We have previously reported the presence of sulfate in brain glycoproteins2. When sulfate-labeled glycopeptides were treated with 0.2 M NaOH for 48 h at 25 °C, 3-5 % of the total sulfate radioactivity was released in the form of lower molecular weight material which appeared as a single retarded peak on Sephadex G-25, at an effluent volume of approximately 110-115 ml (Fig. 1). Although no sulfate radioactivity was released by treatment with 0.02 M NaOH containing NaBH 4, a retarded peak of sulfate radioactivity did appear when the glycopeptides were treated first with alkaline borohydride and then with 0.2 M NaOH, indicating that sulfate-labeled oligosaccharides were not produced by peeling. Paper chromatography in Solvent I showed that the radioactivity released by alkali did not migrate with Components B-I, B-II or B-Ill, but remained at the origin, while chromatography in Solvents V, VI and VII demonstrated that the released radioactivity migrated identically with 35SO~-. It therefore appears that a small percentage of the sulfate in these glycopeptides is released by alkaline hydrolysis in 0.2 M NaOH but not by 0.02 M NaOH under the conditions used in our experiments. No low molecular weight labeled oligosaccharides were detected after treatment of glycopeptides labeled with [3H]fucose with 0.2 M NaOH, in confirmation of our chemical analyses which demonstrated that fucose was not present in the alkali-labile oligosaccharides. DISCUSSION When rat or rabbit brain glycopeptides were treated with alkali alone, several oligosaccharides could be separated from the alkali-stable glycopeptides by gel filtration and preparative paper chromatography. Our data indicate that Component B-I consists of the linkage N-acetylgalactosamine (after conversion to Morgan-Elson chromogen) substituted in the 6-position by sialic acid. This can be concluded since the major peak of Morgan-Elson chromogen also contains bound sialic acid and is eluted from Sephadex G-25 at a position corresponding to a disaccharide. Treatment with neuraminidase changes the elution position of the Morgan-Elson chromogen to that of a monosaccharide. The sialic acid must be linked by an alkali-stable bond at C-6 since a substituent at C-3 would be eliminated during alkali treatment, and substitution at C-4 would prevent chromogen formation 16,z o. The minor Component B-II has galactosamine at the reducing end, and con-
428
R.K. MARGOLIS, R. U. MARGOLIS
tains sialic acid as well as smaller amounts of nonreducing galactose and glucosamine. Although Component B-II could not be resolved into more than one component by paper chromatography in many solvent systems, because of the molar ratios of the constituent sugars (sialic acid : galactose : glucosamine : galactosamine --~ 1.2 : 0.35 : 0.6 : 1.0) it would appear that it contains more than one oligosaccharide. Periodate oxidation and NaBH4 reduction demonstrated that the galactosamine in Component B-II is linked at only the 6-position, while the glucosamine is either 1,6-1inked or nonreducing terminal. The molar ratios given above and the results of NaBH4 reduction and periodate oxidation are consistent with the presence of approximately equal amounts of N-acetylneuraminylgalactosyl-(l~6)-N-acetylgalactosamine, and N-acetylneuraminyl-(2--,6)-N-acetylglucosaminyl-(l ~6)-N-acetylgalactosamine. These tentative structures are also suggested by the almost I : 1 : I molar ratios of sialic acid: (galactose~-glucosamine): galactosamine, and the substitution of galactose by glucosamine in some of the molecules is in accord with the known microheterogeneity of oligosaccharides in glycoproteins. However, larger amounts of material will be required in order to obtain more direct evidence for the structure(s) of Component B-II. Component B-Ill is a trisaccharide containing sialic acid and galactose, in a molar ratio of 2 : 1. The galactose was converted to galactitol by NaBH 4 reduction of the trisaccharide, and Component B-Ill was obtained only under conditions of alkali treatment which permit peeling to occur. When rat or rabbit brain glycopeptides were treated with alkaline NaBH4 under conditions which prevent peeling of a 3-substituent on the linkage N-acetylgalactosamine, large alkali-labile oligosaccharides were produced which were mostly excluded from Sephadex G-25. Although we did not succeed in separating the complete, reduced alkali-labile pentasaccharide from the much larger amount of alkalistable glycopeptide, we were able to obtain the expected yield of the