THE JOURNAL OF BIOLOGICAL. CKE~~I~TRY Vol. 248, No. 17, Issue of September 10, pp. 6232-6238, 1973

Printed in U.S.A.

Blood Group A Active Glycolipids of Hog Gastric Mucosa

ISOLATION AnTD PARTIAL CHARACTERIZATION*

(Received for publication, April 27, 1973)

AMALIA SLOMIANY A?;D MARTIN I. HOROWITZ

From the ITeu York A[edical College, Basic Science Building, Department of Biochendry, I;alhalla, New York 10595

SUMMARY

Glycolipids with blood group A activity were purified from hog stomach mucosa powder. The active lipids were iso- lated by chloroform-methanol extraction, partition with aqueous KC1 followed by precipitation of the polar lipids with acetone. Then the A active glycolipids were purified by DEAE-cellulose and Florisil column chromatography. Final purification of the A active compounds was achieved by sequential preparative thin layer chromatography in two sol- vent systems. The homogeneity of the purified fractions was confirmed by thin layer chromatography in neutral, acidic, and basic solvent systems, thin layer chromatography of the acetylated derivatives of the purified fractions and by osmium- catalyzed periodate oxidation followed by paper chromatog- raphy of the released oligosaccharide chains. Two similar but distinct A active glycolipid fractions (L and U) were iso- lated. Fraction U contained high amounts of long chain hydroxy fatty acids, while Fraction L was rich in C&C18 fatty acids. Sphingenine and heptadecasphinganine were the major long chain bases found in both fractions. The carbo- hydrate composition of Fractions L and U was identical and was found to be (in moles per 1 mole of glucose): galactose, 3.04; fucose, 1.09, N-acetylglucosamine, 0.99, N-acetyl- galactosamine, 0.93. Partial acid hydrolysis, mild acid hy- drolysis, enzymatic digestion, and immunological assays established the following carbohydrate sequence for the A active glycosphingolipid isolated from hog stomach mucosa powder: GalNAc-Gal-GlcNac-Gal-Gal-Glc-Ceramide.

I

Fuc

Blood group A and H glycoproteins (2-7) and sulfated A and H glycoproteins (8) from hog st.omach mucosa have been isolated

and characterized. The early observations of Friedenreich and Hartmann (9), Holborow (10) and Szulman (11) were sug-

* This research was supported by Grants AM15565 and AM15475 from the National Institute of Arthritis, Metabolism and Diges- tive Diseases. This work is taken in part from a dissertation sub- mitted by A. Slomiany to New Yori Medical College in partial fulfillment of the requirements for the Doctor of Philosophy de- gree. Preliminary results of t,his work have been presented (1).

gestive of the presence also of lipoidal blood group substances in mucosal linings of the alimentary tract. Hakomori et al. (12, 13) reported the presence and structures of Lewis-active gllyco- lipids isolated from gastric adenoma.

The presence of blood group A and B activities in alcohol extracts of human stomach (14, 15) and of A activity in dog intestinal glycolipids (16) also have been observed but the sub- stances responsible for these activities have not been charac- terized nor have their structures been elucidated. We report here the isolation, purification, and partial characterization of the blood group active glycolipids from hog stomach mucosa powder.

EXPERIMEYTAL PROCEDURES

Materials

Stomach substance powder (lot No. 119023), washed with hexane, was purchased from Wilson Lab, Chicago, Ill. Human red cells Al, A*, B, 0 types, human blood grouping serum anti-l,

anti-B, and anti-H (Ulex europeous extract) from Schering Diagnostics, Port Reading, N. J.

