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Immunochemical studies on blood groups L structures of some oligosaccharides obtained by mild acid hydrolysis of human ovarian cyst blood group A substance
ARCHIVES OF B I O C H E M I S T R Y ANI) B I O P H Y S I C S
145,490-501 (1971)
Immunochemical Studies on Blood Groups L Structures of Some Oligosaccharides Obtained by Mild Acid Hydrolysis of Human Ovarian Cyst Blood Group A Substance~ B Y R O N A N D E R S O N } E L V I N A. K A B A T , S H E R M A N B E Y C H O K , AND F L A V I O G R U E Z O
Departments of Microbiology, Neurology, and Human Genetics and Development, College of Physicians and Surgeons and the Departments of Biological Sciences and Chemistry, Columbia University and the Neurological Institute, Presbyterian Hospital, New York Received February 23, 1971; accepted April 13, 1971 Mild acid hydrolysis of a human ovarian cyst blood group A substance followed by preparative chromatography resulted in the isolation of two disaceharides, r --* 3)DGNAc, r --* 4)DGNAc, a trisaccharide, ~DGal(1 ~ 3)BDGNAe(1 --* 3)DGal, and two tetrasaceharides, ~nGal(1 ~ 3)f~DGNAe(1 ~ 3)~DGaI(1 --* 3)DGNAe, and BDGal(1 --~ 3)/~DGNAe(1 --* 3)r --* 4)bGNAe. Their structures were established by analyses, periodate oxidation, methylation, alkaline degradation, and ORD spectra. The oligosaecharides appear to be derived from the interior of the megalosaeeharide chains of the blood group active glyeoprotein. A procedure for establishing linkages of internal DGNAc residues in oligosaeeharides by gas chromatography following BaO methylation, methanolysis, and O-acetylation has been developed. Methylation analyses are also presented for four oligosaceharides obtained from alkaline borodeuteride degradation of a precursor blood group substance, OG, which, unlike the other oligosaeeharides, contained 4-substituted internal DGNAe residues. Although the two tetrasaeeharide fractions from A substance were only obtained as mixtures, the methods developed for methylation analysis and base-borohydride degradation were capable of elucidating each structure in the mixtures. The soluble blood group substances are glyeoproteins whose oligosaccharide chains contain the antigenic speeifieities, A, B, H, Le% Le b, and I. Acid or base degradation has been used to obtain oligosaeeharides of a size amenable to structural studies and free from the protein portion of the parent molecule. These d a t a have led to a proposed composite structure for the oligosaeeharide portion of the blood group substance (1-5). 1 Aided by grants from the National Science Foundation (GB-8341, GB-25686, and GB-22847), a General Research Support Grant,, and Grant GM-10576 from the United States Public Health Service. Helen Hay Whitney Fellow and National Cystic Fibrosis Research Foundation Fellow 1968-1971.
Partial acid hydrolysis, using either mineral acid or polystyrene suIfonic acid, has been employed extensively to obtain from blood group substances several A active oligosaeeharides (6-8); B active oligosaeeharides (9-11); and m a n y inactive oligosaceharides containing DGal, DGNAe or DGalNAe 3 or both (7, 10, 12-15). The present study describes the isolation and characterization of two disaeeharides, a trisaeeharide, and two tetrasaccharides, each containing only DGal and DGNAe. These oligosaecharides were obtained from 3 Abbreviations: Gal = galaetose, GalNAe = N-acetylgalactosamine, GNAc = N-acetylglucosamine, Glc = glucose, GalNH~ = galactosamine.
490
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS a partial acid hydrolysate of a h u m a n blood group A substance which had been previously ehromatographed and from which A active di- and trisaeeharides had been isolated (7). During this study it became necessary to identify linkages to internal GNAc residues b y methylation procedures. The method developed has also been applied to four reduced oligosaecharides previously isolated by alkaline-borodeuteride degradation of a h u m a n ovarian cyst, precursor substance (3, 16). MATERIALS AND METHODS Monosaceharides were obtained from Mann Research Laboratories or Nutritional Bioehemicals Co. r --~ 4)DGNAc was obtained from Dr. F. Zilliken; f/DGal(1 --~ 3)DGNAe, BDGal(1 --~ 4) I)GNAc, BDGal(1 --* 6)DGNAe (17), r --~ 4) DGal(18), and laeto-:V-tetraose were kindly provided by Dr. A. Gauhe. flDGal(1 --~ 6)nGal(19) was from Dr. A.M. Stephen; /~nGle(1 --~ 3)-Naeetyl-D-galaetosaminitol from Dr. D. M. Carlson; 3-deoxy-D-Gal from Dr. S. Svensson. Nitrogen, methylpentose (fucose), hexosamine, N-acetylhexosamine, and hexose (galaetose) were determined by colorimetric methods (20, 21). Periodate uptake, formaldehyde, and formic acid were measured as previously described (20). Paper chromatography was carried out in three solvent systems: 1-propanol-ethyl acetate-water (7:1:2), solvent 1; 1-butanol-pyridine-water (6:4:3), solvent 2; 1-butanol-pyridine-water (6:1:1), solvent 3. Schleicher and Schiill 589 green ribbon paper was used for analytical and preparative and Whatman 3 MM for preparative studies. The sodium borohydride reduction, the baseborohydride degradation with gas-liquid chromatographic quantitation of galactitol, 3-deoxygalactirol and reduced chromogen, and the methylation procedure using BaO as catalyst (22) were described previously (23). Formation of the methyl glycosides with methanolic HC1 followed by methylation was used to prepare the tri-O-methyl methyl glycosides of N-acetyl-D-glucosamine and N-acetyl-D-galacto9samine. The preparation of the O-acetyl-tetra-Omethyl-N-acetyl-D-glucosaminitol and N-acetylD-galactosaminitol derivates has been described (23). The methyl glycoside of 3-O-acetyl-4,6-diO-methyl-N-acetyl-D-glucosamine was obtained from Dr. A. Gauhe. The 3,6-di-O-methyl-, 3,4-die-methyl-, 3-O-methyl-, and 6-O-methyl-Nacetyl-D-glueosamines were obtained from Drs. A. Webb and M. B. Perry (24); the 3,6-di-O-methyland 4,6-di-O-methyl-N-methyl-D-glucosamine hy-
491
drochlorides were supplied by Dr. P. A. J. Gorin (25); these were converted into methyl glycosides by methanolic-HC1 and the free hydro)tyls were then 0-acetylated. Gas-liquid chromatography was carried out using an F and M (Hewlett Packard) 810 gas chromatograph. ORD spectra were measured in a Bendix Ericcsen or in a Cary Model 60 spectropolarimeter. Optical rotations were measured in a Perkin-Elmer polarimeter Model 141 at five wavelengths. EXPERIMENTAL AND RESULTS Four oligosaccharide fractions obtained previously (7) b y chromatography on charcoal and elution with an ethanol gradient from the dialysate of a h u m a n ovarian cyst A substance (McDon), which had been subjected to mild acid hydrolysis, were studied. T h e y were: Ai, 25 mg; A2, 343 mg; As, 83 rag; A4, 59 mg; and A 6 , 1 1 9 mg. Of the A2 fraction, 91 mg was chromategraphed by three descending developments in solvent 1 to give 49 mg of a. compound which had moved one-third down the paper. Three components were obtained on chrom a t o g r a p h y in solvent 2: RL 4 1.67 (26.3 rag), RL 1.44 (14.9 rag), and RL 1.22 (3.1 rag). The latter was discarded. The RL 1.67 component, was chromatographed in solvent 3 to yield 14.7 mg of RL 2.29 and 10.9 mg of RL 1.94. The RL 2.29 component was purified through Bio-Gel P-2 to yield 7.7 mg (oligosaccharide RL 2.29). The RL 1.44 material was also chromategraphed in solvent 3 to yield RL 1.7 (4.2 rag), which was discarded, and RL 1.9 (6.1 mg), which was combined with the above R~ 1.94 material, and purified through BioGel P-2 to yield 12 mg (oligosaccharide RL
1.9). The A1, As, A4, and A~ fractions were each chromatographed in solvent 2 to give the following broad cuts: RL 0.2, 83.9 mg; RL 0.52, 74.6 rag; RL 0.79, 37.3 mg; RL 0.98, 24.5 mg; RL 1.2, 49.2 mg; and Rc~l 1.0, 55.7 rag. The R~ 0.2, RL 0.98, and RG~, 1.0 materials were apparently very heterogeneous, and further a t t e m p t s to purify t h e m b y gel filtration and paper chromatogr a p h y resulted in fractions of insufficient quantity for structural determinations. 4 RL
~
t~Lactose
-
ANDERSON ET AL.
492
were still impure. T h e 15.9-mg fraction was purified t h r o u g h Bio-Gel P-2 to give 14.2 m g (oligosaeeharide IlL 0.61); its a c l u a l IlL as i s o l a t e d was 0.61 in solvent 2. E a c h of the five oligosaeeharides s t u d i e d g:tve a single spot on p a p e r in solvent 2.
T h e RL 0.52 was passed t h r o u g h Bio-Gel P-2 to y i e l d two fractions of 29.0 a n d 24.1 mg. T h e l a t t e r was again e h r o m a t o g r a p h e d on Bio-Gel P-2 to give 19.0 rag. This comp o u n d h a d a n RL of 0.54 in solvent 2 (oligosaecharide R~, 0.54). T h e 29.0-mg fraction was purified t h r o u g h Bio-Gel P-2 a n d b y p r e p a r a t i v e p a p e r c h r o m a t o g r a p h y twice in solvent 2 s e p a r a t i n g o u t t h r e e m i n o r a n d one m a j o r c o m p o n e n t , RL 0.63 (12.2 rag). T h i s l a t t e r c o m p o n e n t was p a s s e d t h r o u g h BioGel P-2 to y i e l d 8.5 m g (oligosaceharide RL 0.63). T h e RL 0.79 m a t e r i a l was purified t h r o u g h Bio-Gel P-2 to give two fractious of 15.9 a n d 8.3 mg. T h e l a t t e r gave two more fractions on B i o - G e l P-2, 3.1 a n d 4.7 rag, each of which
Determination of Structures of Oligosaccharides RL 2.29. A n a l y t i c a l d a t a (Table I) showed e q u i m o l a r a m o u n t s of glucosamine a n d galactose, a n d t h e glucosamine was des t r o y e d on r e d u c t i o n w i t h N a B H 4 . A d i r e c t N - a c e t y l h e x o s a m i n e (26) r e a c t i o n on t h e disaccharide gave a color yield of 100% as compared with a 3-0-substituted N-acetylglucosamine. T h e r e d u c e d disaccharide con-
TABLE I ANALYTICAL PROPERTIES OF ~SOLATED OLIGOSACCHARIDES Percentage composition Oligosaccharide
~L 2.29 Calcd ~educed RL 2.29 Calcd ~L 1.9 Calcd reduced RL 1.9 Calcd
Yield (rag)
7.7
12
N 2.6 3.65
3.3 3.65
14.2 tL 0.61 Calcd ~educed RL 0.61 Calcd
3.5 3.65
~L0.63 Calcd ~educed RL 0.63 Calcd
3.2 3.65
8.5
~L0.54 19 Calcd Leduced RL 0.54 Calcd
2.8 2.4
Molar ratios
Hexos.~ Hexos(Gal) aminea
NAc GalHexos. amine' NH2
42.2 46.7 33.5 46.7
28.6 46.7 8.2 0
36.7 57.3 0.7 0
- 2.7 0 ~ 7 8 ' 1.00
47.0 46.7 44.5 46.7
33.4 46.7 9.6 0
46.8 57.3 0.9 0
0.91 1.0
45.0 46.7 40.3 46.7
37.6 46.7 22.3 23.4
44.8 57.3 20.4 28.7
48.3 46.7 46.3 46.7
41.5 46.7 25.8 23.4
50.0 57.3 24.1 28.7
67.0 62 30.8 31
27.0 31 22.8 31
32.3 38 28.4 38
N
1.0
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NAc- NAcHexos Hexos- amine/ Hexosamine aminea
Hexos-
amine 0.68 1.0
0.71
0.71 1.0
0.81
1.14
1.0
1.0
1.00 1.0 1.00 1.0
0.83 1.0 0.52 0.50
0.81
0.97
1.0
1.0
1.00 1.0 1.00 1.0
0.86 1.0 0.56 0.50
0.84
0.98
1.0
1.0
1.00 1.0 1.00 1.0
0.41 0.50 0.74 1.0
0.40 0.50 0.75 1.0
I1.0
1.0
1.04 ~ 1.04
I 1.00 1.0
! 4.2
1.00 1.0
0.85 1.0
0.7
0.54 0.50
0.39 0.50
0.42 0.50
I
0.97 1.0
DGNAc and DGaINAc give equal colors in the hexosamine assay while DGalNAc gives only onethird the color of DGNAc in the N-aeetylhexosamine color reaction. A ratio of 1.0 indicates that the oligosaecharides contain only DGNAc and an equimolar mixture of DGNAc and DGalNAc gives a ratio of 0.67.