reduced disaccharide galactosyl-(l-~3)-N-acetylgalactosaminitol (Component M) by N a O H - N a B H 4 treatment of the desialylated glycopeptides. Since the molar yield of Component B-III was approximately 80 ~, that of Component B-I (and more than three times that of Component B-II, based on its galactosamine content), it appears that the di-N-acetylneuraminylgalactose (Component B-1II) was originally present as a 3-substituent on the linkage N-acetylgalactosamine in Component B-I. Components B-I1 and B-Ill are apparently not directly linked to serine or threonine residues since they are not obtained if peeling is prevented. A small portion of the alkali-labile carbohydrate may be present as a pentasaccharide in which Component B-I! is linked at the C-3 position of N-acetylgalactosamine in Component B-I, in place of di-N-acetylneuraminylgalactose (Component B-III). From the yield of Component M, and the relative amounts of Components B-I, B-II and B-111 obtained by preparative paper chromatography, it can be estimated that approximately 70-80 o~ of the alkali-labile carbohydrate was originally present as a pentasaccharide linked to serine and threonine residues, and having the structure: di-N-acetylneuraminyl-D-galactopyranosyl-( 1 ~ 3) [N-acetylneuraminyl-(2 --* 6)]-0-2acetamido-2-deoxy-D-galactose. Thomas and Winzler 21 have described an alkalilabile tetrasaccharide from human erythrocyte glycoproteins which resembles the structure proposed above for the major alkali-labile pentasaccharide from brain, except that the tetrasaccharide from erythrocyte glycoproteins has only one rather
OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
429
t h a n two sialic acid residues linked to galactose. A l t h o u g h most of our structural studies have been carried out using glycopeptides from r a b b i t b r a i n glycoproteins, preliminary investigation of rat b r a i n glycoproteins indicates that similar alkali-labile oligosaccharides are also present in this species. ACKNOWLEDGEMENTS We t h a n k Regina Santella a n d D o n n a A t h e r t o n for their skillful technical assistance. This work was supported by research grants from the U. S. Public Health Service (NS-09348 a n d MH-17018).
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Margolis, R. U., Margolis, R. K. and Atherton, D. M. (1972) J. Neurochem. 19, 2317-2324 Margolis, R. K. and Margolis, R. U. (1970) Biochemistry 9, 4389-4396 Crimmin, W. R. C. (1957) J. Chem. Soc. 2838 Bray, B. A., Lieberman, R. and Meyer, K. (1967) J. Biol. Chem. 242, 3373-3380 Warren, L. (1959) J. Biol. Chem. 234, 1971-1975 Jourdian, G. W., Dean, L. and Roseman, S. (1971) J. Biol. Chem. 246, 430-435 Spencer, B. (1960) Biochem. J. 75, 435--440 Slein, M. W. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 117-123, Academic Press, New York Finch, P. R., Yuen, R., Schachter, H. and Moscarello, M. A. (1969) Anal. Biochem. 31,296-305 Kuhn, R. and Kriiger, G. (1956) Chem. Bet. 89, 1473-1486 Reissig, J. L., Strominger, J. L. and Leloir, L. F. (1955) J. Biol. Chem. 217, 959-966 Bella, Jr, A. M. and Kim, Y. S. (1970) J. Chromatogr. 51,314-315 Weber, P. and Winzler, R. J. (1969) Arch. Biochem. Biophys. 129, 534-538 Carlson, D. M., Iyer, R. N. and Mayo, J. (1970) in Bloodand Tissue Antigens (Aminoff, D., ed.), pp. 229-247, Academic Press, New York Mayo, J. W. and Carlson, D. M. (1970) Carbohydr. Res. 15, 300-303 Jeanloz, R. W. and Tr6m~ge, M. (1956) Fed. Proc. 15, 282 Cifonelli, J. A. and Dorfman, A. (1957) J. Biol. Chem. 228,547-557 Lee, Y. C. and Scocca, J. R. (1972) J. Biol. Chem. 247, 5753-5758 I-[uang, C. C. and Aminoff, D. (1972) Fed. Proc. 31,466 (Abstr.) Kuhn, R., Gauhe, A. and Baer, H. 1-[. (1954) Chem. Ber. 87, 1138-1141 Thomas, D. B. and Winzler, R. J. (1969) J. Biol. Chem. 244, 5943-5946
ALKALI-LABILE OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
RENI~E K. MARGOLIS and R I C H A R D U. MARGOLIS
Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn, N.Y. 11203 and Department of Pharmacology, New York University School of Medicine, New York, N.Y. 10016 (U.S.A.) (Received November 13th, 1972)
SUMMARY