Wheat germ hemagglutinin was a gift, from Dr. Berger, Princeton University, Princeton, N. J. Human blood group -1 substance from ovarian cyst was kindly supplied by Dr. E. -1. Kabat, Columbia University College of Physicians and Surgeons, New York, N. Y.; cr-N-acetylgalactosaminidase was a gift from Dr. 0. P. Bahl, Buffalo, N. Y. Eicosasphingenine was from Professor M. Prostenik, Zagreb, Yugoslavia; hydrosysphing- anine was obtained as a gift from Dr. H. E. Carter, Urbana, Ill.; sphingenine and sphinganine were purchased from Miles Labora- tories Inc., Elkhart, Indiana. St,andard fatty acids methyl esters mixtures: L-202, L-205, L-207, BC-1, UC-2, UC-3, and methyl arachidate were purchased from Applied Science Labora- tory, State College, Pennsylvania. Standard dodecyl, tetra- decyl, hexadecyl, octadecyl wcrc from Aldrich Chemical Com- pany, Cedar Knolls, N. J. DEAE-cellulose-Cellex D, Silicic acid-Bio Sil (-325 mesh) from Bio-Rad Laboratories, Richmond, California; Florisil (100 to 200 mesh) from Fisher Scientific Co., Fair Lawn, N. J.

Extraction of Lipids from Hog Stomach Mucosa Powder

Portions of 100 g of hog stomach mucosa powder were extracted with 4 liters of chloroform-methanol (2:1, v/v) mixture at room temperature for 24 hours. The water soluble fraction of the

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extracts was removed by partitioning with aqueous 0.74y0 KC1 according to the Folch procedure (17). The lower organic layer was evaporated to dryness, dissolved in acidified ethyl ether (1 ml of glacial acetic acid per 25 ml of ethyl ether), and the polar lipids w-ere precipitated with an excess of acetone (18).

The collected precipitate was dried under nitrogen, dissolved in chloroform, and chromatographed on DEAE-cellulose (19).

The fractions of the fucose-containing glycolipids were col- lected and blood group activity was determined on aliquots of those fractions by hemagglutination-inhibition assay. The A active glycolipid fractions were fractionated subsequently on a Florisil column (20). Prior to application on the column, the lipids were acetylated for 24 hours at room temperature in an acetic anhydride-pyridine (2 : 3, v/v) mixture. After removal of the acetylating mixture by evaporation with an excess of toluene, the derivatized lipids were dissolved in 1,2-dichloro- ethane-hexane (4:1, v/v) and applied to the column in the proportion of 20 mg of lipid for each gram of dry Florisil. Elu- t,ion volume for each solvent mixture was in the ratio of 30 ml of solvent per 1 gram of adsorbant.

The blood group active lipids and inactive glycolipids eluted with 1,2-dichloroethane-acetone (1: 1) were collected, dried in vacuum evaporator, and deacetylated with 0.6% sodium meth- oxide in chloroform-methanol-sodium methoxide (2: 1:0.6) mix- ture for 30 min (20). After neutralization with acetic acid, the glycolipids (fractions with blood group activity) were emulsified with water and dialyzed against ice-cold water for 20 hours. Salt-free dialyzate was evaporated to dryness and dissolved in chloroform-methanol (2 : 1, v/v). Further purification of blood group actjive glycolipids was accomplished by thin layer chroma- tography. Silica Gel HR plates, 500 pm coating thickness, were activated for 2 hours at 130”. The glycolipid fraction with blood group A activity was separated in two solvent systems: first with chloroform-methanol-water (65 :25:4) and then with chloroform- methanol-water (65 : 30 : 8 lower phase) on nonactivated plates developed twice with intermittent drying. The l-propanol- water (7 :3) also was used instead of chloroform-methanol-water (65:25:4) mixture. Individual bands were visualized with iodine vapors, and then extracted from silica gel with chloroform- methanol-water (1:4:0.1).

Lipid Homogeneity Analyses

The homogeneity of the blood group active lipids was ex- amined by thin layer chromatography on Silica Gel HR 250 pm plates in the following systems: chloroform-methanol-water (65 : 30 : 8 lower phase) ; chloroform-methanol-acetic acid-water (55 :45 : 5 : 5) ; chloroform-methanol-ammonia (40 : 80 : 25) ; chloro- form-methanol-water (90 : 10 : 1) (for acetylated glycolipids).