493
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS TABLE II SPECIFIC OPTICAL ROTATIONS OF ISOLATED OLIGOSACCHARIDES Oligosaccharide
RL 2.29 RL 1.9 RL 0.61 RE 0.63 RL 0.54
Wavelength (nm) 589
578
+19.6 a +35. I s +14.0 +12.8 +14.2
+18.3 +35.1 +14.7 +12.8 +13.5
546
+20.8 +38.3 +16.8 +14.2 +15.6
435
365
+30.0 +57.8 +21.7 +19.8 +22.0
+37.3 +76.0 +25.8 +21.2 +26.8
A value of +14.0 has been reported (17). b A value of +30.9 has been reported (17).
sumed 4 moles of periodate (theory 4) and yielded 1.2 moles of formic acid (theory 1) and 2.1 moles of formaldehyde (theory 2). The compound had a small positive optical rotation (Table II). Methylation analysis of the reduced disaccharide yielded peaks on gas-liquid chromatography (glc) corresponding to methyl 2,3,4,6-tetra-O-methyl-o-Gal and 3-O-acetyl - 1,4,5,6 - tetra - 0 - methyl - N - acetyl D-glucosaminitol (Tables III and IV). With 0.2 N NaOH in 1% sodium borohydride, RL 2.29 yielded reduced chromogen in an amount corresponding to a 3-1inked terminal reducing N-aeetylglucosamine (Table VI). These data indicate the compound is $I)Gal (1 -* 3)DGNAc. RL 1.9. RL 1.9 was identified as BDGal (1 --~ 4)oGNAc by the following criteria. It contained equimolar amounts of galactose and glucosamine; the latter was at the reducing end as shown by reduction with NaBH4. The reduced compound consumed 3.3 moles of periodate (theory 3) with the formation of 1.1 moles of formic acid (theory 1) and 1.1 moles of formaldehyde (theory 1) and after the methylation procedure methyl 2,3,4,6-tetra-0-methyl-D-Gal and 4-O-acetyl - 1,3,5,6 - tetra - 0 - methyl - N - acetyl D-glucosaminitol were identified by glc. A minor peak was detected with a retention time corresponding to 3-0-acetyl-l,4,5,6tetra-O-methyl-N-acetyl-D-glucosaminitol. Treatment with base-borohydride (Table VI) produced only 8 % of the reduced chromogcn and indicated the compound probably contained a small amount of ~DGal (1 --~ 3) DGNAc.
RL 0.54. Analyses (Table I) indicated that this compound was a trisaccharide containing 1 mole of glucosamine and 2 moles of galactose, with 1 mole of galactose being reduced by NaBH4. Base-borohydride treatment (Table VI) yielded 3-deoxygalactitol in an amount corresponding to a 3-1inked reducing D-Gal residue. Periodate oxidation of the reduced compound yielded 1.8 moles of formaldehyde (theory 2) indicating that the reduced galactose was linked 1 --* 3 or 1 --~ 4. Periodate uptake was rapid with overoxidation to 7.6 moles and with the formation of 3.1 moles of formic acid, as expected from the overoxidation of the 3-carbon intermediate formed from carbons 2, 3, and 4, or 3, 4, and 5 of the reduced galactose residue (27, cf. 20). 5~[ethylation analysis yielded peaks on gle corresponding to a 3-1inked reducing D-Gal residue, methyl 2,3,4,6-tetra-O-methyl-DGal and methyl 3-O-acetyl-4,6-di-O-methylD-GNAc. Minor peaks corresponding to the N-methyl instead of the N-acetyl derivatives were found for the internal DGNAc residues of the tri- and tetrasaccharides (see Discussion). These data and the specific optical rotations and ORD spectrum indicate that the compound is ~DGal(1 --~ 3)BDGNAc(1 --~ 3)nGal. RL 0.63. This oligosaccharide was shown to be flDGal(1 --~ 3)~DGNAc(1 -~ 3)r --* 3)DGNAc, which contained some r --~ 3)r --+ 3)BDGal(1 --* 4)DGNAc, by the following criteria. The analytical data showed equimolar amounts of galactose and glucosamine and half the glucosamine was reduced with sodium borohydride. The re-
494
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duced compound initially consumed 2.9 moles of periodat.e and rose to 3.8 moles after 48 hr, with the formation of 1.5 moles of formic acid and 1.1 moles of formaldehyde. Methylation analysis yielded peaks in gle corresponding to methyl 2 , 3 , 4 , 6-tetra-O-methyl-D-Gal, methyl 2,4,6-tri-0methyl-D-Gal, methyl 3-0-acetyl-4,6-di-0methyl - D - GNAe, and a mixture of 3 - 0 aeetyl - 1 , 4 , 5 , 6 - tetra - 0 - methyl - N acetyl - U - glueosaminitol and 4- 0 - aeetyl 1 , 3 , 5 , 6 - tetra - 0 - methyl - N - aeetyl - D glueosaminitol. Calculation of the peak areas of the latter two gave amounts equal to 66 and 33%, respectively. Based on the amount of GNAe at. the reducing end the compound on treatment with base-borohydride gave 74% of reduced ehromogen. Thus, at the reducing end, the main component contains a 1 ~ 3 linked GNAc while the minor component (about 30%, average of both methods) contains 1 - ~ 4 linked GNAe. The specific optical rotations and O R D curve supported the assignment of $-anomerie linkages. Paper chromatography in solvent 3 of a partial a d d hydrolysate (21) showed r --+ 3)DGNAc, a trace of $DGal(1 --+ 4)DGNAc and DGal. RL 0.61. Analyses indicated equimolar amounts of galaetose and glueosamine with half of the glucosamine being reduced with NaBH4. Periodate oxidation of the reduced oligosaceharide resulted in the initial uptake of 4 moles of periodate rising to 4.9 moles after 48 hr, with the release of 1.3 moles of formaldehyde and 2.5-3 moles of formic add. ~DGal(1 -+ 3)DGNAe, a trace of/3DGal(1 4)DGNAc and DGal were identified b y paper chromatography in solvent 3 of a partial acid hydrolysate of RL 0.61. Gas-liquid chromatography of the reduced and methylated oligosaccharide gave methyl - 2 , 3 , 4 , 6 - tetra - 0 - methyl - D Gal, methyl - 2 , 4 , 6 - tri - 0 - methyl - D Gal, methyl - 3 - 0 - acetyl - 4,6 - di - 0 methyl - 9 - GNAe, 4 - 0 - acetyl - 1 , 3 , 5 , 6 tetra - 0 - methyl - N - acet,yl - D - glucosaminitol, and 3 - 0 - a c e t y l - l , 4 , 5 , 6 - t e t r a - O methyl-N-acetyl-D-glueosaminitol. Base-borohydride treatment (Table VI) produced 48 % of reduced chromogen based on the calculated weight of the terminal
GNAc residue. The low positive optical rotation and lhe ORD curve indicated ~-anomerit linkages. From methylation and baseborohydride degradation, the compound is a mixture of tetrasaceharides; about one-half each of 5nGal(1 -+ 3)~DGNAc(1 --+ 3) 5DGal(1 --+ 4)DGNAe and 5DGal(1 --+ 3)~DGX*Ae(1 -+ 3)SDGal(1 ---+3)DGNAe. The methylation data alone do not exclude the following sequence for the tetrasaeeharides: r --+ 3) ~ D G a l ( 1 -+ 3) B D G N A e ( 1 - - + 3) or (1 --+ 14) D G N A e . However, the amount of 3-0-aeetyl- 1 , 4 , 5 , 6 tetra - 0 - methyl - N - aeetyl - ~ - glueosaminitol compared to the total permethylated D-glucosaminitol derivatives found on gle after permethylation was approximately equal to the quantity of reduced ehromogen formed on base-borohydride degradation, 74 % for RL 0.63 and 48 % for R~ 0.61 (Table VI). If the two DGNAc residues were linked 1 --+ 3 as above almost twice as much reduced chromogen would have been expected as a consequence of the elimination of the two DGNAc residues at the reducing end (28), i.e., 120 and 85 % of reduced chromogen for RL 0.63 and RL 0.61, respectively.