Treatment of glycopeptides prepared from rabbit brain glycoproteins with NaOH or NaOH-NaBH4 released approximately 10 7o of the carbohydrate as low molecular weight oligosaccharides. After treatment with alkali alone, a disaccharide and a trisaccharide were isolated by gel filtration and preparative paper chromatography, and have been identified as N-acetylneuraminyl-(2--,6)-O-2-acetamido-2deoxy-D-galactose (Component B-I) and di-N-acetylneuraminyl-D-galactose (Component B-III). The N-acetylgalactosamine in Component B-I was obtained as preformed Morgan-Elson chromogen, and Components B-I and B-III were not obtained when the glycopeptides were treated with alkaline NaBH4 under conditions which prevent peeling. These results indicate that the trisaccharide (Component B-III) was originally present as a substituent at C-3 of the N-acetylgalactosamine involved in the carbohydrate-peptide linkage, and was eliminated as a result of alkali treatment. A disaccharide (Component M) was isolated after alkaline NaBH4 treatment of the desialylated glycopeptides, and yielded equimolar amounts of galactose and galactosaminitol after hydrolysis. Smith degradation of Component M gave galactose and threosaminitol, indicating that the disaccharide was galactopyranosyl-(1 --, 3)-0-2aeetamido-2-deoxy-D-galactitol. From the isolation of Components B-I, B-III and M after NaOH and N a O H NaBH4 treatment of the glycopeptides, it is concluded that the major alkali-labile oligosaccharide of brain glycoproteins is the pentasaccharide di-N-acetylneuraminylD- galactopyranosyl- (1 --, 3) [N- acetylneuraminyl- (2 ~ 6) ]- O-2-acetamido-2-deoxy-Dgalactose.
INTRODUCTION"
Treatment of glycopeptides prepared from glycoproteins of rat and rabbit brain with NaOH-NaBH4 leads to the destruction of a portion of the serine, threonine and galactosamine present, and the appearance in acid hydrolysates of alanine, ~-aminobutyric acid and galactosaminitol ~. These results indicate that N-acetylgalaetosamine at the reducing end of oligosaccharide chains in brain glycoproteins is
422
R.K. MARGOLIS, R. U. MARGOLIS
linked O-glycosidically to the hydroxyl groups of serine and threonine residues. Although most of the carbohydrate in brain glycoproteins appears to be linked by N-acetylglucosaminylasparagine linkages, it was found that approximately 10 "~, of the sialic acid, hexose and hexosamine in rat and rabbit brain glycoproteins is present in alkali-labile oligosaccharides. This report describes the partial structure of a pentasaccharide which accounts for the major portion of the alkali-labile carbohydrate in rabbit brain glycoproteins. MATERIALS AND METIffODS Materials
Rabbit brains were carefully cleaned of blood, superficial blood vessels, and meninges, and glycopeptides were prepared from brain glycoproteins as described previouslyL 2. The procedure consists of digestion of the lipid-free protein residue of brain with pronase, and precipitation of nucleic acids and acid mucopolysaccharides with cetylpyridinium chloride. Excess cetylpyridinium chloride is precipitated with KSCN, and the glycopeptides are recovered from the dialyzed supernatant by lyophilization. Glucosaminitol was a gift of Dr P. Weber, and galactosaminitol was prepared by NaBH 4 reduction of N-acetylgalactosamine 3 followed by acid hydrolysis (3 M HCI, 3 h, 100 °C). Chondrosine was obtained from Miles Laboratories, Kankakee, Ill., and N-acetylchondrosine was prepared as described by Bray et aL 4. N-acetylchondrosinitol was obtained by reduction of N-acetylchondrosine with N a B H , , and threosaminitol was prepared by Smith degradation (see below) of N-acetylchondrosinitol. Clostridium perfringens neuraminidase was from Worthington Biochemical Corp., Freehold, N. J., and Eseheriehia coli fl-galactosidase was from Boehringer Mannheim Corp., New York, N.Y. Methods
Free and bound sialic acid were determined differentially either by the thiobarbituric acid method 5 or by the periodate-resorcinol method of Jourdian et al. 6. For the release of bound sialic acid, samples were hydrolyzed for 1 h in 0.05 M H2SO, at 80 °C, and the values obtained were corrected for destruction during hydrolysis. Sulfate was measured by the barium chloranilate method of Spencer 7. Methods for the determination of total neutral sugar, galactose (using D-galactose dehydrogenase), fucose and uronic acid have been described previously 2. Glucose was measured spectrophotometrically in glycopeptide hydrolysates (1 M HCI, 3 h, 100 °C) by means of yeast hexokinase and glucose-6-phosphate dehydrogenase a, and mannose was determined enzymatically 9 after adsorption of hexosamines on a short column of Dowex
50 (H +). Preformed Morgan-Elson chromogen t° was determined by a modification of the method of Reissig et aL 11 in which the heating step after the addition of potassium tetraborate was omitted 4. Standards of N-acetylgalactosamine were carried through the test as described by Reissig et al. l l Hexosamines and amino sugar alcohols were separated on a 56-cm column of Beckman UR-30 resin using a Beckman Model 120C amino acid analyzer. The columns were eluted either with a 0.35 M sodium citrate buffer (pH 5.06) containing 0.3 M