Oligosaccharides were released from blood group active, acetylated glycolipids by osmium-catalpzed periodate oxidation (21) followed by alkaline treatment and paper chromatography in two solvent systems on Whatman No. 1 paper, developed by descending chromatography. The following solvent systems were used : A, ethyl acetate-pyridine-water (2 : 1: 2) (upper phase) and B (12 : 5 : 4). The chromatograms were developed for 18 and 40 hours, respectively. The oligosaccharides and simple sugars were detected with alkaline silver nitrate or by the periodate- benzidine staining technique.

Acid Methanolysis

The blood group A active lipids were subjected to acid meth- anolysis (2 N methanolic HCI, 18 hours at 80”). The fatty acid methyl esters were removed into petroleum ether and then

analyzed for their quantitative and qualitative composition on gas-liquid chromatography. The 1570 diethyleneglycolsucci- nate and SE-30 column were used in temperature range 120-180” and 120-220”, respectively. The elution patterns were compared with the appropriate fatty acid methyl esters standard mixtures.

The remaining solution of the methanolyzate was evaporated to dryness and the residue was dissolved in 2 N NaOH. Long chain bases were extracted from the alkaline solution with diethyl ether. The ether extracts were combined, washed with water, and the long chain bases were determined calorimetrically ac- cording to the Lauter and Trams procedure (22).

The alkaline-aqueous phase was filtered through a mixed bed resin; the neutral sugars were recovered from the eluate and calorimetrically determined (23). The molar ratios of long chain base to neutral sugars were determined.

The same hydrolysis conditions were used for the estimation of the long chain bases by gas-liquid chromatography (24). The crude etherial extract of long chain bases was purified on a silicic acid column, treated with periodate and the aldehydes obtained from the long chain bases were analyzed on 15% DEGS column isothermally at 140” and 160”. The unsaturated long chain bases were revealed by catalytic hydrogenation of the mixture of aldehydes followed by comparison of the gas-liquid chromatog- raphy patterns before and after hydrogenation.

Sugar Composition

The ratios of the sugars present in the native and in the par- tially degraded blood group active glycosphingolipids were de- termined as the reduced and acetylated compounds by gas-liquid chromatography on a 3% ECNSS-M column according to the procedure of Yang and Hakomori (25).

Degradation Study

Hydrolysis of Fucosyl Residue-The A active glycolipids were hydrolyzed in aqueous 0.1 N HCI for 1 hour at 80”. Liberation of the fucosyl residue from oligosaccharides (obtained after osmium-catalyzed periodate oxidation of the native A active glycolipids) was performed according to Yang and Hakomori (25) (0.05 N HCI, 1 hour at SOO).

Study of Carbohydrate-Sequence in Blood Group A Active Lipids

Purified glycolipids were subjected to partial acid hydrolysis (26). About 5 mg of the substance were dissolved in chloroform- methanol (2:1, v/v) containing 0.3 N HCI and then was heated at 60” for 50 min. The reaction mixture was neutralized with 0.3 N methanolic KaOH, shaken with water three times, and centrifuged to separate layers. The oligosaccharide ceramides obtained in the organic layer were separated according to their migration on thin layer plates (Silica Gel HR, 250 pm coating thickness) in the solvent system: chloroform-methanol-water (65:25:4). The resulting bands were visualized with iodine vapors, extracted from silica gel with the solvent mixture chloro- form-methanol-water (1: 1 :O.l), and hydrolyzed (25). The sugar components were identified and molar ratios obtained by gas-liquid chromatography. The mixture of mono- and oligo- saccharides was recovered and partially separated on a P-2 col- umn (1.7 x 230 cm). Each fraction was separated further on thin layer plates in the solvent system, I-propanol-water (7:l) and as acetylated derivative in benzene-methanol (96:4) (27). Monosaccharides were identified by comparison with appropriate standards. The isolated oligosaccharides were reduced with NaBH4, hydrolyzed (S), N-acetylated, derivatized with silylat-

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ing reagent (28), and chromatographed on the gas-liquid chro- matograph using a SE-30 column isothermally at 160”.