Identification of the Methyl Glycosides of the Di-O-methyl Ethers of DGNAc Obtained from Permethylated Oligosaccharides Tables IV and V give retention times of some of the 0-methyl ether derivatives of DGNAc and DGalNAc, as their methyl glycosides and hexosaminitols. Details of the BaO methylation procedure (22) and retention times for the O-acetyl-tetra-O-methyl-Nacetyl-D-glucosaminitols have been described earlier (23). Because the main peak for methyl 3-0-acetyl-4,6-di-0-methyl-D-GNAc is eluted faster from the columns than the other di- or m o n o - 0 - m e t h y l - > G N A c derivatives, the retention times are reported for the lower column temperatures (165 and 190 ~ for the E C N S S - M and N P G S columns, respectively). Peak retention times are also ineluded in Table IV for the OG Ro,~ 0.87 compound. This disaeeharide was shown previously to consist of galaetose and Nacetyl-n-galaetosaminitol, and periodate oxidation results suggested the galaetose was linked to the 3-position of the galaetosamini-
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS tol (3). The retention times correspond to 3O-acetyl - 1,4,5,6 - tetra - 0 - methyl - N acetyl-D-galactosaminitol and further confirm the assignment of the 1 --~ 3 linkage. This linkage has been encountered in pig submaxillary mucin (28a). The retention times at 185~ on the ECNSS-M column are given for all peaks detected. The values underlined represent the major pe~ks and others represent minor or very weak peaks, i.e., those with an are~ less than 20 or 5%, respectively, of the major peaks in ~ny sample. For the NPGS column, run at 210 ~, only the major peaks are given. These major peaks represent the a-anomer of the methyl glycoside because methanolysis yields predominantly the aproduct (29), and the minor peaks in the standards are probably the /~-anomer. The reference compound, methyl 3-0-acetyl4,6-di-0-methyl-o-GNAc, has a peak at 2.53 which corresponds to the 6-0-methyl derivative and is most likely a contaminant. The peaks obtained with the methyl glycosides of the known 0-acetyl-di-O-methylN-methyl-D-GNAc account for the minor peaks observed in the BaO methylated compounds. Thus methyl 3-O-acetyl-4,6di-0-methyl-N-methyl-D-GNAc has a major peak at 1.95, two medium-sized peaks at 1.47 and 5.42, and a weak peak at 1.29, and methyl 4 - 0 - acetyl - 3,6-di - O-methyl - Nmethyl-D-GNAc has two major peaks at 1.13 and 1.63 ~nd one weak peak at 2.02. It is not known which of these peaks represent the a- or f~-anomers, or which may represent N-methyl or N-methyl-N-acetyl derivatives, however, these peaks can be clearly seen in and are characteristic for the linkage type in the oligosaccharide structures studied (Table V). For the oligosaccharides RL 0.61 and R~ 0.63 in Table V, the 0.80 and 0.97 (or 0.99) retention times correspond to the 4-0- and the 3-O-~cetyl- derivatives of the tetra-0methyl-N-acetyl-D-glucosaminitols. The 1.13 or 1.17 and the 3.44-3.65 retention times indicate the methyl 3-0-acetyl-4,6-di-0methyl-D-GNAc. The 2.68-2.77 peak corresponds to methyl 4-0-acetyl-3,6-di-0methyl-n-GNAc and indicates the presence of less than 5 % of a 1 --~ 4 linked internal
497
DGNAc moiety. In addition, minor peaks were obtained at retention times of 1.40-1.46 and 1.89-1.97. These were also found in the permethylated, methanolyzed sample of reduced lacto-N-tetraose which contains the /~DGal(1 --~ 3)DGNAc- sequence (17), and corresponds to the methyl 3-O-acetyl-4,6di-0-methyl-N-methyl-n-GNAc.5 The isolation and identification of the structures of three additional oligosaccharides isolated from a "precursor" blood group glycoprotein has been previously described (3). The results of their permethylation are also give in Table V. In each of these oligosaccharides two major peaks were found with retention times of 1.64-1.65 and 2.75-2.80. The latter corresponds to methyl 4-0-acetyl-3,6-di-0-methyl-D-GNAc. The area of the former peaks varied between 25 and 50% of the maior peak, and with the peak at 1.13-1.16 and 1.96-2.03 correspond to methyl 4-O-acetyl-3,6-di-0methyl-N-methyl-o-GNAc. The amount of N-methylation that occurs is therefore somewhat variable under the conditions used for permethylation of different oligosaccharides. Because the 1.13-1.16 peak of the latter reference compound has the same retention time as methyl 3-O-acetyl-4,6-diO-methyl-D-GNAc, it cannot be ascertained whether the OG oligosaccharides contain minor amounts of a 3-substituted internal DGNAc residue. In each sample minor peaks were found which corresponded to the known values for the di- or mono-O-methyl-D-GiNTAc derivatives. For OG RL 0.44 a major peak is found at 0.80 retention time which is presumed to be the derivative formed from the 3,6-disubstituted N-acetyl-D-galactosaminitol, a reference compound which was not available. Minor peaks were not found in the solvent reaction blank. In addition the minor peaks are not attributable to incomplete methylation of DGal as suggested by the following experiment. Each of the permethylated, methanolyzed and O-acetylated samples was 5The N-methyl derivative is written as Nmethyl-D-GNAc although it is uncertain whether these derivatives are N-methyl- or N-methyl-Nacetyl-glucosamines.
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ANI-)EIISON ET AL.
hydrolyzed in 2 N HC1 for 2 hr. The HC1 was removed in vacuo, the samples treated with Dowex 50-H + resin, and the resin washed with water. The charged amino compounds were eluted with 1 N HC1, the HC1 removed in vacuo, and the samples Naeetylated, methanolyzed, and O-acetylated. Each sample gave exactly the same pattern on the ECNSS-M column at 185 ~ with respect to relative peak areas and retention times, as those in Table V. Moreover, no peaks were found at a lower temperature setting at which permethylated DGal derivatives would have been observed, indicating complete separation of neutral and amino sugars. The O R D spectra of RL 1.9, RL 0.54, R~ o.63, and RL 0.61 were recorded. Table V I I lists values of molar rotations at the troughs of the longest wavelength Cotton effect (n-~r*) and at 300 nm. The differences [re]trough - - [m]s00 are also tabulated for the four compounds. I n earlier papers, these differences have been utilized empirically as an aid in determining sequences, substitutions, and configuration of glycosidic linkages (30-33, 23). The table also gives values, from other work, for three related disaccharides and two tetrasaccharides, lacto-N-tetraose and lacto-N-neotetraose. From results cited above (Table VI), it is
clear that IlL 1.9 is a mixture of r --~ 4)DGNAc with a small amount of ~DGal (1 -~ 3)DGNAc. The value of [re]trough -[m]a00 is intermediate between those of the authemic disaecharldes. The calculated percentage of the minor disaccharide is 38cI: from this value. The measurement at 300 nm is, however, subject to great uncertainty when very small amounts of material are available, as was tl~e here. From the trough rotations alone, which are more precisely measured, the minor component is 2 5 ~ of the total. The position of the trough was recorded at 220 nm in IlL 1.9 but is at 218 nm in both authentic disaccharides. If all are compared at the same wavelength, 218 nm, the minor component is but 1 4 - 1 6 ~ . This is still higher than the reduced chromogen value of 8 %, but the results are reasonable considering errors of both procedures with such small samples. The trisaccharide, RL 0.54, gives a value in close agreement ~x~ith that expected from additivity of component residues in the sequence /~DOal(1 ~ 3)/~DGNAc(1 --~ 3)DGal or by comparison with lacto-Ntetraose. The computations are discussed. The two tetrasaccharide solutions, IlL 0.63 and RL 0.61, are both mixtures, as shown above. Calculations of the expected rotations of the component tetrasaccharides
TABLE VI IDENTIFICATION OF LINKAGE TO ]~EDUCING END BY THE ACTION OF SODIUM HYDROXIDESODIUM BOROHYDRIDE ON THE ISOLATED OLIGOSACCHARIDESa Compound
a-D-Gal-(1 --~ 3)-D-Gal fl-D-Glc-(1 ~ 4)-D-Gal fl-D-Gal-(1 --~ 6)-D-Gal I~L 0.54 ~-D-Gal-(1 ---* 3)-D-GNAc
/~-D-Gal-(1 --* 4)-D-GNAc ~-D-Gal-(I ~ 6)-D-GNAc RL 2.29
RL 1.9 RL 0.61 RL 0.63
Scission products obtained Galactitol
3-Deoxygalactitol
5.2
5.2 0.4 0 4.2
06
Reduced chromogen
100 0 0
104~ 8
48 74
" Compounds were treated with 0.2 N NaOH-I% NaBH4 for 24 hr at 24~ (23). b Less than 0.1% of the product was detected. ~,Percentage of free reduced chromogen relative to that obtained with/!l-D-Gal-(1 --* 3)-D-GNAc.
501
IMMUNOCHEMICAL STU1)IES ()N BLO01) GR()UPS
TABLE V11 ORD PARAMETERSOF RL 1.9, RL 0.54, RL 0.63, RL 0.61, AND RELATEDOLIGOSACCHAR1DES Compound
[,tdt r(,ugh Trough [mla~. deg.cm=/decimoIe wavelength(nm) dccimaledCg'cm2/deg.cm2/decimole[m]trough - [m]aa0
RL 1.9 RL 0.54 RL 0.63
--3250 --3900 -- 6400
220 219 222
250 75 -- 80
RL 0.61
--6360 -4440 -2860 --800 -- 3140
222 218 218 222 218
160 110 80 500 580
--6510 -4550 -2940 --1300 -- 3720
-2510
218-220
575
--3140
~3DGal(1 ---+ 3)DGNAc~ /~DGal(1 - + 4)DGNAc~
~DGNAc(1 --+ 3)DGalb Lacto-N-tetraose~, ~DGal(1 --+ 3)~DGNAc(1 -+ 3)~DGal(1 -+ 4)DGlc Lacto-N-neotetraose", /~DGal(1 --+ 4)~DGNAc(1 -+ 3)BDGal(1 -+ 4)DGlc
--3500 --3975 -- 6320
From (30). 9 ~'From (33). in the mixtures did not accord well with the observed values. The main discrepancy is that the observed values of [m]tro,gh [re]a00 are less negative by about 2000 deg. cm~/decimole than anticipated by summing the separate contributions fl'om the residues with the indicated glycosidic linkages. The discrepancies are accounted for below. DISCUSSION H u m a n ovarian cyst A substance treated b y mild acid hydrolysis had previously yielded A-active di- and trisaccharide (7). The further purification of other fractions obtained from charcoal chromatography is described with the isolation of five additional oligosaccharides. The two disaccharides RL 2.29 and RL 1.9 have been found in many sources including B, H, and Le ~ substances (12) and in hum'm milk (17, 34). Thc trisaccharide, ~DGal(1 --~ 3)tSDGNAc(1 ---+ 3)DGal, has also been isolated from A, B, H, and Le ~ substances (15). The two tetrasaccharides, RL 0.63 and RL 0.61, each contained the above trisaccharidc sequence linked either to the 3 or to the 4 position of a reducing DGNAc; each tetrasaccharide fraction was a mixture of two tetrasaccharides. The use of the baseborohydride degradation and especially the methylation analyses provided substantial proof of the structures despitc the fractions
being mixtures. Considering the difficulties in separating similar oligosaccharides, these technics should be of value in structural studies of mixtures of other oligosaccharides. The sequence ~DGal(1 -+ 3 ) ~ G N A c ( 1 3)/~DGal(1 ~ 3)I)GNAc is represented ill the proposed composite megalosaccharide structure of the carbohydrate portion of tile blood group substances (1-5). The tetrasgmcharide with the 1 --+ 4 linked ,)GNAt at, the reducing end does ,tot, occur in the proposed structure and is either a nalurally occurring sequence or is an acid reversion product,. For the biosynthesis of the oligosaccharide chains there either may be distinct glycosyl transferases for each separate sugar moiety added, or a limited number of tranferases may possibly transfer a given sugar to more than one position in the chain or lo different hydroxyls of the terminal acceptor carbohydrate. For lhe first possibility, separate genes would code for the enzymes for each carbohydrate position and linkage in the megalosaccharide, analogous to tile genes responsible for the structures of the A, B, H, Le ~, or Le t' determinants (cf. 35). However, if the glyeosyl transferases are not strictly specific, this might account for the isolation of the two tetrasaeeharides containing 3- and 4-substituted terminal reducing DGNrAc residues. The l etrasaccharide containing the 4-substituted reducing DGNAc could also have been formed by reversion during the initial
502
ANDERSON E T AL.