O L I G O S A C C H A R I D E S OF BRAIN GLYCOPROTEINS
423
boric acid 12, or with the pH 5.29 citrate-borate buffer described by Weber and Winzler 13. For the quantitation of amino sugars, samples were hydrolyzed for 8 h at 100 °C in 4 M HCI, evaporated to dryness in a rotary evaporator, and redissolved in 0.02 M HC1 for application to the amino acid analyzer. These hydrolysis conditions gave greater than 95 9/0 recovery of hexosamine and hexosaminitol standards. When NaBH4 reduction was used to determine the nature of the reducing end in oligosaccharides, treatment was carried out for 6 h at 4 °C with 0.5 M NaBH4, after which the solution was adjusted to pH 6 with acetic acid and passed through a small column of Dowex 50 (H ÷). The eluate was concentrated to dryness under reduced pressure and boric acid was removed as trimethylborate by repeated evaporation with HCl-methanol (1 : 1000, v/v). For periodate oxidation or Smith degradation, glycopeptides or oligosaccharides, at a concentration of less than 3 mg/ml, were treated with 0.03 M sodium metaperiodate in 0.1 M sodium acetate buffer, pH 4.0, at 4 °C in the dark. After 48 h excess periodate was destroyed with ethyleneglycol. To measure the extent of destruction of hexosamines, samples were evaporated to dryness under vacuum, hydrolyzed, and analyzed for glucosamine and galactosamine using the amino acid analyzer. For Smith degradations, periodate-treated samples (1-10 mg/ml) were reduced with 1 M NaBH4 (24 h at 4 °C). Excess NaBH4 was destroyed with glacial acetic acid, and the solution was desalted by gel filtration on a 1 cm × 50 cm column of Sephadex G-10. After concentration in a rotary evaporator, the residue was hydrolyzed for 45 rain in 4 M HC1 at 100 °C and neutralized with NaOH. Paper chromatography was performed both on Whatman No. 1 and on prewashed Whatman No. 3 MM paper (for preparative separations) using the following solvent systems: I, butanol-acetic acid-water (4 : 1 : 5, by vol., upper phase); II, ethyl acetate-acetic acid-water ( 6 : 3 : 2 , by vol.); III, propanol--ethyl acetate-water (7 : 1 : 2, by vol.); IV, ethyl acetate-pyridine-butanol-butyric acid-water (10 : 10 : 5 : 1 : 5, by vol.); V, pyridine-acetic acid-butanol-water (20 : 6 : 30 : 24, by vol.); VI, ethanol-pyridine-water-ammonia (30 : 3 0 : 4 0 : 2 . 5 , by vol.); and VII, glycerol-butanolacetone-water-ammonia (5 : 35 : 30 : 30 : 2.5, by vol.). Oligosaccharides were hydrolyzed for 2 h in 2 M trifluoroacetic acid at 120 °C, and sugars on paper chromatograms were detected with alkaline AgNO 3. RESULTS
The composition of the rabbit brain glycopeptides used in this study is given in Table I. When these glycopeptides were treated with 0.2 M NaBH 4 in 0.2 M NaOH for 48 h at 25 °C and analyzed for amino acids and hexosamines 1, a peak was observed on the amino acid analyzer at the same elution volume as Peak 3 described by Bray et aL 4, which they tentatively identified as a reduction product of the unsaturated chromogen I postulated by Kuhn and Kriiger I o. However, when the glycopeptides were treated at 45 °C with 0.4 M NaBH4 in 0.02 M NaOH, Peak 3 was much reduced in size or absent. Similar alkaline NaBH4 treatment conditions have been shown to favor reduction of N-acetylgalactosamine residues at the reducing ends of oligosaccharides released by fl-elimination, in preference to alkali-mediated "peeling" of substituents from C-3 (refs 14, 15). It therefore appears likely that the difference between the galactosamine destroyed (7 pmoles/100 mg) and the galactosaminitol produced
424
R. K. M A R G O L I S , R. U. M A R G O L I S
TABLE I COMPOSITION
OF GLYCOPEPTIDFS
Component
RABBIT BRAIN
GLYCOPROTEINS
of moisture-free weight*
N-Acetylglucosamine** N-Acetylgalactosamine** Galactose Mannose Fucose Glucose N-Acetylneuraminic acid A m i n o acids Sulfate*** Phosphorus Ash
19.5 3. I 14.6 8.4 4.2 0.2 20.6 21.4 I. I 0.3 1.5
Total
94.9
* ** during ***
FROM
W a t e r content was 7.0 ~0, as d e t e r m i n e d by drying to c o n s t a n t weight in vacuo at 60 °C. D e t e r m i n e d using an a u t o m a t e d a m i n o acid analyzer. Values are corrected for destruction hydrolysis a n d expressed as acetylated sugars. Uronic acid was n o t present in these glycopeptide preparations (cf. ref. 2t.