Enzyme Study

cr-N-Acetylgalactosaminidase Assay (29)-Enzyme activitywas checked with synthetic substrate phenyl a-N-acetylgalactosami- nide according to the procedure of Ressig et al. (30) modified by the addition of 0.6% sodium taurocholate. While using blood group A active glycolipid as a substrate, 100 ~1 of O.S$!$ sodium taurocholate was added to 500 pg of the native A active lipid, and the mixture was suspended in 25 ~1 of 0.05 M sodium citrate buffer pH 4.6. To this was added 75 ~1 of the enzyme in the same buffer and a drop of toluene. The digesting mixture was incubated for 24 hours at 37”. The enzymatic reaction was stopped by addition of 4 volumes of chloroform-methanol (2:1), mixed, and centrifuged. The upper layer was evaporated to a small volume and spot,ted on paper along with controls and standard sugars. Paper chromatograms were developed by descending technique on Whatman No. 1 in the solvent system: 1-butanol-acetic acid-water-ethanol (5:1.5:3.5:0.5) for 24 hours. The sugars were visualized with silver nitrate reagent.

The appropriate controls containing lipid plus heat inactivated enzyme and sodium taurocholate, and enzyme plus taurocholate were carried through the entire procedure.

Immunological Assays

Hemagglutination and Hemagglutination-inhibition Assays- Assays were performed with the Takatsy microtiter (Cooke Engineering Co., Alexandria, Va.) using 0.025-ml loops and 2% suspension of A, B, and H positive human red cells. The anti-A, anti-B serums were used diluted to contain 4 units/O.025 ml; the anti-H (Ulex europeous extract) was not diluted.

Phytohemagglutinin Reaction-The ability of the various hexo- sides ceramides (obtained after partial acid hydrolysis of A active glycosphingolipid) to inhibit agglutination was tested according to Hakomori et al. (31).

Gas-Liquid Chromatography-Gas-liquid chromatography anal- yses were performed with Perkin-Elmer model 801 apparatus equipped with a hydrogen flame ionization detector. Glass columns (180 X 0.4 cm, imzer diameter) were used packed with 15% DEGS on CHROM-W-HMDS, 80 to 100 mesh; 3% SE-30 on CHROM-W-DMCS 80 to 100 mesh; 3yo ECNSS-M on Gas Chrom Q, 100 to 120 mesh. Helium was used as the carrier gas at 50 p.s.i.

RESULTS

Extraction of lipids from 500 g of dry hog stomach mucosa powder with chloroform-methanol (2 : 1, v/v) and separation from gangliosides and non-lipid components by partition with aqueous 0.74% KC1 afforded 16 g of lipid. Precipitation of the lipids with acetone resulted in the isolation of 8.5 g of polar lipids which were recovered in the precipitate. Subsequent fractionation on DEAE-cellulose column gave three A active fractions eluted with chloroform-methanol-water 7 :3 :O.l, 6:4:0.1, and 4:6:0.1, respectively (Table I). The yield of frac- tions containing A active compounds from this column was 1.900 g. Acetylation followed by fractionation of the acetylated lipids on the Florisil column yielded 857 mg of the A active frac- tion which was eluted with 1,2-dichloroethane-acetone (1: 1). Final purification of the A active lipids by preparative thin layer chromatography (Figs. 1 and 2) resulted in isolation of two lipid fractions of equal A activity (0.015 pg/O.l ml) designated as L (lower band) and U (upper band) in yields of 55.6 mg and 49.2

TABLE I

Elution of polar lipid fraction of hog stomach mucosa substance from DEAE-cellulose column

Dry DEAE-cellulose (250 g) was packed into the column (5.0 X 55.0 cm); lipid (8.5 g) was applied.