mild acid hydrolysis. Th(~ formation of oligosaeeharides by 'tci(1 reversion is described by Jones "rod Nieholson (36), Neuberger and Nl.trshall (:/7), and I~arker et aL (38, 39); both ~- and r glycosides can be formed and substitutions may occur at different hydroxyls of the aeeeptor earbohydrate. There is insufficient data to indieate a preferential formation of part ieul'tr linkages. It is also possible that different, types of oligosaeeharide chains, as defined by their linkage sequences, are attached to the protein core, and the two tetrasaeeharides could then have been derived from separate megalosaeeharides in the original ovarian cyst material. Two new determinant-containing oligosaeeharides, c~DGNAe(1 -+ 4) ~/DGal(1 --~ 4)DGNAe and aDGNAc(1 4)flDGal(1 ~ 3)DGalNAe (23), were recently isolated from hog muein A q- H substance. The second of these was presumably attached directly to the polypeptide core through the terminal reducing GalNAe residue, while the former oligosaeeharide was probably originally part of a larger oligosaceharide. The isolation of these oligosaceharides indieates that several types of chains occur in blood group glycoproteins. A definitive answer to the heterogeneity and speeifieity of synthesis of the oligosaeeharide ehains will be obtained when the intact ehains are isolated, fraetionated, and eharaeterized. Various techniques for the separation of the 0-methyl ether derivatives have been deseribed including paper chromatography (37), the gle identification of the fully aeetylated 2-aeetamido-2-deoxy-D-glueitol derivatives (24), and ion-exchange eolumns (40). Some methyl ethers have been identiffed as the methyl glycosides of I)GNAe (41, 42). Table V shows the utility of the BaO methylation and methanolysis method as described for establishing the linkage to the imernal I)GNAe of the isolated oligosaeeh-~rides. Only 200 ug of each substituted DGNAe of the original oligosaeeharide is needed for the methylation procedure and for gle analyses. The additional minor peaks generally
found on glc of methylatcd amino sugars have been shown to be due to N-methylation and are determined by the link'tge to the internal amino sugar in the oligosaceharide. Thus they help to provide unequivoeM identification of the internal linkages to amino sugars in oligosaecharides. The ORD spectra of four of the oligosaeeharides reported here showed the eharaeteristic trough near 220 nm associated with S-linked and reducing terminal DGNAe residues. While some fractions are mixtures, and may have small amounts of impurities, they were nonetheless useful to eontinued attempts to seek out elements of additivity or regularity in ORD spectra of oligosaeeharides. The ORD data on the trisaeeharide (RL 0.54) were examined in several ways. If to the [m]~ro~h -- [m]~00value of ~oGNAe(1 ~ 3)DGal is added that of methyl SDGal(-- 135), the difference between this sum and the observed value for RL 0.54 should represent the effeet of substitution on carbon 3 of the r DGNAe residue. That difference is -2540 deg. em-~/deeimole. The disaeeharide, ~DGNAe(1 -+ 3)DGal, may, in turn, be considered as comprised of methyl f3DGNAe (-2140) and DGal (875). The sum of these is --1255, which agrees well with the observed value of --1300 for the disaceharide (Table VII). The trisaeeharide may thus be thought of as comprising three residue contributions and an additional effect due to the carbon-3 substitution on the internal I)GNAe residue. The sum of these four eontributions is -3930; the observed value is -3975 deg. em=/deeimole. Alternatively, one may begin with the laeto-N-tetraose value, subtract the DGle (960) and methyl ~DGal residues and add the value for DGal. This gives -3670 deg. em2/deeimole. Although the trisaeeharide eomains only a very small amount of material ( < 5 % ) with earbon-4 substituted, rather than carbon-3 substituted, ~dinked DGNAe residues, it was of interest to evaluate the difference expected from mixtures of these two eompounds. Comparison of laeto-Ntetraose with laeto-N-neotetraose reveals
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS t h a t such a change results in a difference of just under 600 deg- em2/deeimole. Two other oligosaccharides derived from A substance, also differing by just the one change in substitution on an internal ~-linked DGNAc residue have previously been reported to show a difference of just under 500 deg. cm2/ deeimole in the value of [m]t:o~gh - [m]300
(3~).
The tetrasaccharides present a quite different problem. If one sums the elements as in the trisaccharide using any of the disaccharide combinations or adding the monosaceharides separately, the computed values of [rn]t~o~gh - [m]~00 are more t h a n 2000 deg. cm~/decimole more negative than the observed values. This difference is well outside the uncertainties involved in estimating the various contributions to the sequence. The two fractions, in addition, differ considerably in the ratios of the two tetrasaccharides which each contains, as noted above, but the observed values of tin]trough -- [mJa00 are very close. The discrepancy of the observed value compared to the computed value for each oligosaccharide probably arises for different reasons. T h e total quantity of RL 0.63 isolated was 8.5 rag. I t has been shown previously (3, 21), t h a t when such small amounts are isolated as much as 2 mg m a y be inert material. Such a 25 % error in concentration based on dry weight would account for the O R D anomaly. With RL 0.61, the yield was 14.2 mg and the proportion of inert material would be lower. In this sample, however, analysis showed 4.2% galactosamine which probably is due to oligosaccharide containing a nonreducing terminal aDGalNAc residue. The latter would add very substantial positive rotation in the ultraviolet (30). Together with any concentration error, this could account for the anomalous value of [rn]trough -- [m]a00 for RL 0.61. Thus when substantial discrepancies between calculated and observed O R D values are noted, these m a y indicate impurities or inexact concentrations and the analytical data should be carefully scrutinized. In this case the discrepancies were satisfactorily resolved.
503
REFERENCES 1. LLOYD, K. O., AND KARAT, E. A., Proc. Nat. Acad. ~ei. U.S,A. 61, 1470 (1968). 2. KADAT,E. A., in "Blood and Tissue Antigens" (D. Aminoff, ed), p. 187. Academic Press, New York (1970). 3. VICARI,G., ANDKABAT,E. A., Biochemistry 9, 3414 (1970). 4. FEIzI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARSH, W. L., J. Exp. Med. 133, 39 (1971). 5. FEIZI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARGIn, W. L., J. Immunol., 106, 1578 (1971). 6. COTE, R. H., AND MORGAN, W. T. J., Nature London 178, 1171 (1956). 7. SCHIFFMAN, G., I4~ABAT, E. A., AND LESKOWlTZ, S., J. Amer. Chem. Soc. 84, 73 (1962). 8. CHEESE, I. A. F. L., AND MORGAN, W. T. J., Nature London 191, 149 (1961). 9. PAINTER, T. ,l., WATKINS, W. M., AND MORGAN, W. T. J., Nature London 193, 1042
(1962). 10. PAINTER, T. J., WATKINS, W. M., AND MORGAN,W. T. J., Nature London 199, 282 (1963). 11. ASTON, W. P., I'IAGUE, G. M., DONALD, A. S. R., AND MORGAN,W. T. J., Biochem. J.
110, 157 (1968). 12. PAINTER, T. J., REGE, V. P., AND MORGAN, W. T. J., Nature London 199, 569 (1963). 13. ASTON,W. P., DONALD,A. S. R., ANDMORGAN, W. T. J., Biochem. Biophys. ReG. Commun.
30, 1 (1968). 14. ASTON,W. P., DONALD,A. S. R., ANDMORGAN, W. T. J., Biochem. J. 107, 861 (1968). 15. REGE, V. P., PAINTER, T. J., WATKINS,W. M., AND MORGAN,W. T. J., Nature London 200, 532 (1963). 16. VICARI, G., AND KADAT, E. A., J. Immunol. 102, 821 (1969). 17. KUHN, R., BAER, H. H., AND GAUHE, A., Chem. Ber. 87, 1553 (1954). 18. KUHN, R., .~ND LOW, I., Chem. Bet. 86, 1027 (1953). 19. SMITH, F., AND STEPHEN, A. M., J. Chem. Soe. 4892 (1961). 20. KABAT, E. A., "Kabat and Mayer's Experiperimental Immunochemistry," 2nd Ed. Thomas, Springfield, Ill. (1961). 21. LLOYD, K. O., KABAT, E. A., LAYUG, E. J., AND GRUEZO,F., Biochemistry 5, 1489 (1966). 22. KUHN, R., BAER, H. H., AND SEELIGER, A., Ann. Chem. 611,236 (1958). 23. ETZLER, M. E., ANDERSON, B., BEYCHOK, S., GRUEZO, F, LLOYD, K. O., RICHARDSON,
504
ANI)EI:(SON ET AL.
N. (;., ~xl) K.~J~.xT. 1':. A., Ar~'h. Bio,'bem. 33. K.\n.~'v, E. A., L[.oYo, K. O., AND BEYGHOK, S., Biochemistry 8, 747 (1969). Biod~e,~. Biophy.~. 141,588 (1970). 24. [)r;lr M. B., ANDWEan, A. C., Can. J. Chem. 34. KOBATA,A., J. Bioehem. Tokyo 53, 167 (1963). 35. W=~TK~NS,W. M., in "Blood and Tissue Anti~ 47, 4091 (1969), gens" (D. Aminoff, ed.), p. 441. Academic 25. GORIN, P. A. J., AND FINLAYSON, i~.. J., CarPress, New York (1970). bohyd. Re,~., in press. 26. I~)EISSIG, J. L., STI%OMtNGER) J. L.) AND 36. JONES, J. K. N., ANn NICHOLSON, W. I t , J. Chem. Soc. 27 (1958). LELOIE, L. F., J. Biol. Chem. 217, 959 (1955). 27. WHELAN,W. J., in "Haudbuch der Pflanzen- 37. NEUBERGER, A., ANn MARSHALL, R. ])., in "Glycoproteins" (A. Gottschalk, ed.), p. physiologie," Vol. 6, p. 154. Springer, Berlin 235. Elsevier, New York (1966). (1958). 28. LLOYD, K. O., AND KABAT, E. A., Carbohyd. 38. BARKER,S. A., MURRAY,K., AND STACEY,M., Nature London 191, 142 (1961). Res. 9, 41 (1969). 28a CARLSON, ]). M., J. Biol. Chem. 243, 616 39. BARKER,S. A., MURRAY,K., STACEY,M., AND (1968). STROUD, D. B. E., Nature London 191, 143 29. KUHN, R., ZILLIKEN, F., AND GAUHE, A., (1961). Chem. Ber. 66, 466 (1953). 40. ADAMS, G. A., YAGUCHI, M., AND PERRY, 30. BEYCHOE,S., AND KABAT, E. A., Biochemistry M. B., Carbohyd. Res. 12, 267 (1970). 4, 2565 (1965). 31. LLOYD, K. O., BEYCHOK, S., AND K.~BAT, 41. KUHN, R., AND EGGS, H., Chem. Ber. 96, 3338 (1963). E. A., Biochemistry 6, 1448 (1967). 42. KOBATA, A., AND GINSBURG,V., J. Biol. Chem. 32. LLOYD, K. O., BEYCHOK, S., A.ND KABAT, 244, 5496 (1969). E. A., Biochemistry 7, 3762 (1968).