(2/~moles/100 mg) by alkaline NaBH 4 treatment of brain glycopeptides under the milder reducing conditions I can be accounted for as reduced Morgan-Elson chromogen (i.e., reduced chromogen I of Kuhn and Krfiger).
Components B-I, B-H and B-Ill Rat and rabbit brain glycopeptides were also treated at a concentration of 10-30 mg/ml with 0.2 M NaOH at 25 '~C under nitrogen, and neutralized with HCI after 48 h. This alkali treatment without NaBH~ resulted in cleavage of O-glycosidic carbohydrate-peptide linkages and yielded preformed Morgan-Elson chromogen in an amount 10-20 ~ greater than that of the N'acetylgalactosamine destroyed. Since only N-acetylhexosamines which are substituted in the 3-position (and unsubstituted at C-4) react in the modified Morgan-Elson reaction used 4, it would appear that most of the N-acetylgalactosamine linked to serine and threonine, and destroyed by alkali treatment, is also 3-substituted. The results of periodate oxidation studies are consistent with this assumption, insofar as only 17 ~ and 20'3o of the N-acetylgalactosamine in glycopeptides from rat and rabbit brain, respectively, was destroyed by periodate. When glycopeptides prepared from rat or rabbit brain glycoproteins were treated with 0.2 M N a O H (withot, t NaBH4) and submitted to gel filtration on Sephadex G-25, several retarded peaks containing sialic acid were observed, and fractions were pooled as indicated in Fig. 1. The major peak of alkali-stable glycopeptides (A) was followed by two retarded peaks containing bound sialic acid (B), as well as a peak of free sialic acid (C). The free sialic acid was evidently released by the nonspecific alkaline hydrolysis of a small percentage of the total sialic acid in the glycopeptides, rather than by peeling, since retreatment of glycopeptides with 0.2 M NaOH after prior alkaline NaBH4 treatment under conditions shown to prevent peeling (0.4 M
OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
425
1.2 1.0 A
I,--- B--'l'-c--I-----
i 0.8 0.6
0 - --'1
M-EIC
-
0,4 0.2
0
I
90 102
I
I
I
I
ll4
126
138
150
I
16;) EFFLUENT VOLUME (mi)
Fig. 1. Rabbit brain glycopeptides were treated with 0.2 M NaOH for 48 h at 25 °C under nitrogen, neutralized with I-[C1, and aliquots containing 130 mg of alkali-treated glycopeptides were eluted with water from a 2 cm x 58 cm column of Sephadex G-25. Free (O-O) and bound (Q-Q) sialic acid were determined by the thiobarbituric method. Free sialic acid was also identified by its chromatographic mobility in Solvents I, II and III. Peak A, containing the alkali-stable glycopeptides, emerged mostly with the void volume as a single symmetrical peak. Preformed Morgan-Elson chromogen (M-E C) was measured as described under Methods, and appeared as two peaks (90 ~o in B and 10 ~o in D) at the positions designated by arrows. NANA -- N-acetylneuraminicacid. NaBH4, 0.02 M N a O H , 16 h, 45 °C) resulted in the release of approximately the same amount of free sialic acid. 90 % of the preformed Morgan-Elson chromogen was eluted at a position coinciding with the second retarded peak of bound sialic acid, while the remaining 10 ~ appeared in Fraction D, slightly ahead of a glucose marker. When Fraction A, obtained by 0.2 M N a O H treatment of the glycopeptides, was subsequently treated with 0.5 M NaBH4, no galactosaminitol was detected after acid hydrolysis. This finding demonstrates that no large alkali-labile oligosaccharides containing N-acetylgalactosamine at the reducing end were present in the fraction of alkali-stable glycopeptides excluded from Sephadex G-25, and implies that all of the alkali-labile carbohydrate is present in oligosaccharide chains which undergo at least some degree of peeling. The tubes comprising Fraction B were pooled and separated into three components by preparative paper chromatography in Solvent I. The most rapidly migrating component had an Rtactos e of 1.7, and was designated Component B-I. It contained only preformed Morgan-Elson