THE Jona~u OB BIOLOGICAL CH~~TBY Vol. 236, No. 11, November 1961

P&tad in U.S.A

Isolation and Characterization of Glycolipids from Erythro- cytes of Human Blood A (Plus) and B (Plus)*

SEN-ITIROH HAKOMORI~ AND ROGER W. JEANLOZ

From the Departments of Biological Chemistry and Medicine, Harvard Medical School, and the Massachusetts General Hospital, Boston 14, Massachusetts

(Received for publication, March 30, 1961)

The presence in horse erythrocytes of a glycolipid composed of sphingosine, fatty acids, hexoses, and neuraminic acid was reported by Yamakawa and Suzuki (1). A similar product was isolated by Klenk and Lauenstein (2) from human erythrocytes; it contained galactosamine, but no neuraminic acid. Further studies by Klenk and Lauenstein (3) and by Yamakawa and Suzuki (4) and Matsumoto (5) showed that the composition of the glycolipids varies with the source of the erythrocytes.

Blood group activity has generally been associated with substances of glycoprotein nature, isolated from glandular tissues and secretions (6). The chemical nature of the sub- stances responsible for blood group activity in erythrocytes has not been clearly established, and contradictory statements have been made in the past concerning their chemical nature (7-9). Recently, Yamakawa et al. reported blood group activity in a glycolipid fraction (lo), but it could not be related to a definite chemical structure (11).

The purpose of the present work was to purify glycolipid fractions possessing blood group activity starting from two different types of human blood, A ( +) and B ( +), and further to study the chemical differences between the purified substances. In agreement with the evidence presented by Yamakawa and Iida (lo), it was shown that each glycolipid isolated was able to inhibit the hemagglutination of its parent erythrocyte by the corresponding antibody.

After the present work had been completed, independent investigations by I&din (12), and by Koscielak and Zakrzewski (13), as well as a reinvestigation by Klenk of substances pre- viously isolated (14), led to the same conclusions.

EXPERIMENTAL PROCEDURE

Analytical Met-The presence of the major constituents was established after hydrolysis of a 1% solution of the sample with 1 N sulfuric acid for 5 hours. The fatty material was filtered off or extracted with chloroform, and the water solution

* This is publication XXVIII of the Amino sugars, and No. 299 of the Robert W. Lovett Memorial Unit for the Study of Crippling Disease, Harvard Medical School, at the Massachusetts General Hospital. This investigation was supported by grants of the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service (Grant A-148 C3), and of Eli Lilly and Company. A preliminary report was presented at the 132nd meeting of the American Chemical Society, New York, September 1957.-

t Fulbrizht Fellow. Present address. Tohoku Pharmaceutical School, Odiwara Nankozawa, Sendai, Japan.

was neutralized with barium carbonate, filtered, and evaporated to dryness under nitrogen.

Hexose, hexosamine, and N-acetylhexosamine were detected, respectively, by the Molisch test, the Elson and Morgan (15), and the Morgan and Elson (16) reactions. Differentiation be- tween glucosamine and galactosamine was obtained by nin- hydrin degradation, followed by chromatography (17). Sphm- gosine was identified by reaction with ninhydrin, or with Ehrlich’s reagent after oxidation with hypobromite (18), and the fatty acids by the hydroxamate-ferric ion test (19).

The hexosamines were quantitatively estimated by the procedure of Rondle and Morgan (20), after hydrolysis with 4 N

hydrochloric acid for 5 hours. The hexoses were quantitatively estimated by the reaction of Dische with tryptophan (21), after hydrolysis with 2 N sulfuric acid for 6 hours, followed by extrac- tion of fatty acids and sphingosine-like material with chloroform, and centrifugation of the fine precipitate of sphmgosine sulfate. It was necessary to remove sphingosine, because it reacted with carbohydrates in the presence of sulfuric acid. After separation by paper chromatography, the hexoses were quantitatively estimated by the benzidine method of Jones and Pridham (22).

The total reducing power was measured by the modified Hagedorn and Jensen reaction (23), after hydrolysis with 6 N

hydrochloric acid for 30 minutes (24). The quantitative estimation of the fatty acids was kindly

performed by Professor H. Klenk by gas chromatography with a Barber-Colman instrument.

Optical rotations were determined in semimicro tubes 200 mm long with a polarimeter equipped with a Rudolph model 200 photoelectric attachment, on solutions of water or of chloroform- methanol (1: 1).