145,490-501 (1971)
Immunochemical Studies on Blood Groups L Structures of Some Oligosaccharides Obtained by Mild Acid Hydrolysis of Human Ovarian Cyst Blood Group A Substance~ B Y R O N A N D E R S O N } E L V I N A. K A B A T , S H E R M A N B E Y C H O K , AND F L A V I O G R U E Z O
Departments of Microbiology, Neurology, and Human Genetics and Development, College of Physicians and Surgeons and the Departments of Biological Sciences and Chemistry, Columbia University and the Neurological Institute, Presbyterian Hospital, New York Received February 23, 1971; accepted April 13, 1971 Mild acid hydrolysis of a human ovarian cyst blood group A substance followed by preparative chromatography resulted in the isolation of two disaceharides, r --* 3)DGNAc, r --* 4)DGNAc, a trisaccharide, ~DGal(1 ~ 3)BDGNAe(1 --* 3)DGal, and two tetrasaceharides, ~nGal(1 ~ 3)f~DGNAe(1 ~ 3)~DGaI(1 --* 3)DGNAe, and BDGal(1 --~ 3)/~DGNAe(1 --* 3)r --* 4)bGNAe. Their structures were established by analyses, periodate oxidation, methylation, alkaline degradation, and ORD spectra. The oligosaecharides appear to be derived from the interior of the megalosaeeharide chains of the blood group active glyeoprotein. A procedure for establishing linkages of internal DGNAc residues in oligosaeeharides by gas chromatography following BaO methylation, methanolysis, and O-acetylation has been developed. Methylation analyses are also presented for four oligosaceharides obtained from alkaline borodeuteride degradation of a precursor blood group substance, OG, which, unlike the other oligosaeeharides, contained 4-substituted internal DGNAe residues. Although the two tetrasaeeharide fractions from A substance were only obtained as mixtures, the methods developed for methylation analysis and base-borohydride degradation were capable of elucidating each structure in the mixtures. The soluble blood group substances are glyeoproteins whose oligosaccharide chains contain the antigenic speeifieities, A, B, H, Le% Le b, and I. Acid or base degradation has been used to obtain oligosaeeharides of a size amenable to structural studies and free from the protein portion of the parent molecule. These d a t a have led to a proposed composite structure for the oligosaeeharide portion of the blood group substance (1-5). 1 Aided by grants from the National Science Foundation (GB-8341, GB-25686, and GB-22847), a General Research Support Grant,, and Grant GM-10576 from the United States Public Health Service. Helen Hay Whitney Fellow and National Cystic Fibrosis Research Foundation Fellow 1968-1971.
Partial acid hydrolysis, using either mineral acid or polystyrene suIfonic acid, has been employed extensively to obtain from blood group substances several A active oligosaeeharides (6-8); B active oligosaeeharides (9-11); and m a n y inactive oligosaceharides containing DGal, DGNAe or DGalNAe 3 or both (7, 10, 12-15). The present study describes the isolation and characterization of two disaeeharides, a trisaeeharide, and two tetrasaccharides, each containing only DGal and DGNAe. These oligosaecharides were obtained from 3 Abbreviations: Gal = galaetose, GalNAe = N-acetylgalactosamine, GNAc = N-acetylglucosamine, Glc = glucose, GalNH~ = galactosamine.
490
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS a partial acid hydrolysate of a h u m a n blood group A substance which had been previously ehromatographed and from which A active di- and trisaeeharides had been isolated (7). During this study it became necessary to identify linkages to internal GNAc residues b y methylation procedures. The method developed has also been applied to four reduced oligosaecharides previously isolated by alkaline-borodeuteride degradation of a h u m a n ovarian cyst, precursor substance (3, 16). MATERIALS AND METHODS Monosaceharides were obtained from Mann Research Laboratories or Nutritional Bioehemicals Co. r --~ 4)DGNAc was obtained from Dr. F. Zilliken; f/DGal(1 --~ 3)DGNAe, BDGal(1 --~ 4) I)GNAc, BDGal(1 --* 6)DGNAe (17), r --~ 4) DGal(18), and laeto-:V-tetraose were kindly provided by Dr. A. Gauhe. flDGal(1 --~ 6)nGal(19) was from Dr. A.M. Stephen; /~nGle(1 --~ 3)-Naeetyl-D-galaetosaminitol from Dr. D. M. Carlson; 3-deoxy-D-Gal from Dr. S. Svensson. Nitrogen, methylpentose (fucose), hexosamine, N-acetylhexosamine, and hexose (galaetose) were determined by colorimetric methods (20, 21). Periodate uptake, formaldehyde, and formic acid were measured as previously described (20). Paper chromatography was carried out in three solvent systems: 1-propanol-ethyl acetate-water (7:1:2), solvent 1; 1-butanol-pyridine-water (6:4:3), solvent 2; 1-butanol-pyridine-water (6:1:1), solvent 3. Schleicher and Schiill 589 green ribbon paper was used for analytical and preparative and Whatman 3 MM for preparative studies. The sodium borohydride reduction, the baseborohydride degradation with gas-liquid chromatographic quantitation of galactitol, 3-deoxygalactirol and reduced chromogen, and the methylation procedure using BaO as catalyst (22) were described previously (23). Formation of the methyl glycosides with methanolic HC1 followed by methylation was used to prepare the tri-O-methyl methyl glycosides of N-acetyl-D-glucosamine and N-acetyl-D-galacto9samine. The preparation of the O-acetyl-tetra-Omethyl-N-acetyl-D-glucosaminitol and N-acetylD-galactosaminitol derivates has been described (23). The methyl glycoside of 3-O-acetyl-4,6-diO-methyl-N-acetyl-D-glucosamine was obtained from Dr. A. Gauhe. The 3,6-di-O-methyl-, 3,4-die-methyl-, 3-O-methyl-, and 6-O-methyl-Nacetyl-D-glueosamines were obtained from Drs. A. Webb and M. B. Perry (24); the 3,6-di-O-methyland 4,6-di-O-methyl-N-methyl-D-glucosamine hy-
491
drochlorides were supplied by Dr. P. A. J. Gorin (25); these were converted into methyl glycosides by methanolic-HC1 and the free hydro)tyls were then 0-acetylated. Gas-liquid chromatography was carried out using an F and M (Hewlett Packard) 810 gas chromatograph. ORD spectra were measured in a Bendix Ericcsen or in a Cary Model 60 spectropolarimeter. Optical rotations were measured in a Perkin-Elmer polarimeter Model 141 at five wavelengths. EXPERIMENTAL AND RESULTS Four oligosaccharide fractions obtained previously (7) b y chromatography on charcoal and elution with an ethanol gradient from the dialysate of a h u m a n ovarian cyst A substance (McDon), which had been subjected to mild acid hydrolysis, were studied. T h e y were: Ai, 25 mg; A2, 343 mg; As, 83 rag; A4, 59 mg; and A 6 , 1 1 9 mg. Of the A2 fraction, 91 mg was chromategraphed by three descending developments in solvent 1 to give 49 mg of a. compound which had moved one-third down the paper. Three components were obtained on chrom a t o g r a p h y in solvent 2: RL 4 1.67 (26.3 rag), RL 1.44 (14.9 rag), and RL 1.22 (3.1 rag). The latter was discarded. The RL 1.67 component, was chromatographed in solvent 3 to yield 14.7 mg of RL 2.29 and 10.9 mg of RL 1.94. The RL 2.29 component was purified through Bio-Gel P-2 to yield 7.7 mg (oligosaccharide RL 2.29). The RL 1.44 material was also chromategraphed in solvent 3 to yield RL 1.7 (4.2 rag), which was discarded, and RL 1.9 (6.1 mg), which was combined with the above R~ 1.94 material, and purified through BioGel P-2 to yield 12 mg (oligosaccharide RL
1.9). The A1, As, A4, and A~ fractions were each chromatographed in solvent 2 to give the following broad cuts: RL 0.2, 83.9 mg; RL 0.52, 74.6 rag; RL 0.79, 37.3 mg; RL 0.98, 24.5 mg; RL 1.2, 49.2 mg; and Rc~l 1.0, 55.7 rag. The R~ 0.2, RL 0.98, and RG~, 1.0 materials were apparently very heterogeneous, and further a t t e m p t s to purify t h e m b y gel filtration and paper chromatogr a p h y resulted in fractions of insufficient quantity for structural determinations. 4 RL
~
t~Lactose
-
ANDERSON ET AL.
492
were still impure. T h e 15.9-mg fraction was purified t h r o u g h Bio-Gel P-2 to give 14.2 m g (oligosaeeharide IlL 0.61); its a c l u a l IlL as i s o l a t e d was 0.61 in solvent 2. E a c h of the five oligosaeeharides s t u d i e d g:tve a single spot on p a p e r in solvent 2.
T h e RL 0.52 was passed t h r o u g h Bio-Gel P-2 to y i e l d two fractions of 29.0 a n d 24.1 mg. T h e l a t t e r was again e h r o m a t o g r a p h e d on Bio-Gel P-2 to give 19.0 rag. This comp o u n d h a d a n RL of 0.54 in solvent 2 (oligosaecharide R~, 0.54). T h e 29.0-mg fraction was purified t h r o u g h Bio-Gel P-2 a n d b y p r e p a r a t i v e p a p e r c h r o m a t o g r a p h y twice in solvent 2 s e p a r a t i n g o u t t h r e e m i n o r a n d one m a j o r c o m p o n e n t , RL 0.63 (12.2 rag). T h i s l a t t e r c o m p o n e n t was p a s s e d t h r o u g h BioGel P-2 to y i e l d 8.5 m g (oligosaceharide RL 0.63). T h e RL 0.79 m a t e r i a l was purified t h r o u g h Bio-Gel P-2 to give two fractious of 15.9 a n d 8.3 mg. T h e l a t t e r gave two more fractions on B i o - G e l P-2, 3.1 a n d 4.7 rag, each of which
Determination of Structures of Oligosaccharides RL 2.29. A n a l y t i c a l d a t a (Table I) showed e q u i m o l a r a m o u n t s of glucosamine a n d galactose, a n d t h e glucosamine was des t r o y e d on r e d u c t i o n w i t h N a B H 4 . A d i r e c t N - a c e t y l h e x o s a m i n e (26) r e a c t i o n on t h e disaccharide gave a color yield of 100% as compared with a 3-0-substituted N-acetylglucosamine. T h e r e d u c e d disaccharide con-
TABLE I ANALYTICAL PROPERTIES OF ~SOLATED OLIGOSACCHARIDES Percentage composition Oligosaccharide
~L 2.29 Calcd ~educed RL 2.29 Calcd ~L 1.9 Calcd reduced RL 1.9 Calcd
Yield (rag)
7.7
12
N 2.6 3.65
3.3 3.65
14.2 tL 0.61 Calcd ~educed RL 0.61 Calcd
3.5 3.65
~L0.63 Calcd ~educed RL 0.63 Calcd
3.2 3.65
8.5
~L0.54 19 Calcd Leduced RL 0.54 Calcd
2.8 2.4
Molar ratios
Hexos.~ Hexos(Gal) aminea
NAc GalHexos. amine' NH2
42.2 46.7 33.5 46.7
28.6 46.7 8.2 0
36.7 57.3 0.7 0
- 2.7 0 ~ 7 8 ' 1.00
47.0 46.7 44.5 46.7
33.4 46.7 9.6 0
46.8 57.3 0.9 0
0.91 1.0
45.0 46.7 40.3 46.7
37.6 46.7 22.3 23.4
44.8 57.3 20.4 28.7
48.3 46.7 46.3 46.7
41.5 46.7 25.8 23.4
50.0 57.3 24.1 28.7
67.0 62 30.8 31
27.0 31 22.8 31
32.3 38 28.4 38
N
1.0
[ Gal
NAc- NAcHexos Hexos- amine/ Hexosamine aminea
Hexos-
amine 0.68 1.0
0.71
0.71 1.0
0.81
1.14
1.0
1.0
1.00 1.0 1.00 1.0
0.83 1.0 0.52 0.50
0.81
0.97
1.0
1.0
1.00 1.0 1.00 1.0
0.86 1.0 0.56 0.50
0.84
0.98
1.0
1.0
1.00 1.0 1.00 1.0
0.41 0.50 0.74 1.0
0.40 0.50 0.75 1.0
I1.0
1.0
1.04 ~ 1.04
I 1.00 1.0
! 4.2
1.00 1.0
0.85 1.0
0.7
0.54 0.50
0.39 0.50
0.42 0.50
I
0.97 1.0
DGNAc and DGaINAc give equal colors in the hexosamine assay while DGalNAc gives only onethird the color of DGNAc in the N-aeetylhexosamine color reaction. A ratio of 1.0 indicates that the oligosaecharides contain only DGNAc and an equimolar mixture of DGNAc and DGalNAc gives a ratio of 0.67.