chromogen and sialic acid, but no hexose or hexosamine. F r o m its composition and its elution position on G-25 between free sialic acid and the first retarded peak of bound sialic acid (which was shown to contain trisaccharides), Component B-I appeared to be a disaccharide. The molar ratio of Morgan-Elson chromogen to bound sialic acid (measured by the periodate-resorcinol method) was 1.82 : 1.00. When an aliquot of B-I was treated with neuraminidase and reapplied to Sephadex G-25, all of the Morgan-Elson chromogen was eluted in Fraction D, at an effluent volume slightly less than that of glucose. The high apparent molar ratio of Morgan-Elson chromogen to sialic acid is in agreement with previous findings that substitution at the C-3 position greatly enhances color formation in the Morgan-Elson reaction 16-19. Two other more slowly-migrating components of the pooled B fractions could also be resolved, and were designated Component B-II (Rt,c,os ~ 1.06) and Component
426
R.K. MARGOLIS, R. U. MARGOLIS
B-III (R,.c,ose 0.80). Component B-II contained sialic acid, galactose, glucosamine and galactosamine, in molar ratios of 1.2 : 0.35 : 0.6 : 1.0. After treatment with NaBH4, all of the galactosamine was reduced to galactosaminitol, while the glucosamine was unaffected. No galactitol was detected by paper chromatography of the reduced, hydrolyzed oligosaccharides in Solvents II or III, and all of the hexosamine was destroyed by periodate oxidation. Component B-Ill contained only sialic acid and galactose, in a molar ratio of 1.8 : 1 (traces of glucosamine and galactosamine present in Component B-Ill could be removed by rechromatography in Solvent I). When sialic acid was removed from Component B-Ill by mild acid hydrolysis, chromatography in Solvents I, I!. and IlI revealed only galactose and sialic acid. No mannose or fucose were found in hydrolysates of Components B-I, B-II or B-Ill when examined by paper chromatography in Solvents II, Ill, and IV, and Components B-II and B-Ill contained no preformed Morgan-Elson chromogen.
Component M When rat or rabbit brain glycopeptides were treated with N a O H - N a B H ~ under conditions which prevent peeling and submitted to gel filtration on Sephadex G-25, the carbohydrate was eluted as a single peak, with most of the material appearing in the void volume. These results were obtained whether the elution was carried out in water, 0.1 M NaCI or 1 ~,,,acetic acid, and several other chromatographic and electrophoretic methods also failed to completely separate the reduced alkali-labile oligosaccharides from the much larger amount of alkali-stable carbohydrate. In order to obtain further evidence in support of the structures suggested by the results of treatment with alkali alone, 200 mg of rabbit brain glycopeptides were desialylated by mild acid hydrolysis (0.05 M H2SO4, I h, 80 °C). The desialylated glycopeptides were separated from free sialic acid by gel filtration on a 2 cm ~: 60 cm column of Sephadex G-25, and eluted with water as a single peak which was only slightly retarded. The fractions containing the desialylated glycopeptides were pooled and lyophilized, and the glycopeptides were then treated with 0.4 M NaBH4 in 0.02 M N a O H as described above. When the alkaline NaBH4-treated, desialylated glycopeptides were again submitted to gel filtration on Sephadex G-25, a retarded peak of carbohydrate was detected as well as the major peak of desialylated alkali-stable glycopeptides. The retarded carbohydrate-containing fractions were pooled and examined by paper E
La
o
v
o ¢n I
I
I
I
I
I
I
I
I
BO
100
t20
140
160
180
200
220
240
minutes
Fig. 2. Elution of amino sugars and amino sugar alcohols using Beckman UR-30 resin and pH 5.29 citrate-borate buffer (see Materials and Methods). Lower tracing: 1, galactosaminitol; 2, glucosamine; 3, galactosamine; 4, threosaminitol; and 5, serinol. Upper tracing: amino sugar alcohol obtained by Smith degradation of Component M.