Detewkation of Blood Croup Activity-A solution of 2 mg of glycolipid per ml in 0.85% NaCl solution was prepared. When the material was sparingly soluble in the NaCl solution, it was first dissolved in water, then diluted with the same volume of 1.9% NaCl solution. A volume of 1.2 ml of this solution was used in a a-fold serial dilution. To each tube was added 0.2 ml of a standard anti-A or anti-B serum’ with a standard titer of 16 hemagglutinin units. The mixture was shaken well, in- cubated at 37” for 2 hours, and then left at 5” overnight. Two drops of a 5% suspension of A or B red cells were added to each tube containing the anti-A and anti-B sera, respectively, and the mixture was incubated at 37” for 30 minutes. Then the tubes

1 The anti-A and anti-B sera were obtained from the Blood Grouping Laboratory, Bost,on 16, Massachusetts.

2827

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2828 Glycolipids from Erythrocytes of Human Blood Vol. 236, No. 11

400-

P

s

2 5 200-

B

d 0 500 looo 1500 ml

ttt tt tt CHLOROFORM: IO0 75 65 50 35 25 0

METHANOL: 0 25 35 50 65 0 loo

MOLISCH: -Ht t t

ELSON-MORGAN: tt tt 0

PHOSPHORIJS: t it itt

FIG. 1. Purification of 555 mg of glycolipid, isolated from human blood A (+), by adsorpt.ion chromatography on 100 g of silica gel. Each fraction of effluent contained 50 ml. The Molisch and phos- phorus determinations were carried out on 366 to 566 fig, and the Elson and Morgan test on 1 to 2 mg of substance.

were gently shaken two or three times. The limit of dilution was determined by visual observation. One or two large clumps of red cells were read as + + +, several large clumps as + + , a fine flocculation with free cells as +, no flocculation as 0.

No standard blood group substances were used for comparison, since the various fractions were compared between themselves. The activity of the most potent fractions was kindly determined by Dr. E. A. Kabat and Professor W. ‘I’. J. Morgan.

Preparation of Glycolip&--The extraction was carried out conventionally (25). Sedimented red cells from human blood groups, A ( +) and B ( +), were suspended in 5 volumes of 0.85 % NaCl solution. After sedimentation, the supernatant solution was siphoned off and the sediment was centrifuged. The packed red cells (20 and 18 liters of packed red cells, A (+) and B (+), respectively) were stirred with 5 volumes of 0.5% acetic acid (pH 4 to 5) and left at 0” for 1 day. The aggregated stroma particles were centrifuged in a Sharples centrifuge, and the resulting paste was stored at -20’ (yield, 1.68 kg of A (+) and 1.07 kg of B (+)).

The paste was shaken successively with 8 volumes, 5 volumes, and finally 3 volumes of acetone, each time for 24 hours. The extracts, containing cholesterol and neutral fats, were filtered, combined, and evaporated. The residue (470 g of A ( +) ; 359 g of B (+)) was shaken twice with 5 volumes of hexane. This second extract, containing glycerophospholipids, was filtered, evaporated under reduced pressure to a small volume, and precipitated with an excess of acetone.

The insoluble residue was dried in a vacuum over calcium chloride, then extracted under reflux with 5 volumes of chloro- form-methanol (1: 1). It was filtered while hot and washed with the hot solvents. This extraction was repeated three times. The combined extracts were concentrated under reduced pressure to 300 ml, and an equal amount of chloroform was added. The solution was treated with Darco G-60, filtered, and concentrated to 150 ml. An equal volume of methanol was added; the mixture was cooled overnight at 0” and centrifuged at 0”. The precipi- tate gave a strong Molisch reaction, whereas the supernatant

reacted only faintly. The latter, containing glycerophospho- lipids, was evaporated under reduced pressure.

The precipitate was purified by dissolution in hot methanol and, after precipitation by cooling at 0”, it was filtered, washed with acetone, and dried in a vacuum. The acetone washings gave a negative Molisch reaction. The precipitate was ex- tracted twice with 100 ml of dry ether to remove the last traces of glycerophospholipids. A few milliliters of acetone were added to the ether before centrifugation, to facilitate the deposit of the sediment. The centrifugate containing the sphingolipid fraction was dried in a vacuum.

The sphingolipid fraction of both types of blood (8.5 g of A (+); 5.7 g of B (+)) was fractionated into sphingophospholipids and sphingoglycolipids, according to Klenk and Renkamp (26). It was dissolved in 60 ml of pyridine and allowed to stand overnight at room temperature. The solution was filtered through Celite, then through a column of activated alumina (20 g; diameter, 0.8 mm) prewashed with 30 ml of pyridiie. The filtrate was evaporated under reduced pressure to a sirup, which crystallized by addition of a large volume of acetone. Recrystallization from acetone gave 2.5 g of glycolipid from A (+) blood and 2.0 g from B (+) blood. Both substances were soluble in warm methanol, chloroform, and benzene, but sparingly soluble in acetone, hexane, and ether. They showed a strong Elson and Morgan reaction after hydrolysis, a strong Molisch reaction, but only a faint reaction for phosphate.