Fraction Eluent composition I ! Quantity of lipid

Nmxn:,“f elyted IllllllgKi~

VOlUIlXP Der 500 z

n/s I

Chloroform Chloroform-methanol 95:5 Chloroform-methanol 8:2 Chloroform-methanol-water 7:3:0.1

Chloroform-methanol-water 6:4:0.1

Chloroform-methanol-water 4:6:0.1 Chloroform-glacial acetic acid 1: 1 Glacial acetic acid

5 12 10 10 10 10 5 5

720 1505 1950 1234 368 312

1140 800

a One column volume = 950 ml.

mg, respectively. These fractions were homogenous when chromatographed in the acidic system, chloroform-methanol- acetic acid-water (55 :45 : 5 : 5) (Fig. 3)) in chloroform-methanol- water (65:30:8 lower phase), and as acetylated derivatives in chloroform-methanol-water (9: 1 :O.l) (Fig. 4).

Homogeneity of the oligosaccharide moiety of the A active preparations (L and U) m--as established by osmium-catalyzed periodate oxidation and paper chromatography of the liberated oligosaccharides. Oligosaccharides from both fractions (L and U) possessed identical mobility in both chromatography systems and they migrated 4.8 cm from the origin after 48 hours of de- scending chromatography in system A, and 0.5 cm after 18 hours in system B. Complete hydrolysis of the liberated oligosac- charides from L and U samples (2 x HCl, 2 hours at 100”) fol- lowed by neutralization with Ag&Os and paper chromatography in solvent B showed the presence of glucose, galactose, fucose, N-acetylglucosamine, and N-acetylgalactosamine in both L and U samples. The liberated oligosaccharides (from L and U) were highly active in hemagglutination-inhibition; they were able at concentrations of 0.02 to 0.04 pgjO.1 ml to inhibit 4 hemagglutinating units of anti-A.

Methanolysis of the purified A glycolipids (L and U) and calorimetric determination of the neutral sugars and long chain bases showed their molar relation as 4 : 1.

Gas-liquid chromatography of the alditol acetate derivatives formed from A active glycolipid established the presence of glucose, galactose, fucose, N-acetylglucosamine, and N-acet;vl- galactosamine in a molar proportion of 1: 3 : 1: 1: 1 (Fig. 5, Table II).

Mild acid hydrolysis of the native A active glycolipids and their free oligosaccharides released the fucosyl residue which was detected by paper chromatography (for 16 hours in Solvent a).

The partial acid hydrolysis of the A active lipids produced mono-, di-, tri-, tetra-, and hexahexosides ceramides. Gas-liquid chromatography of their reduced, acetylated derivatives gave evidence that the sugar sequence in the A active glycolipid (Table III) is GalNAc-Gal-GlcNAc-Gal-Gal-Glc-Cer.

To support the results obtained with various hexoside ceram- ides resulting from partial acid hydrolysis, the aqueous phase of this hydrolyzate was examined for released oligosaccharides.

Homogenous fractions of oligosaccharides were reduced,

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FIG. 1. First preparative thin layer chromatography of the A

active glycolipid fraction eluted from Florisil with 1,2-dichloro- ethane-acetone (l:l, v/v). Condition: Silica Gel HR, 500 pm

coating thickness, activated for 1 hour at 130”, solvent system: 1-propanol-water (7:3).

FIG. 2. Second preparative thin layer chromatography sep- aration of the A active band obtained in first thin layer chroma- tography system (Fig. 1). Condition: Silica Gel HR, 500 coating thickness, nonactivated, solvent system: chloroform-methanol-

FIG. 5. Gas-liquid chromat’ogram of the alditol acetate deriva- tives of monosaccharides derived from A active glycolipid, l-fu- case, 2-arabinitol (internal st’andard), 3-galactose, -I-glucose, 5-N- acetylglucosamine, 6-N-acetylgalactosamine. Condition: 3y0 ECNSS-M on Gas-chrom Q glass column (180 X 0.4 cm, inner diameter), temperature 180” for 28 min then programmed 4” per min to 220”. Helium was used as carrier gas at 50 psi.

water (65:30:8) (lower phase) developed twice with intermediate drying.