493
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS TABLE II SPECIFIC OPTICAL ROTATIONS OF ISOLATED OLIGOSACCHARIDES Oligosaccharide
RL 2.29 RL 1.9 RL 0.61 RE 0.63 RL 0.54
Wavelength (nm) 589
578
+19.6 a +35. I s +14.0 +12.8 +14.2
+18.3 +35.1 +14.7 +12.8 +13.5
546
+20.8 +38.3 +16.8 +14.2 +15.6
435
365
+30.0 +57.8 +21.7 +19.8 +22.0
+37.3 +76.0 +25.8 +21.2 +26.8
A value of +14.0 has been reported (17). b A value of +30.9 has been reported (17).
sumed 4 moles of periodate (theory 4) and yielded 1.2 moles of formic acid (theory 1) and 2.1 moles of formaldehyde (theory 2). The compound had a small positive optical rotation (Table II). Methylation analysis of the reduced disaccharide yielded peaks on gas-liquid chromatography (glc) corresponding to methyl 2,3,4,6-tetra-O-methyl-o-Gal and 3-O-acetyl - 1,4,5,6 - tetra - 0 - methyl - N - acetyl D-glucosaminitol (Tables III and IV). With 0.2 N NaOH in 1% sodium borohydride, RL 2.29 yielded reduced chromogen in an amount corresponding to a 3-1inked terminal reducing N-aeetylglucosamine (Table VI). These data indicate the compound is $I)Gal (1 -* 3)DGNAc. RL 1.9. RL 1.9 was identified as BDGal (1 --~ 4)oGNAc by the following criteria. It contained equimolar amounts of galactose and glucosamine; the latter was at the reducing end as shown by reduction with NaBH4. The reduced compound consumed 3.3 moles of periodate (theory 3) with the formation of 1.1 moles of formic acid (theory 1) and 1.1 moles of formaldehyde (theory 1) and after the methylation procedure methyl 2,3,4,6-tetra-0-methyl-D-Gal and 4-O-acetyl - 1,3,5,6 - tetra - 0 - methyl - N - acetyl D-glucosaminitol were identified by glc. A minor peak was detected with a retention time corresponding to 3-0-acetyl-l,4,5,6tetra-O-methyl-N-acetyl-D-glucosaminitol. Treatment with base-borohydride (Table VI) produced only 8 % of the reduced chromogcn and indicated the compound probably contained a small amount of ~DGal (1 --~ 3) DGNAc.
RL 0.54. Analyses (Table I) indicated that this compound was a trisaccharide containing 1 mole of glucosamine and 2 moles of galactose, with 1 mole of galactose being reduced by NaBH4. Base-borohydride treatment (Table VI) yielded 3-deoxygalactitol in an amount corresponding to a 3-1inked reducing D-Gal residue. Periodate oxidation of the reduced compound yielded 1.8 moles of formaldehyde (theory 2) indicating that the reduced galactose was linked 1 --* 3 or 1 --~ 4. Periodate uptake was rapid with overoxidation to 7.6 moles and with the formation of 3.1 moles of formic acid, as expected from the overoxidation of the 3-carbon intermediate formed from carbons 2, 3, and 4, or 3, 4, and 5 of the reduced galactose residue (27, cf. 20). 5~[ethylation analysis yielded peaks on gle corresponding to a 3-1inked reducing D-Gal residue, methyl 2,3,4,6-tetra-O-methyl-DGal and methyl 3-O-acetyl-4,6-di-O-methylD-GNAc. Minor peaks corresponding to the N-methyl instead of the N-acetyl derivatives were found for the internal DGNAc residues of the tri- and tetrasaccharides (see Discussion). These data and the specific optical rotations and ORD spectrum indicate that the compound is ~DGal(1 --~ 3)BDGNAc(1 --~ 3)nGal. RL 0.63. This oligosaccharide was shown to be flDGal(1 --~ 3)~DGNAc(1 -~ 3)r --* 3)DGNAc, which contained some r --~ 3)r --+ 3)BDGal(1 --* 4)DGNAc, by the following criteria. The analytical data showed equimolar amounts of galactose and glucosamine and half the glucosamine was reduced with sodium borohydride. The re-
494
ANDEI{SON
of
~'~
~
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ET AL.
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IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS
495
%
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i~
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496
ANDERSON ET AL.
duced compound initially consumed 2.9 moles of periodat.e and rose to 3.8 moles after 48 hr, with the formation of 1.5 moles of formic acid and 1.1 moles of formaldehyde. Methylation analysis yielded peaks in gle corresponding to methyl 2 , 3 , 4 , 6-tetra-O-methyl-D-Gal, methyl 2,4,6-tri-0methyl-D-Gal, methyl 3-0-acetyl-4,6-di-0methyl - D - GNAe, and a mixture of 3 - 0 aeetyl - 1 , 4 , 5 , 6 - tetra - 0 - methyl - N acetyl - U - glueosaminitol and 4- 0 - aeetyl 1 , 3 , 5 , 6 - tetra - 0 - methyl - N - aeetyl - D glueosaminitol. Calculation of the peak areas of the latter two gave amounts equal to 66 and 33%, respectively. Based on the amount of GNAe at. the reducing end the compound on treatment with base-borohydride gave 74% of reduced ehromogen. Thus, at the reducing end, the main component contains a 1 ~ 3 linked GNAc while the minor component (about 30%, average of both methods) contains 1 - ~ 4 linked GNAe. The specific optical rotations and O R D curve supported the assignment of $-anomerie linkages. Paper chromatography in solvent 3 of a partial a d d hydrolysate (21) showed r --+ 3)DGNAc, a trace of $DGal(1 --+ 4)DGNAc and DGal. RL 0.61. Analyses indicated equimolar amounts of galaetose and glueosamine with half of the glucosamine being reduced with NaBH4. Periodate oxidation of the reduced oligosaceharide resulted in the initial uptake of 4 moles of periodate rising to 4.9 moles after 48 hr, with the release of 1.3 moles of formaldehyde and 2.5-3 moles of formic add. ~DGal(1 -+ 3)DGNAe, a trace of/3DGal(1 4)DGNAc and DGal were identified b y paper chromatography in solvent 3 of a partial acid hydrolysate of RL 0.61. Gas-liquid chromatography of the reduced and methylated oligosaccharide gave methyl - 2 , 3 , 4 , 6 - tetra - 0 - methyl - D Gal, methyl - 2 , 4 , 6 - tri - 0 - methyl - D Gal, methyl - 3 - 0 - acetyl - 4,6 - di - 0 methyl - 9 - GNAe, 4 - 0 - acetyl - 1 , 3 , 5 , 6 tetra - 0 - methyl - N - acet,yl - D - glucosaminitol, and 3 - 0 - a c e t y l - l , 4 , 5 , 6 - t e t r a - O methyl-N-acetyl-D-glueosaminitol. Base-borohydride treatment (Table VI) produced 48 % of reduced chromogen based on the calculated weight of the terminal
GNAc residue. The low positive optical rotation and lhe ORD curve indicated ~-anomerit linkages. From methylation and baseborohydride degradation, the compound is a mixture of tetrasaceharides; about one-half each of 5nGal(1 -+ 3)~DGNAc(1 --+ 3) 5DGal(1 --+ 4)DGNAe and 5DGal(1 --+ 3)~DGX*Ae(1 -+ 3)SDGal(1 ---+3)DGNAe. The methylation data alone do not exclude the following sequence for the tetrasaeeharides: r --+ 3) ~ D G a l ( 1 -+ 3) B D G N A e ( 1 - - + 3) or (1 --+ 14) D G N A e . However, the amount of 3-0-aeetyl- 1 , 4 , 5 , 6 tetra - 0 - methyl - N - aeetyl - ~ - glueosaminitol compared to the total permethylated D-glucosaminitol derivatives found on gle after permethylation was approximately equal to the quantity of reduced ehromogen formed on base-borohydride degradation, 74 % for RL 0.63 and 48 % for R~ 0.61 (Table VI). If the two DGNAc residues were linked 1 --+ 3 as above almost twice as much reduced chromogen would have been expected as a consequence of the elimination of the two DGNAc residues at the reducing end (28), i.e., 120 and 85 % of reduced chromogen for RL 0.63 and RL 0.61, respectively.