Purijkation of Glycolipid by Adsorption Chrmnatography-A sample of 0.5 to 0.6 g of glycolipid was dissolved in 50 ml of chloroform and passed through a column containing 100 g of activated silica gel (Davison Company, Grade 950, 60 to 100 mesh) suspended in chloroform. Elution was carried out with fractions of 50 ml. The main peak was eluted in the first five fractions. Solvent mixtures of increasing methanol concentra- tion eluted the remaining substance (see Fig. 1). The main fraction was recrystallized from methanol, and its properties are reported in Table I. The yield varied from 65 to 80%.

Puri$catiun of Glycolipid by Partition Chromabgraphy-The main fraction (300 to 400 mg) was further purified according to Svennerholm (27), dissolved in 30 ml of chloroform saturated with water, and adsorbed on a cellulose column (3 x 15 cm, Solka-Floe), which had been previously washed with water, then with methanol, and finally with chloroform, and equilibrated for 1 day with chloroform saturated with water. The column was eluted at room temperature with chloroform saturated with water (Solvent I), then with the following mixtures of chloro- form-methanol-water : Solvent II, 270 : 30 : 5; Solvent III, 40 : 10 : 1; Solvent IV, 2 : 2 : 1. Aliquots of 15 ml were collected at a rate of 20 to 40 drops per minute. The fractions were evaporated under reduced pressure, and fractions of identical peaks were combined and crystallized from methanol.

Properties and Composition of Various Fractions of Glycolipids- The main peaks were analyzed for hexosamines and hexoses, and the optical rotations were determined. The results for the fractions obtained from blood type A (+) are reported in Fig. 2 and from blood type B (+) in Fig. 3.

Qualitative analysis by paper chromatography showed the presence of gala&se, glucose, galactosamine, and a trace of glucosamine in the thiid peaks of both blood samples (Fractions P-3-A and P-3-B).

The elementary analysis, fatty acids, hexose, hexosamine, and sphingosine content, reducing value, optical rotation, and

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November 1961 S. I. Halannori and R. W. Jeanlox 2829

melting point of Fractions P-3-.4 and P-3-B are reported in Table II.

The quantitative estimation of the fatty acids of Fractions P-l-A, P-3-8, P-l-B, and P-3-B is reported in Table III.

For both types of blood, the two first peaks (Fractions P-l-A, P-2.4, P-l-B, and P-Z-B) gave opalescent, nonviscous solu- tions in water, and they were readily soluble in chloroform, but sparingly in methanol. The third peaks (Fractions P-3-.4 and P-3-B), in both cases, gave a clear and viscous solution in water, but were sparingly soluble in salt solution. In organic solvents, their solubility decreased with the decrease of polarity.

The results of the inhibition test of the various fractions are reported in Fig. 2 and Fig. 3. Both peaks, P-3-A and P-3-B, showed maximal inhibition against anti-A and anti-B sera respectively, but no reaction at amounts up to 500 I.rg against anti-B and anti-A sera, respectively. The activity of these two fractions was compared to standard substances, isolated from mucous secretions, and is reported in Table IV.

Determination of Hexose Components of P-3-A and P-3-B-A sample of 3 to 5 mg of glycolipid was dissolved in 0.5 ml of 1 N

sulfuric acid in a sealed tube and heated at 100” for 5 hours. After neutralization with barium carbonate and filtration, the pH was adjusted to 6 with 0.01 N sulfuric acid and the solution was evaporated to dryness under nitrogen. The residue was dis- solved in 50 ~1 of water and 5 ~1 portions were applied to What- man So. 54 paper. Qualitative standard spots and quantitative spots of 20,40, 60, and 80 fig of glucose and galactose were also applied. Descending chromatography was performed for 24 hours with n-butanol-pyridine-water, 5:3:2. After drying, the procedure was repeated twice. The standard spots were cut off the paper and detected with the modified silver nitrate method (17,28). The areas containing the remaining spots were eluted separately with water by the capillary method, and the solutions were concentrated to dryness with nitrogen. The residues were dissolved in 1 .O ml of water and then treated with benzidine (22). The amount of gala&se and glucose was determined by com- parison with the standards. The results are reported in Table II.