FIG. 3. Thin layer chromatography of the purified L and U preparations in chloroform-methanol-glacial acetic acid-water (55:45:5:5). Condition: Silica Gel G, 250 pm coating thickness, activation 1 hour at 130”.

FIG. 4. Thin layer chromatography of the purified L and U A active preparations in their acetylated form (1) in chloroform- methanol-water (90: 10: l), and nonacetylated form (2) (partial separation L from U) in chloroform-methanol-water (65:35:8).

methanolyzed (1 N methanolic HCI, 80” for 18 hours), N-acety- lated, trimethylsilyl derivatized and analyzed on gas-liquid chro- matography on SE-30 column run isothermally at 160” The analyses of those fractions were in accord with the presence of disaccharides, trisaccharide, and tetrasaccharide. The chemical composition of these saccharides and molar ratios of their sugars are given in Table IV.

The phytohemagglutinin reaction was inhibited by the glyco- lipid containing glucose to galactose to N-acetylglucosamine 1:2 : 1 which was isolated from the partial acid hydrolyzate.

Treatment of the native A active glycolipid with a-N-acetyl-

galactosaminidase resulted in the liberation of N-acetylgalactos- amine into the aqueous phase of the incubation mixture. This

was shown by paper chromatography of the aqueous phase of the incubation mixture (see “Enzyme Study”). The long

chain base compositions of the isolated lipid preparation (L and U) were almost identical (Table V). The most abundant bases were sphingenine and heptadecasphinganine.

Gas-liquid chromatography of the fatty acid methyl esters showed that CA&I& were the major fatty acids of L, while C&-C24 a-hydroxy were the major fatty acids found in U (Table VI).

DISCUSSION

Extraction of lipids from hog stomach mucosa powder followed by partition with aqueous KC1 and precipitation with acetone

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T.4BLlI II TABLE V

Composition and molar ratios of purified L and U preparations Long chain base composition of blood group A active

Mean value and rang& glycosphingolipids (U and L samples)

Carbohydratea The cumulative areas under the aldehyde peaks derived from

L I

u long chain bases are taken as 100% and compared with the area under each aldehyde peak on the same chromatogram. These

P&f are the average values of 5 chromatograms. -

Glucose.................. 1.00 1.00 Galactose.. 3.04 (2.96-3.14) 3.05 (2.89-3.17) Fucose................... 1.08 (0.95-1.17) 1.10 (1.05-1.14) - A--Acetylglucosamine.. 0.99 (0.94-1.10) 1.02 (0.88-1.12)

X-Acetylgalactosamine. 0.93 (0.83-0.99) 1.04 (0.94-1.12)

a Carbohydrate expressed as micromoles per 1 hmole of glucose. b Determined by gas-liquid chromatography on 4 samples each

of I, and U which were hydrolyzed, reduced, and acetylated ac-

cording to the Yang and Hakomori (25) procedure.

TABLE III

Products of partial acid hydrolysis of native A active glycosphingolipids (L alrd U fractions)Q

Hexoside ceramide Molar ratios

dc gal glcXAc galNAc Fuc

Native*........................... 1.0 3.04 0.99 0.93 1.08 Glc-Cer.. 0.3c

Gal-Glc-Cer.. 1.0 1.01

Gal-Gal-Glc-Cer.. 1 .O 1.82 GlcNAc-Gal-Gal-Glc-Cer.. 1.0 1.85 0.98

GaliX;Bc-Gal-GlcNAc-Gal-Gal-Glc- Cer.. 1.0 3.15 1.05 1.09

a Determined by gas-liquid chromatography on the hydrolyzed, reduced, and acetylated aliquots of the purified products of the partial acid hydrolysis of native A active glycolipid.

b Mean values from Table II. c Determined with respect to internal standard of arabinitol.