OLIGOSACCHA.RIDESOF BRAIN GLYCOPROTEINS
427
chromatography. In most of the solvent systems tested there was a major, rapidly migrating component, as well as a minor component of lower mobility or which remained at the origin. The major component (M) had an Rtacto~e of 1.44 in Solvent I, and was isolated in approximately 8 ~ yield (based on the hexose and hexosamine content of the glycopeptides) by preparative paper chromatography using this solvent system. Component M contained equimolar amounts of galactose and galactosaminitol. Threosaminitol was obtained as the amino sugar fraction after Smith degradation (Fig. 2). These results support the proposed structure of Component M as galactopyranosyl-(1--+3)O-2-acetamido-2-deoxy-D-galactitol. The disaccharide was not cleaved by a fl-galactosidase from E. coli, suggesting a possible ~ configuration of the glycosidic bond.
Sulfate and fucose We have previously reported the presence of sulfate in brain glycoproteins2. When sulfate-labeled glycopeptides were treated with 0.2 M NaOH for 48 h at 25 °C, 3-5 % of the total sulfate radioactivity was released in the form of lower molecular weight material which appeared as a single retarded peak on Sephadex G-25, at an effluent volume of approximately 110-115 ml (Fig. 1). Although no sulfate radioactivity was released by treatment with 0.02 M NaOH containing NaBH 4, a retarded peak of sulfate radioactivity did appear when the glycopeptides were treated first with alkaline borohydride and then with 0.2 M NaOH, indicating that sulfate-labeled oligosaccharides were not produced by peeling. Paper chromatography in Solvent I showed that the radioactivity released by alkali did not migrate with Components B-I, B-II or B-Ill, but remained at the origin, while chromatography in Solvents V, VI and VII demonstrated that the released radioactivity migrated identically with 35SO~-. It therefore appears that a small percentage of the sulfate in these glycopeptides is released by alkaline hydrolysis in 0.2 M NaOH but not by 0.02 M NaOH under the conditions used in our experiments. No low molecular weight labeled oligosaccharides were detected after treatment of glycopeptides labeled with [3H]fucose with 0.2 M NaOH, in confirmation of our chemical analyses which demonstrated that fucose was not present in the alkali-labile oligosaccharides. DISCUSSION When rat or rabbit brain glycopeptides were treated with alkali alone, several oligosaccharides could be separated from the alkali-stable glycopeptides by gel filtration and preparative paper chromatography. Our data indicate that Component B-I consists of the linkage N-acetylgalactosamine (after conversion to Morgan-Elson chromogen) substituted in the 6-position by sialic acid. This can be concluded since the major peak of Morgan-Elson chromogen also contains bound sialic acid and is eluted from Sephadex G-25 at a position corresponding to a disaccharide. Treatment with neuraminidase changes the elution position of the Morgan-Elson chromogen to that of a monosaccharide. The sialic acid must be linked by an alkali-stable bond at C-6 since a substituent at C-3 would be eliminated during alkali treatment, and substitution at C-4 would prevent chromogen formation 16,z o. The minor Component B-II has galactosamine at the reducing end, and con-
428
R.K. MARGOLIS, R. U. MARGOLIS
tains sialic acid as well as smaller amounts of nonreducing galactose and glucosamine. Although Component B-II could not be resolved into more than one component by paper chromatography in many solvent systems, because of the molar ratios of the constituent sugars (sialic acid : galactose : glucosamine : galactosamine --~ 1.2 : 0.35 : 0.6 : 1.0) it would appear that it contains more than one oligosaccharide. Periodate oxidation and NaBH4 reduction demonstrated that the galactosamine in Component B-II is linked at only the 6-position, while the glucosamine is either 1,6-1inked or nonreducing terminal. The molar ratios given above and the results of NaBH4 reduction and periodate oxidation are consistent with the presence of approximately equal amounts of N-acetylneuraminylgalactosyl-(l~6)-N-acetylgalactosamine, and N-acetylneuraminyl-(2--,6)-N-acetylglucosaminyl-(l ~6)-N-acetylgalactosamine. These tentative structures are also suggested by the almost I : 1 : I molar ratios of sialic acid: (galactose~-glucosamine): galactosamine, and the substitution of galactose by glucosamine in some of the molecules is in accord with the known microheterogeneity of oligosaccharides in glycoproteins. However, larger amounts of material will be required in order