Determination of Hexosamine Components of Fractions P-3-A and P-3-B-A sample of 1.0 to 1.5 mg of glycolipid was hy- drolyzed with 0.1 ml of 4 N hydrochloric acid in a sealed tube for 5 hours. After cooling, the hydrolysate was extracted twice with chloroform, and the chloroform extract was washed with water. The water extracts were evaporated under nitrogen and the residue was dissolved in 50 ~1 of water. Spots of 15 ~1 of solution were applied to Whatman No. 54 paper with quanti- tative standards of 20, 40, 60, and 80 pg of galactosamine and one reference standard.

The ehromatogram was run, descending, for 24 hours with butyl acetate-acetic acid-ethanol-water, 3:2 : 1: 1. After the spots were located, the remaining strips of paper were cut off into small pieces and treated for 20 minutes with 2 ml of water and 2 ml of a solution containing 1 ml of acetylacetone in 50 ml of 0.7 N sodium carbonate. After addition of Ehrlich’s reagent, the amount of hexosamine was determined as usual and the results are reported in Table II.

Periodate Oxidutirm of Fractions P-b-A, P-S-A, P-2-B, and P-3-B

1. Quuntitative Estimation-In a first experiment, samples of 22 to 23 mg of the glyeolipid, and of 6.5 mg of methyl CY-n-

TABLE I

Properties of glycolipids purified by silica gel adsorption

Glycolipid

A (+) B (+I

Ial ; Cc, 0.6) in chloro-

form-meth- anol (1: 1)

+21"

+28"

-

__

-

Melting point

210-215" 212-218'

Hexosamine Reducing substance

I

%

7 30 7 31

t t t t SOLVENT: I JI III lx

FRACTION: P-l-A P-2-A P-3-A

ACTIVITY IN ,ug: 1125 1500 SO0 l-2

w,: +l9' +lo"

HEXOSE x: 31 34 48

HEXOSAMINE %: 7 8

FIQ. 2. Purification of 380 mg of glycolipid, isolated from hu- man blood A (+), by partition chromatography on a cellulose column (3 X 15 cm). The solvents used for elution (I, ZZ, ZZZ, and IV) are described in “Purification of Glycolipid by Partition Chromatography.” Each fraction contained 30 ml of efhuent.

I 1 I . I

0 500 1000 1500 ml

t t t SOLVENT: I It III

FRACTION: P-l-B P-Z-B P-3-B

GTIVITY lN,ug:, 1200 1200 5-10

C-J,: tl6O t170 +270

HEXOSE 9.: 32 42

HEXOSAHlNE%: 6 0

FIG. 3. Purification of 340 mg of glycolipid, isolated from hu- man blood B (+), by partition chromatography on a cellulose column (3 X 15 cm). The solvents used for elution (I, ZZ, and ZZZ) are as in Fig. 2. Each fraction cont,ained 50 ml of effluent.

glucopyranoside were dissolved in 4 ml of water. To this solu- tion were added 2 ml of 2 xz sodium acetate-acetic acid buffer solution and 3.0 ml of 0.05 M sodium metaperiodate solution. The solution was diluted to 25 ml and left in the dark at about 5”. Aliquots of 5 ml were taken at 30 minutes, 2 hours, 12 hours, 48 hours, and 96 hours, and the excess periodate was determined by the arsenite method (29).

In a second experiment, only 1.5 ml of 0.05 M sodium meta-

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2830 GlycoZipids from Erythrocytes of Human Blood Vol. 236, No. 11

TABLE II Properties and analytical data of the glycolipids fractions P-S-A and P-3-B

3 8

se 2 _ %

1.86 1.89 1.89

I

.

[a]:: in water [(21: in chloroform- methanol (1: 1)

Melting point

% ------~

% % % % % % %

12 48 42 60 8 48 53 44 4-6 22-27 10 8 42 56 40 443 30-32 10

Calculated for C&r- HmOt$Jd . . . . . .

Fraction P-3-A.. . . . 215-225’ Fraction P-3-B.. . . . 229-230’

%

52.25 59.04 59.13

-6” (c, 0.8) +lo” (c, 1.0) +38” (c, 1.0) +26” (c, 0.2)

~1 These microanalyses were carried out by Dr. K. Ritter, Base;, Switzerland. * Direct Rondle and Morgan procedure (20). 0 Direct Dische procedure (21). d Jones and Pridham procedure (22) after hydrolysis and paper chromatography. 6 Ronclle and Morgan procedure (20) after hydrolysis and paper chromatography. f Corresponds to a N-lignoceryl-O-(tetrahexosyl-N-acetylhexosaminyl)-sphingosine.