Identification of the Methyl Glycosides of the Di-O-methyl Ethers of DGNAc Obtained from Permethylated Oligosaccharides Tables IV and V give retention times of some of the 0-methyl ether derivatives of DGNAc and DGalNAc, as their methyl glycosides and hexosaminitols. Details of the BaO methylation procedure (22) and retention times for the O-acetyl-tetra-O-methyl-Nacetyl-D-glucosaminitols have been described earlier (23). Because the main peak for methyl 3-0-acetyl-4,6-di-0-methyl-D-GNAc is eluted faster from the columns than the other di- or m o n o - 0 - m e t h y l - > G N A c derivatives, the retention times are reported for the lower column temperatures (165 and 190 ~ for the E C N S S - M and N P G S columns, respectively). Peak retention times are also ineluded in Table IV for the OG Ro,~ 0.87 compound. This disaeeharide was shown previously to consist of galaetose and Nacetyl-n-galaetosaminitol, and periodate oxidation results suggested the galaetose was linked to the 3-position of the galaetosamini-
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS tol (3). The retention times correspond to 3O-acetyl - 1,4,5,6 - tetra - 0 - methyl - N acetyl-D-galactosaminitol and further confirm the assignment of the 1 --~ 3 linkage. This linkage has been encountered in pig submaxillary mucin (28a). The retention times at 185~ on the ECNSS-M column are given for all peaks detected. The values underlined represent the major pe~ks and others represent minor or very weak peaks, i.e., those with an are~ less than 20 or 5%, respectively, of the major peaks in ~ny sample. For the NPGS column, run at 210 ~, only the major peaks are given. These major peaks represent the a-anomer of the methyl glycoside because methanolysis yields predominantly the aproduct (29), and the minor peaks in the standards are probably the /~-anomer. The reference compound, methyl 3-0-acetyl4,6-di-0-methyl-o-GNAc, has a peak at 2.53 which corresponds to the 6-0-methyl derivative and is most likely a contaminant. The peaks obtained with the methyl glycosides of the known 0-acetyl-di-O-methylN-methyl-D-GNAc account for the minor peaks observed in the BaO methylated compounds. Thus methyl 3-O-acetyl-4,6di-0-methyl-N-methyl-D-GNAc has a major peak at 1.95, two medium-sized peaks at 1.47 and 5.42, and a weak peak at 1.29, and methyl 4 - 0 - acetyl - 3,6-di - O-methyl - Nmethyl-D-GNAc has two major peaks at 1.13 and 1.63 ~nd one weak peak at 2.02. It is not known which of these peaks represent the a- or f~-anomers, or which may represent N-methyl or N-methyl-N-acetyl derivatives, however, these peaks can be clearly seen in and are characteristic for the linkage type in the oligosaccharide structures studied (Table V). For the oligosaccharides RL 0.61 and R~ 0.63 in Table V, the 0.80 and 0.97 (or 0.99) retention times correspond to the 4-0- and the 3-O-~cetyl- derivatives of the tetra-0methyl-N-acetyl-D-glucosaminitols. The 1.13 or 1.17 and the 3.44-3.65 retention times indicate the methyl 3-0-acetyl-4,6-di-0methyl-D-GNAc. The 2.68-2.77 peak corresponds to methyl 4-0-acetyl-3,6-di-0methyl-n-GNAc and indicates the presence of less than 5 % of a 1 --~ 4 linked internal
497
DGNAc moiety. In addition, minor peaks were obtained at retention times of 1.40-1.46 and 1.89-1.97. These were also found in the permethylated, methanolyzed sample of reduced lacto-N-tetraose which contains the /~DGal(1 --~ 3)DGNAc- sequence (17), and corresponds to the methyl 3-O-acetyl-4,6di-0-methyl-N-methyl-n-GNAc.5 The isolation and identification of the structures of three additional oligosaccharides isolated from a "precursor" blood group glycoprotein has been previously described (3). The results of their permethylation are also give in Table V. In each of these oligosaccharides two major peaks were found with retention times of 1.64-1.65 and 2.75-2.80. The latter corresponds to methyl 4-0-acetyl-3,6-di-0-methyl-D-GNAc. The area of the former peaks varied between 25 and 50% of the maior peak, and with the peak at 1.13-1.16 and 1.96-2.03 correspond to methyl 4-O-acetyl-3,6-di-0methyl-N-methyl-o-GNAc. The amount of N-methylation that occurs is therefore somewhat variable under the conditions used for permethylation of different oligosaccharides. Because the 1.13-1.16 peak of the latter reference compound has the same retention time as methyl 3-O-acetyl-4,6-diO-methyl-D-GNAc, it cannot be ascertained whether the OG oligosaccharides contain minor amounts of a 3-substituted internal DGNAc residue. In each sample minor peaks were found which corresponded to the known values for the di- or mono-O-methyl-D-GiNTAc derivatives. For OG RL 0.44 a major peak is found at 0.80 retention time which is presumed to be the derivative formed from the 3,6-disubstituted N-acetyl-D-galactosaminitol, a reference compound which was not available. Minor peaks were not found in the solvent reaction blank. In addition the minor peaks are not attributable to incomplete methylation of DGal as suggested by the following experiment. Each of the permethylated, methanolyzed and O-acetylated samples was 5The N-methyl derivative is written as Nmethyl-D-GNAc although it is uncertain whether these derivatives are N-methyl- or N-methyl-Nacetyl-glucosamines.
Z
Z<
<
Z
~.~
~o Z
9
o
~
Z
9
9
498
~4
,--4
>r
2 t~
z
O
& 9
& g~
.g
gg
499
500
ANI-)EIISON ET AL.
hydrolyzed in 2 N HC1 for 2 hr. The HC1 was removed in vacuo, the samples treated with Dowex 50-H + resin, and the resin washed with water. The charged amino compounds were eluted with 1 N HC1, the HC1 removed in vacuo, and the samples Naeetylated, methanolyzed, and O-acetylated. Each sample gave exactly the same pattern on the ECNSS-M column at 185 ~ with respect to relative peak areas and retention times, as those in Table V. Moreover, no peaks were found at a lower temperature setting at which permethylated DGal derivatives would have been observed, indicating complete separation of neutral and amino sugars. The O R D spectra of RL 1.9, RL 0.54, R~ o.63, and RL 0.61 were recorded. Table V I I lists values of molar rotations at the troughs of the longest wavelength Cotton effect (n-~r*) and at 300 nm. The differences [re]trough - - [m]s00 are also tabulated for the four compounds. I n earlier papers, these differences have been utilized empirically as an aid in determining sequences, substitutions, and configuration of glycosidic linkages (30-33, 23). The table also gives values, from other work, for three related disaccharides and two tetrasaccharides, lacto-N-tetraose and lacto-N-neotetraose. From results cited above (Table VI), it is
clear that IlL 1.9 is a mixture of r --~ 4)DGNAc with a small amount of ~DGal (1 -~ 3)DGNAc. The value of [re]trough -[m]a00 is intermediate between those of the authemic disaecharldes. The calculated percentage of the minor disaccharide is 38cI: from this value. The measurement at 300 nm is, however, subject to great uncertainty when very small amounts of material are available, as was tl~e here. From the trough rotations alone, which are more precisely measured, the minor component is 2 5 ~ of the total. The position of the trough was recorded at 220 nm in IlL 1.9 but is at 218 nm in both authentic disaccharides. If all are compared at the same wavelength, 218 nm, the minor component is but 1 4 - 1 6 ~ . This is still higher than the reduced chromogen value of 8 %, but the results are reasonable considering errors of both procedures with such small samples. The trisaccharide, RL 0.54, gives a value in close agreement ~x~ith that expected from additivity of component residues in the sequence /~DOal(1 ~ 3)/~DGNAc(1 --~ 3)DGal or by comparison with lacto-Ntetraose. The computations are discussed. The two tetrasaccharide solutions, IlL 0.63 and RL 0.61, are both mixtures, as shown above. Calculations of the expected rotations of the component tetrasaccharides
TABLE VI IDENTIFICATION OF LINKAGE TO ]~EDUCING END BY THE ACTION OF SODIUM HYDROXIDESODIUM BOROHYDRIDE ON THE ISOLATED OLIGOSACCHARIDESa Compound
a-D-Gal-(1 --~ 3)-D-Gal fl-D-Glc-(1 ~ 4)-D-Gal fl-D-Gal-(1 --~ 6)-D-Gal I~L 0.54 ~-D-Gal-(1 ---* 3)-D-GNAc
/~-D-Gal-(1 --* 4)-D-GNAc ~-D-Gal-(I ~ 6)-D-GNAc RL 2.29
RL 1.9 RL 0.61 RL 0.63
Scission products obtained Galactitol
3-Deoxygalactitol
5.2
5.2 0.4 0 4.2
06
Reduced chromogen
100 0 0
104~ 8
48 74
" Compounds were treated with 0.2 N NaOH-I% NaBH4 for 24 hr at 24~ (23). b Less than 0.1% of the product was detected. ~,Percentage of free reduced chromogen relative to that obtained with/!l-D-Gal-(1 --* 3)-D-GNAc.
501
IMMUNOCHEMICAL STU1)IES ()N BLO01) GR()UPS
TABLE V11 ORD PARAMETERSOF RL 1.9, RL 0.54, RL 0.63, RL 0.61, AND RELATEDOLIGOSACCHAR1DES Compound
[,tdt r(,ugh Trough [mla~. deg.cm=/decimoIe wavelength(nm) dccimaledCg'cm2/deg.cm2/decimole[m]trough - [m]aa0
RL 1.9 RL 0.54 RL 0.63
--3250 --3900 -- 6400
220 219 222
250 75 -- 80
RL 0.61
--6360 -4440 -2860 --800 -- 3140
222 218 218 222 218
160 110 80 500 580
--6510 -4550 -2940 --1300 -- 3720
-2510
218-220
575
--3140
~3DGal(1 ---+ 3)DGNAc~ /~DGal(1 - + 4)DGNAc~
~DGNAc(1 --+ 3)DGalb Lacto-N-tetraose~, ~DGal(1 --+ 3)~DGNAc(1 -+ 3)~DGal(1 -+ 4)DGlc Lacto-N-neotetraose", /~DGal(1 --+ 4)~DGNAc(1 -+ 3)BDGal(1 -+ 4)DGlc
--3500 --3975 -- 6320
From (30). 9 ~'From (33). in the mixtures did not accord well with the observed values. The main discrepancy is that the observed values of [m]tro,gh [re]a00 are less negative by about 2000 deg. cm~/decimole than anticipated by summing the separate contributions fl'om the residues with the indicated glycosidic linkages. The discrepancies are accounted for below. DISCUSSION H u m a n ovarian cyst A substance treated b y mild acid hydrolysis had previously yielded A-active di- and trisaccharide (7). The further purification of other fractions obtained from charcoal chromatography is described with the isolation of five additional oligosaccharides. The two disaccharides RL 2.29 and RL 1.9 have been found in many sources including B, H, and Le ~ substances (12) and in hum'm milk (17, 34). Thc trisaccharide, ~DGal(1 --~ 3)tSDGNAc(1 ---+ 3)DGal, has also been isolated from A, B, H, and Le ~ substances (15). The two tetrasaccharides, RL 0.63 and RL 0.61, each contained the above trisaccharidc sequence linked either to the 3 or to the 4 position of a reducing DGNAc; each tetrasaccharide fraction was a mixture of two tetrasaccharides. The use of the baseborohydride degradation and especially the methylation analyses provided substantial proof of the structures despitc the fractions
being mixtures. Considering the difficulties in separating similar oligosaccharides, these technics should be of value in structural studies of mixtures of other oligosaccharides. The sequence ~DGal(1 -+ 3 ) ~ G N A c ( 1 3)/~DGal(1 ~ 3)I)GNAc is represented ill the proposed composite megalosaccharide structure of the carbohydrate portion of tile blood group substances (1-5). The tetrasgmcharide with the 1 --+ 4 linked ,)GNAt at, the reducing end does ,tot, occur in the proposed structure and is either a nalurally occurring sequence or is an acid reversion product,. For the biosynthesis of the oligosaccharide chains there either may be distinct glycosyl transferases for each separate sugar moiety added, or a limited number of tranferases may possibly transfer a given sugar to more than one position in the chain or lo different hydroxyls of the terminal acceptor carbohydrate. For lhe first possibility, separate genes would code for the enzymes for each carbohydrate position and linkage in the megalosaccharide, analogous to tile genes responsible for the structures of the A, B, H, Le ~, or Le t' determinants (cf. 35). However, if the glyeosyl transferases are not strictly specific, this might account for the isolation of the two tetrasaeeharides containing 3- and 4-substituted terminal reducing DGNrAc residues. The l etrasaccharide containing the 4-substituted reducing DGNAc could also have been formed by reversion during the initial
502
ANDERSON E T AL.