to obtain more direct evidence for the structure(s) of Component B-II. Component B-Ill is a trisaccharide containing sialic acid and galactose, in a molar ratio of 2 : 1. The galactose was converted to galactitol by NaBH 4 reduction of the trisaccharide, and Component B-Ill was obtained only under conditions of alkali treatment which permit peeling to occur. When rat or rabbit brain glycopeptides were treated with alkaline NaBH4 under conditions which prevent peeling of a 3-substituent on the linkage N-acetylgalactosamine, large alkali-labile oligosaccharides were produced which were mostly excluded from Sephadex G-25. Although we did not succeed in separating the complete, reduced alkali-labile pentasaccharide from the much larger amount of alkalistable glycopeptide, we were able to obtain the expected yield of the reduced disaccharide galactosyl-(l-~3)-N-acetylgalactosaminitol (Component M) by N a O H - N a B H 4 treatment of the desialylated glycopeptides. Since the molar yield of Component B-III was approximately 80 ~, that of Component B-I (and more than three times that of Component B-II, based on its galactosamine content), it appears that the di-N-acetylneuraminylgalactose (Component B-1II) was originally present as a 3-substituent on the linkage N-acetylgalactosamine in Component B-I. Components B-I1 and B-Ill are apparently not directly linked to serine or threonine residues since they are not obtained if peeling is prevented. A small portion of the alkali-labile carbohydrate may be present as a pentasaccharide in which Component B-I! is linked at the C-3 position of N-acetylgalactosamine in Component B-I, in place of di-N-acetylneuraminylgalactose (Component B-III). From the yield of Component M, and the relative amounts of Components B-I, B-II and B-111 obtained by preparative paper chromatography, it can be estimated that approximately 70-80 o~ of the alkali-labile carbohydrate was originally present as a pentasaccharide linked to serine and threonine residues, and having the structure: di-N-acetylneuraminyl-D-galactopyranosyl-( 1 ~ 3) [N-acetylneuraminyl-(2 --* 6)]-0-2acetamido-2-deoxy-D-galactose. Thomas and Winzler 21 have described an alkalilabile tetrasaccharide from human erythrocyte glycoproteins which resembles the structure proposed above for the major alkali-labile pentasaccharide from brain, except that the tetrasaccharide from erythrocyte glycoproteins has only one rather
OLIGOSACCHARIDES OF BRAIN GLYCOPROTEINS
429
t h a n two sialic acid residues linked to galactose. A l t h o u g h most of our structural studies have been carried out using glycopeptides from r a b b i t b r a i n glycoproteins, preliminary investigation of rat b r a i n glycoproteins indicates that similar alkali-labile oligosaccharides are also present in this species. ACKNOWLEDGEMENTS We t h a n k Regina Santella a n d D o n n a A t h e r t o n for their skillful technical assistance. This work was supported by research grants from the U. S. Public Health Service (NS-09348 a n d MH-17018).
REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Margolis, R. U., Margolis, R. K. and Atherton, D. M. (1972) J. Neurochem. 19, 2317-2324 Margolis, R. K. and Margolis, R. U. (1970) Biochemistry 9, 4389-4396 Crimmin, W. R. C. (1957) J. Chem. Soc. 2838 Bray, B. A., Lieberman, R. and Meyer, K. (1967) J. Biol. Chem. 242, 3373-3380 Warren, L. (1959) J. Biol. Chem. 234, 1971-1975 Jourdian, G. W., Dean, L. and Roseman, S. (1971) J. Biol. Chem. 246, 430-435 Spencer, B. (1960) Biochem. J. 75, 435--440 Slein, M. W. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), pp. 117-123, Academic Press, New York Finch, P. R., Yuen, R., Schachter, H. and Moscarello, M. A. (1969) Anal. Biochem. 31,296-305 Kuhn, R. and Kriiger, G. (1956) Chem. Bet. 89, 1473-1486 Reissig, J. L., Strominger, J. L. and Leloir, L. F. (1955) J. Biol. Chem. 217, 959-966 Bella, Jr, A. M. and Kim, Y. S. (1970) J. Chromatogr. 51,314-315 Weber, P. and Winzler, R. J. (1969) Arch. Biochem. Biophys. 129, 534-538 Carlson, D. M., Iyer, R. N. and Mayo, J. (1970) in Bloodand Tissue Antigens (Aminoff, D., ed.), pp. 229-247, Academic Press, New York Mayo, J. W. and Carlson, D. M. (1970) Carbohydr. Res. 15, 300-303 Jeanloz, R. W. and Tr6m~ge, M. (1956) Fed. Proc. 15, 282 Cifonelli, J. A. and Dorfman, A. (1957) J. Biol. Chem. 228,547-557 Lee, Y. C. and Scocca, J. R. (1972) J. Biol. Chem. 247, 5753-5758 I-[uang, C. C. and Aminoff, D. (1972) Fed. Proc. 31,466 (Abstr.) Kuhn, R., Gauhe, A. and Baer, H. 1-[. (1954) Chem. Ber. 87, 1138-1141 Thomas, D. B. and Winzler, R. J. (1969) J. Biol. Chem. 244, 5943-5946