TABLE III tion, and concentration, each hydrolysate was examined by Fatty acid determination o+f glycolipid fractions paper chromatography run with the n-butanol, pyridine, and

by gas chromatography water (5:3 :2) mixture. For both substances, galactose was Column of 20% Reoplex 400 (Geigy) on Celite; temperature of released after a hydrolysis of 13 hours, concurrently with a

column 188-195°.

No. of carbons in chain

faster moving, unknown substance. After 3 hours, galacto- samine release was observed, and the two other spots were more intense; but even after a 15hour hydrolysis, glucose was not released.

Fractions

2. Quantitative Estimation-Samples of 15.5 mg and 15.0 mg of Fractions P-3-A and P-3-B, respectively, were hydrolyzed with 6 ml of 0.025 N sulfuric acid for 5) hours at 100”. The hydrolysates were then dialyzed against many changes of distilled water for 3 days. The combined dialysates of each glycolipid were neutralized with barium carbonate and, after

-

-

-

_-

-

-

.- P-3-A

%

5 75

5 3

12

P-l-B P-2-B

%

5 16 2

13 3

61

% 5

41 11 10

33 I TABLE IV

Crude A

%

7 26 4

12 5

46

P-l-A

%

8 15 3

15 3

56

16 18 20 22 23 24

Tests under II

Blood group activity of P-S-A and P-b-B

Tests under I were carried out by Dr. E. A. Kabat, using anti-A and anti-B sera with 20 hemagglutinin units. were carried out by Dr. W. T. J. Morgan.

I Substance

Minimal inhibiting concentration Dilution end point

of inhibition

periodate was used. The reaction was not estimated at 30 minutes, but at 144 hours. Blank solutions were run also.

The results of these two experiments are reported in Table V. 6. Estimuhn of Oxidized Components-Samples of 8 mg of

Fractions P-3-A and P-3-B were oxidized under the same condi- tions as described in the first experiment above. After 2 and 144 hours, half the amount was mixed with 0.2 ml of ethyleneglycol. The solution was left standing at room temperature for 1 hour, then dialyzed against running tap water for 2 days, and against distilled water for 1 day. The content of the dialyzing tube was concentrated to dryness under reduced pressure, and the residue was dissolved in chloroform-methanol, 1: 1. The solution was centrifuged and evaporated to dryness under nitrogen. Aliquots of the residues were hydrolyzed with sulfuric and hydrochloric acids and analyzed for glucose, gala&se, and hexosamines. The results are reported in Table VI.

When the same oxidation was carried out with half the amount of sodium metaperiodate for 2 hours, similar results were ob- tained.

Anti-A Anti-B

Test I P.c

Fraction P-3-A.. . 10 500 Fraction P-3-B.. . 590 500 Standard hog A. . 0.1 A ,B phenol-insoluble hu-

man saliva A.. 0.5 Standard human saliva B. 0.5 Horse B.................. 1.0

Test II Fraction P-3-A.. . . Fraction P-3-B.. . Standard ovarian cyst , H$To&~s Studies of Fractions P-3-A and P-3-B

1. Qualitative Experiment-A 0.2 y0 solution of each glycolipid in 0.025 N sulfuric acid was heated at 100” for 13, 3, 6, and 15

Standard ovarian cyst

hours. After neutralization with barium carbonate, centrifuga- :z,” c:::::::::::::::::: /

1:6,400 l:l,fXXl

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November 196 I S. I. Hakomori and R. TV. Jeanloz 283 1

filtration, concentrated under reduced pressure to give 6.5 and 6.3 mg of residue (Fractions D-l-A and D-l-11). The solutions inside the dialyzing tubes were concentrated under nitrogen to 1 ml (Fractions R-I-A and R-1-B).

Chromatography of 25 ~1 aliquots of Fractions R-l-h a.nd K-l -I< in ?z-butanol-pyridinc-water (5: 3 :2) gave no reducing spot, but a spot was observed for both substances with the periodate-starch rragrnt (30). It moved with a RF 0.14 in

n-butanol-pyridine-water (10 : 3 : 3). The bulk solutions of Fractions R-l-A and R-A-B were hydrolyzed for 5 hours at 100” after addition of an equal volume of 0.1 N sulfuric arid. The hydrolysates were dialyzed and concentrated in the same way as described for the first hydrolysis. Flocculation appeared inside the dialysis sacs, affording 3.0 and 1.5 mg after centrifuga- tion (Fractions R-~AA and R-2-R). The supernatant fluids inside the sacs (Fractions R-3-A and R-3-B) and the dialysates (Fractions R-4-A and R-4-H) were conccntratcd under reduced pressure or under nitrogen. Attempts to purify the dialysates R-4-A and R-4-13, as described below for Fractions D-l-A and D-l-H, gave only galactose, galactosamine, and a trace of an unknown substance.