mild acid hydrolysis. Th(~ formation of oligosaeeharides by 'tci(1 reversion is described by Jones "rod Nieholson (36), Neuberger and Nl.trshall (:/7), and I~arker et aL (38, 39); both ~- and r glycosides can be formed and substitutions may occur at different hydroxyls of the aeeeptor earbohydrate. There is insufficient data to indieate a preferential formation of part ieul'tr linkages. It is also possible that different, types of oligosaeeharide chains, as defined by their linkage sequences, are attached to the protein core, and the two tetrasaeeharides could then have been derived from separate megalosaeeharides in the original ovarian cyst material. Two new determinant-containing oligosaeeharides, c~DGNAe(1 -+ 4) ~/DGal(1 --~ 4)DGNAe and aDGNAc(1 4)flDGal(1 ~ 3)DGalNAe (23), were recently isolated from hog muein A q- H substance. The second of these was presumably attached directly to the polypeptide core through the terminal reducing GalNAe residue, while the former oligosaeeharide was probably originally part of a larger oligosaceharide. The isolation of these oligosaceharides indieates that several types of chains occur in blood group glycoproteins. A definitive answer to the heterogeneity and speeifieity of synthesis of the oligosaeeharide ehains will be obtained when the intact ehains are isolated, fraetionated, and eharaeterized. Various techniques for the separation of the 0-methyl ether derivatives have been deseribed including paper chromatography (37), the gle identification of the fully aeetylated 2-aeetamido-2-deoxy-D-glueitol derivatives (24), and ion-exchange eolumns (40). Some methyl ethers have been identiffed as the methyl glycosides of I)GNAe (41, 42). Table V shows the utility of the BaO methylation and methanolysis method as described for establishing the linkage to the imernal I)GNAe of the isolated oligosaeeh-~rides. Only 200 ug of each substituted DGNAe of the original oligosaeeharide is needed for the methylation procedure and for gle analyses. The additional minor peaks generally
found on glc of methylatcd amino sugars have been shown to be due to N-methylation and are determined by the link'tge to the internal amino sugar in the oligosaceharide. Thus they help to provide unequivoeM identification of the internal linkages to amino sugars in oligosaecharides. The ORD spectra of four of the oligosaeeharides reported here showed the eharaeteristic trough near 220 nm associated with S-linked and reducing terminal DGNAe residues. While some fractions are mixtures, and may have small amounts of impurities, they were nonetheless useful to eontinued attempts to seek out elements of additivity or regularity in ORD spectra of oligosaeeharides. The ORD data on the trisaeeharide (RL 0.54) were examined in several ways. If to the [m]~ro~h -- [m]~00value of ~oGNAe(1 ~ 3)DGal is added that of methyl SDGal(-- 135), the difference between this sum and the observed value for RL 0.54 should represent the effeet of substitution on carbon 3 of the r DGNAe residue. That difference is -2540 deg. em-~/deeimole. The disaeeharide, ~DGNAe(1 -+ 3)DGal, may, in turn, be considered as comprised of methyl f3DGNAe (-2140) and DGal (875). The sum of these is --1255, which agrees well with the observed value of --1300 for the disaceharide (Table VII). The trisaeeharide may thus be thought of as comprising three residue contributions and an additional effect due to the carbon-3 substitution on the internal I)GNAe residue. The sum of these four eontributions is -3930; the observed value is -3975 deg. em=/deeimole. Alternatively, one may begin with the laeto-N-tetraose value, subtract the DGle (960) and methyl ~DGal residues and add the value for DGal. This gives -3670 deg. em2/deeimole. Although the trisaeeharide eomains only a very small amount of material ( < 5 % ) with earbon-4 substituted, rather than carbon-3 substituted, ~dinked DGNAe residues, it was of interest to evaluate the difference expected from mixtures of these two eompounds. Comparison of laeto-Ntetraose with laeto-N-neotetraose reveals
IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS t h a t such a change results in a difference of just under 600 deg- em2/deeimole. Two other oligosaccharides derived from A substance, also differing by just the one change in substitution on an internal ~-linked DGNAc residue have previously been reported to show a difference of just under 500 deg. cm2/ deeimole in the value of [m]t:o~gh - [m]300
(3~).
The tetrasaccharides present a quite different problem. If one sums the elements as in the trisaccharide using any of the disaccharide combinations or adding the monosaceharides separately, the computed values of [rn]t~o~gh - [m]~00 are more t h a n 2000 deg. cm~/decimole more negative than the observed values. This difference is well outside the uncertainties involved in estimating the various contributions to the sequence. The two fractions, in addition, differ considerably in the ratios of the two tetrasaccharides which each contains, as noted above, but the observed values of tin]trough -- [mJa00 are very close. The discrepancy of the observed value compared to the computed value for each oligosaccharide probably arises for different reasons. T h e total quantity of RL 0.63 isolated was 8.5 rag. I t has been shown previously (3, 21), t h a t when such small amounts are isolated as much as 2 mg m a y be inert material. Such a 25 % error in concentration based on dry weight would account for the O R D anomaly. With RL 0.61, the yield was 14.2 mg and the proportion of inert material would be lower. In this sample, however, analysis showed 4.2% galactosamine which probably is due to oligosaccharide containing a nonreducing terminal aDGalNAc residue. The latter would add very substantial positive rotation in the ultraviolet (30). Together with any concentration error, this could account for the anomalous value of [rn]trough -- [m]a00 for RL 0.61. Thus when substantial discrepancies between calculated and observed O R D values are noted, these m a y indicate impurities or inexact concentrations and the analytical data should be carefully scrutinized. In this case the discrepancies were satisfactorily resolved.
503
REFERENCES 1. LLOYD, K. O., AND KARAT, E. A., Proc. Nat. Acad. ~ei. U.S,A. 61, 1470 (1968). 2. KADAT,E. A., in "Blood and Tissue Antigens" (D. Aminoff, ed), p. 187. Academic Press, New York (1970). 3. VICARI,G., ANDKABAT,E. A., Biochemistry 9, 3414 (1970). 4. FEIzI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARSH, W. L., J. Exp. Med. 133, 39 (1971). 5. FEIZI, T., KABAT, E. A., VICARI, G., ANDERSON, B., AND MARGIn, W. L., J. Immunol., 106, 1578 (1971). 6. COTE, R. H., AND MORGAN, W. T. J., Nature London 178, 1171 (1956). 7. SCHIFFMAN, G., I4~ABAT, E. A., AND LESKOWlTZ, S., J. Amer. Chem. Soc. 84, 73 (1962). 8. CHEESE, I. A. F. L., AND MORGAN, W. T. J., Nature London 191, 149 (1961). 9. PAINTER, T. ,l., WATKINS, W. M., AND MORGAN, W. T. J., Nature London 193, 1042
(1962). 10. PAINTER, T. J., WATKINS, W. M., AND MORGAN,W. T. J., Nature London 199, 282 (1963). 11. ASTON, W. P., I'IAGUE, G. M., DONALD, A. S. R., AND MORGAN,W. T. J., Biochem. J.
110, 157 (1968). 12. PAINTER, T. J., REGE, V. P., AND MORGAN, W. T. J., Nature London 199, 569 (1963). 13. ASTON,W. P., DONALD,A. S. R., ANDMORGAN, W. T. J., Biochem. Biophys. ReG. Commun.
30, 1 (1968). 14. ASTON,W. P., DONALD,A. S. R., ANDMORGAN, W. T. J., Biochem. J. 107, 861 (1968). 15. REGE, V. P., PAINTER, T. J., WATKINS,W. M., AND MORGAN,W. T. J., Nature London 200, 532 (1963). 16. VICARI, G., AND KADAT, E. A., J. Immunol. 102, 821 (1969). 17. KUHN, R., BAER, H. H., AND GAUHE, A., Chem. Ber. 87, 1553 (1954). 18. KUHN, R., .~ND LOW, I., Chem. Bet. 86, 1027 (1953). 19. SMITH, F., AND STEPHEN, A. M., J. Chem. Soe. 4892 (1961). 20. KABAT, E. A., "Kabat and Mayer's Experiperimental Immunochemistry," 2nd Ed. Thomas, Springfield, Ill. (1961). 21. LLOYD, K. O., KABAT, E. A., LAYUG, E. J., AND GRUEZO,F., Biochemistry 5, 1489 (1966). 22. KUHN, R., BAER, H. H., AND SEELIGER, A., Ann. Chem. 611,236 (1958). 23. ETZLER, M. E., ANDERSON, B., BEYCHOK, S., GRUEZO, F, LLOYD, K. O., RICHARDSON,
504
ANI)EI:(SON ET AL.
N. (;., ~xl) K.~J~.xT. 1':. A., Ar~'h. Bio,'bem. 33. K.\n.~'v, E. A., L[.oYo, K. O., AND BEYGHOK, S., Biochemistry 8, 747 (1969). Biod~e,~. Biophy.~. 141,588 (1970). 24. [)r;lr M. B., ANDWEan, A. C., Can. J. Chem. 34. KOBATA,A., J. Bioehem. Tokyo 53, 167 (1963). 35. W=~TK~NS,W. M., in "Blood and Tissue Anti~ 47, 4091 (1969), gens" (D. Aminoff, ed.), p. 441. Academic 25. GORIN, P. A. J., AND FINLAYSON, i~.. J., CarPress, New York (1970). bohyd. Re,~., in press. 26. I~)EISSIG, J. L., STI%OMtNGER) J. L.) AND 36. JONES, J. K. N., ANn NICHOLSON, W. I t , J. Chem. Soc. 27 (1958). LELOIE, L. F., J. Biol. Chem. 217, 959 (1955). 27. WHELAN,W. J., in "Haudbuch der Pflanzen- 37. NEUBERGER, A., ANn MARSHALL, R. ])., in "Glycoproteins" (A. Gottschalk, ed.), p. physiologie," Vol. 6, p. 154. Springer, Berlin 235. Elsevier, New York (1966). (1958). 28. LLOYD, K. O., AND KABAT, E. A., Carbohyd. 38. BARKER,S. A., MURRAY,K., AND STACEY,M., Nature London 191, 142 (1961). Res. 9, 41 (1969). 28a CARLSON, ]). M., J. Biol. Chem. 243, 616 39. BARKER,S. A., MURRAY,K., STACEY,M., AND (1968). STROUD, D. B. E., Nature London 191, 143 29. KUHN, R., ZILLIKEN, F., AND GAUHE, A., (1961). Chem. Ber. 66, 466 (1953). 40. ADAMS, G. A., YAGUCHI, M., AND PERRY, 30. BEYCHOE,S., AND KABAT, E. A., Biochemistry M. B., Carbohyd. Res. 12, 267 (1970). 4, 2565 (1965). 31. LLOYD, K. O., BEYCHOK, S., AND K.~BAT, 41. KUHN, R., AND EGGS, H., Chem. Ber. 96, 3338 (1963). E. A., Biochemistry 6, 1448 (1967). 42. KOBATA, A., AND GINSBURG,V., J. Biol. Chem. 32. LLOYD, K. O., BEYCHOK, S., A.ND KABAT, 244, 5496 (1969). E. A., Biochemistry 7, 3762 (1968).