Fractions R-2-A and R-2-H were readily soluble in chloroform, but sparingly in methanol or water. Chromat,ogra.phed in the solvent system n-butttnol-pyridine-water (10 : 3 : 3), they gave single spots with R, 0.16, that were detected with the periodate- starch reagent (30).

Fractions 11-l-A and D-1-13 were purified by descending paper chromatography on Whatman Ko. 54 paper, which had previously been washed with 2 N sodium carbonate, water, 2 N

acet,ic acid, and finally water. The development was carried out in the solvent system, n-butanol-pyridine-water (10:3:3), for 48 hours, with an intermediate drying. Guide strips corresponding to 10% of the total amount applied were cut on both edges, and detected with the silver nitrate and the pcriodatestarch reagents. Four spots were identified, galactosamine, gala&se and two unknown spots with Realaclaee 1.7 to 1.8 (Fractions D-2-A and D-2-13) and Rralactose 2.5 (Fractions D-3-A and D-3-U).

The arras corresponding to the bulk of Fractions D-2-A, D-2-1$ D-3-X, and D-3-n were cut out, and cst.racted in a Soshlet apparatus with boiling chloroform-methanol (1: 1) for 24 hours. The extracts were evaporattd under reduced pressure to give, respectively, 2.2, 1.2, 2.0, and 1.3 mg.

The various fractions were analyzed for the presence of glucose, gala.ctose, glucosamins, galactosaminc, sphingosine, and fatty acids. They were hydrolyzed with I K sulfuric acid for 5 hours, a.s described above, and the chromatography was por- formed on Whatman Xo. 54 paper with n-butanol-pyridine- water (5:3 :2). The relative amounB were estimated by visual comparison and the results are rcportcd in Table VII.

DISCUSSION

The present work describes an attempt to elucidate the chemi- cal nature of the substance contained in crythrocytes responsible for blood group antigenicity, and to establish a difference in the chemical constitution of the substances isolated from human A (+) and 13 (+) blood with antigenic properties.

During the fractionation, it was found that the biological activity was carried with substances possessing both a lipid and a carbohydrate moiety. The possibility of a contamination of the glycolipids by substances of high molecular weight consisting of a polysaccharidc linked to amino acids, such as the ones isolated

TABLE V

Deternainalion of periodate reduced by P-8-8, P-S-A, P-2-h’, and P-3-R

The amount of periodate added was 3.5 mmoles a.nd 7 mmoles per g of glycolipid. For calculation, a molecular weight of 1500 was assumed for the glycolipids.

Time

hmm

0.5 2

12

48 96

144

When 7 mm&s of periodate were added

1.2 1.4 1.25 0.95 1.65 1.9 2.15

2.35

Fraction Fraction P-3-A P-3-R

..~ molt/mole g1yco1ipia

1.35 1.3 1.85 1.7 2.3 2.0

2.65 2.2 3.1 2.35

TABLE VI

Components of Fractions P-5-A and P-J-B oxidized

by periodate -.-.-~ ~.

Content of carbohydrate components ..-

Time of oxidation

Fraction P-3-A

I Glucose

I Fraction P-3-R

hours %

0 5 2 5

144 4

--. ~. HeXOS- Galactose amine Glucose Galactose ;zn;-

% % % % %

25 10 5 30 10

11 4 5 6 5

6 3 4 5 3 -..

TABLE VII Componenls of degradation products obtained by partial

hydrolysis of Praclions P-S-A and P-3-B

Theconditions of the experiment are reported in “Experimental

Procedure.” The amount of glucosamine was too small to be identified.

Fractions

T I-

D-2-A D-2-B

D-3-A 11-3-B

R-2-A R-2-B

R-3-A R-3-B

R-4-A R-4-B

--- _...~ --

components

Galactose

0 0

0 0

++ 0

-I- +

+ +

GlUCOX

+

+

+

+

++

++

+

+

0 0

--

Galactosa- mine

--

++ ++

.L

Sphingosine and fatty

acids

+ +

+ +

+

+

+ +

0 0

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2832 Glycolipids from Erythrocytes of Human Blood Vol. 236, nTo. 11

from mucous secretions investigated in the past by the groups of Kabat ((6) p. 122) ‘and Morgan (31), is remote. These amino acid-containing polysaccharides would certainly be insoluble in the chloroform-methanol (1: 1) used for the extraction, and any trace amount carried along would be removed by the successive passages through activated alumina. activated silica gel, and by partition chromatography on celluloseS2

These findings are in agreement with the evidence presented in 1953 by Yamakawa and Iida (lo), that a glycolipid substance, isolated from human erythrocyte stroma, was able to inhibit hemagglutination of erythrocytes of a given blood group by its corresponding antibody. Later on, this evidence was questioned by Kabat (6), who suggested the possibility of contamination.

Both substances were found to be specific, in their hemag- glutination properties, to the antibodies of the red cells from which they had been obtained. Their low activity might be explained by the fact that they are present at the surface of the stroma combined in a special structure, partially destroyed during the extraction with hot organic solvents, and during the frac- tionation on columns. Whereas lyophilized B-stroma was active at the 2 fig-dilution, the chloroform-methanol extract was only active at 25 ,ug, but the residue was inactive. A similar ob- servation was made with the A-stroma. The fractionation on columns was also responsible for a loss of activity, and after the removal of a large proportion of inactive components, the final product was less active than the starting material. The high activity of glycolipids at the early stage of purification has been shown by Koscielak and Zakrzewski (13). The loss of activity may be also related to a change in the macromolecular char- acteristics during fractionation, as indicated by the loss in viscosity (32, 33). Compared to the blood group substances of secretion (Table IV)? Fractions P-3-A and P-3-B showed very little activity, but compared to other fractions of the stroma they clearly indicated hemagglutination properties. The reason for these discrepancies may be because a short chain of carbohydrate linked to a large structural complex through a molecule of sphingosine amidified with a long chain fatty acid is less active than the same chain reproduced many times as end groups of a highly branched polysaccharide. The low solubility in water and a strong aggregation, preventing a large part of the glycolipid molecule to react, may also explain this low activity, as pointed out by Klenk (14).

The chemical homogeneity of the two active substances ob- tained from A (+) and B (+) blood, respectively, Fractions P-3-A and P-3-H, could not be ascertained, and lack of material did not allow further attempts at purification. The presence of a complex mixture of fatty acids and of a small amount of gluco- samine is not compatible with a pure material,if it is assumed that a molecule of glycolipid is built of one sphingosine, one fatty acid, and a definite number of sugars. However, if the active sub- stances consist of high polymers, in which a basic unit made of sphingosine, fatty acids, and a variable number of sugars is repeated, it is possible to have variation in the composition of the

* Blood group A substance, obtained from ovarian cyst fluid, was adsorbed on a column of silicic acid-Celite (l:l, weight for weight). Elution was followed by anthrone reaction and blood group activity. No active substance was eluted with organic solvents, but only with pure water. Under identical conditions, the active glycolipid A was eluted with chloroform-methanol (4:6) (personal communication of Dr. T. Yamakawa, Tokyo Univer- sity).

fatty acids and the sugars, and still consider the substance as pure.

Additional evidence for high molecular substances is the fact that they were practically not adsorbed on silica gel, which’ has been shown not to adsorb and degrade high molecular complexes of lipid (34). Thus, the two active glycolipids would be similar to strandin, considered as a high molecular substance by Folch et al. (35) and Rosenberg and Chargaff (36).

On the other hand, it is difficult to reconcile a high molecular structure with the known chemical properties of glycolipids, since no group seems to be present for cross linkage between the basic units. In the case of ox brain glycolipid, cross linkage through neuraminic acid has been proposed by Karkas and Chargaff (37), but no neuraminic acid had been detected in Fractions P-3-A and P-3-B.

The physical properties and the chemical composition of Fractions P-3-A and P-3-B (Table II) show great similarities, the only difference being in their optical rotations. The elemen- tary analyses suggest a N-lignoceryl-O-(tetrahexosyl-N-acetyl- hexosaminyl)-sphingosine with an approximate molecular weight of 1500. This assumption is not in complete agreement with the fatty acid analyses, since the composition of both compounds, especially Fraction P-3-A, shows a large pre- ponderance of acids with a skeleton of 18 carbon atoms. To have this composition agree with the carbon and hydrogen values would require less carbohydrate in the molecule than

GALACTOSAM

GALACTOSE

GLUCOSE

FIG. 4. Determination of the carbohydrate components of Fractions R-2-A and R-2-B, obtained by hydrolysis of the sub- stances P-3-A and P-3-B. The hvdrolvsis was done with 1 N sul- furic acid for 5 hours, and after-removal of the fatty acid and sphingosine, as described in “Experimental Procedure,” the car- bohydrate components were separated by paper chromatography, on Whatman No. 54 paper, in the mixture n-butanol-pyridine- water, 5:3:2.

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r\‘ovember 1961 S. I. Hakomori and R. W. Jeanloz 2833

is suggested by the carbohydrate analysis. The reason for this discrepancy has not been established.

The proportions of glucose to gala&se to galactosamine (not taking into account the small amount of glucosamine present) for Fractions P-3-A and P-3-B is 1:5:2 and 1: 6:2, respectively. The precision of the determination does not warrant great significance to this difference, but both proportions are difficult to reconcile with a pure substance of low molecular weight with a five carbohydrate component skeleton.

In two recent publications, Yamakawa et al. (33, 38) have reported the isolation of active substances from human blood with A-B-O group specificity. Besides an inactive substance with the general composition of a N-lignoceryl-O-(trihexosyl-N- acetylgalactosaminyl)-sphingosine, they reported the presence of active substances containing glucosamine and sialic acid. On the other hand, Koscielak and Zakrzewski (13) found the activity to be linked to a hexosamine-sphingosine-trihexoside component and their determination of the proportion of galacto- samine to glucosamine as 6 : 1 is in agreement with the present work.

Periodate oxidation indicates a remaining core resistant to degradation, composed for both substances of one glucose, two galactoses, and one hexosamine. Since most of the gala&se is already oxidized after 2 hours, the presence of a free n’s-glycol can be assumed in all moieties reacting with periodate, indicating the presence of a linkage at position 2. A linkage at position 6 would require a much larger amount of periodate than observed.

The consumption of periodate of Fraction P-3-A is in agree- ment with the analysis of components resistant to oxidation, and suggests a skeleton composed of one glucose and two galactoses linked at position 3; the other galactoses would be linked at position 2, and one of the galactosamines in the chain linked at position 3 or 4, whereas the second galactosamine would be located at the extremity of the chain.

For Fraction P-3-B, the consumption of periodate is signifi- cantly below the one of Fraction P-3-A, and difficult to reconcile with the amount of carbohydrate components degraded.

Study of the partial hydrolysis of Fractions P-3-A and P-3-B (Table VII) shows that both substances release first some gala&se, then some galactosamine very rapidly. Consequently, both sugars should be at, or near, the extremities of the chains. Glucose is always found with sphingosine, and is most probably directly linked to it in both substances.

The only significant difference observed between the two sub- stances was in the cores resistant to two successive hydrolyses. Fraction P-3-A gave a glycolipid (R-2-A) composed of fatty acid, sphingosine, glucose, and gala&se, whereas the glycolipid (R-2-B) obtained from Fraction P-3-B was composed of fatty acid, sphingosine, glucose, and galactosamine (Fig. 4).

From the above observations, it can be concluded that the two glycolipid fractions, isolated from erythrocytes of human A (+) and B (+) blood, and possessing hemagglutination properties, possess nearly similar chemical structures, differing by the loca- tion or mode of attachment of one unit of galactosamine. Fur- ther work to elucidate this difference will require the preparation of larger amounts of starting material, further purification, and a better knowledge of the physical state of the active substances.

SUMMARY

Extraction of erythrocytes of human A (+) and B ( +) blood afforded glycolipids possessing blood group activity. Purifica-

tion was obtained by adsorption on activated alumina and activated silica gel, followed by partition chromatography on cellulose.

The active substances isolated from the two different blood types and possessing specific activity showed great similarities in their physical properties and chemical composition, the only noticeable difference being in optical rotation.

Periodate oxidation showed some difference in the amount of oxidant consumed, but none in the constituents oxidized.

Partial hydrolysis showed a resistant core composed of fatty acid, sphingosine, glucose, and galactose for the substance iso- lated from A (+) blood, and a resistant core composed of fatty acid, sphingosine, glucose, and galactosamine for the substance isolated from B (+) blood. In both substances, part of the gala&se and of the galactosamine constituents were located at, or near, the extremities of the carbohydrate chain.

Acknowledgments-The authors are greatly indebted to Professor E. Klenk for carrying out the fatty acid determina- tions, and to Dr. E. A. Kabat and Professor W. T. J. Morgan for measuring the biological activities. They wish to thank the American Red Cross, Boston Chapter, for the generous gift of blood.

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Sen-Itiroh Hakomori and Roger W. JeanlozBlood A (Plus) and B (Plus)

Isolation and Characterization of Glycolipids from Erythrocytes of Human

1961, 236:2827-2834.J. Biol. Chem. 

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