TABLE IV

Composition. and molar ratios of oligosaccharides released upon partial acid hydrolysis of native A active

glycosphingolipida

Oligosaccharide I

Molar ratiosb

Cal-Glc-ol GalNAc-Gal-01.. . GalNAc-Gal-GlcNAc-01..

GlcNAc-Gal-Gal-Glc-01..

0.87:l.O

1.11:l.O 1.09:0.96:1.0 1.12:2.15:1.0

a Determined by gas-liquid chromatography on reduced, hy- drolyzed, N-acetylated, and TMSi derivatixed samples, under the conditions listed under “Experimental Procedures.”

b The molar ratios were calculated with respect to the reduced sugar in each oligosaccharide.

resulted in practically complete recovery of blood group A ac- tivity in the precipitate. Further separation of A active com- pounds from t’he various polar lipids was achieved on DEAE- cellulose and Florisil columns. The above preparative scheme facilitated the removal of sialoglycolipid which remained on DEAE-cellulose column under these conditions. Quantitative separation of glycolipids was obtained without contamination from phospholipids or other lipid classes by Florisil chromatog- raphy of the acetate derivatives of glycolipid. The A active

d14:O Tetradecasphinganine dl6:O Hexadecasphinganine d17:O Heptadecasphinganine

d18:O Sphinganine d14: 1 Tetradecasphingenine dl6:l Hexadecasphingenine d17:l Heptadecasphingenine d18:l Sphingenine

%

1.17 2.62 1.50 1.40

13.40 11.20

2.36 2.80 0.48 1.30 0.77 5.42

2.38 2.20 78.00 72.91

TIBLE VI

Patty acid composition of U and L glycolipids with blood yroup A activity

Fatty acids composition n-as calculated on the basis of fraction of the total area under peaks of gas-liquid chromatograms.

Fatty acids U

Cl%. c&4.. ClS.. (316. CIT., ClS.. czz. c24.. c25. C,d-OH.. C16-OH.. C&-OH..

C&-OH. &-OH.. CIG-iso.. C18-iso..

C22-iso.. C&iso..

Unknown.

/ %

2.21 1.47

0.33

., 11.98 10.32

6.88

I 3.47 .I 3.47

5.88

: .i 0.30 6.59

1.19 .‘i 9.43

i 17.91 _.! 2.59

5.16 0.70

.I 3.50

6.32

L

3.55 3.56

1.15 22.11 12.73

12.05 2.50 2.50

1.54 1.63 2.30

2.88 4.60 6.92

14.71 0.50 1.15

3.60

fractions from the DEAE-column (Fractions IV, V, and VI- Table 1) which were applied to and then eluted from the Florisil column with 1,2-dichloroethane-acetone (1: 1) contained A active blood group substances and other neutral glycolipids.

Separation of these neutral glycolipids from the blood group active substances was achieved by thin layer chromatography. The two solvent systems: chloroform-methanol-water (65:25:4) or 1-propanol-water (7:3) were used for the first preparative step on thin layer chromatography (Fig. 1). It was found that the chloroform-met,hanol-water (65:25:4) system was better for resolution of DEAE-Fraction IV, and 1-propanol-water (7 :3) gave better separation and recovery of activity for the two re- maining fractions (DEAE-Fractions V and VI).

The final purification of A active glycolipids was achieved in the solvent system: chloroform-methanol-water (65:30:8 lower phase), and it was satisfactory for all three DEAE-fractions.

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This thin layer chromatographic syst,em was very selective and separated the active material from each of the DEAE-Fractions

IV, V, VI into three zones, of which two of them (with higher mobility) (Fig. 2) were highly A active, giving inhibition of hemagglutination at concentration of 0.015 pg/O.l ml. The two most active blood group components were des’ignated according to their mobilities as L (lower zone) and U (upper zone).

It had been shown earlier that considerable attention must be given to purification and evaluation of homogeneity of blood group glycolipids because they are extremely difficult to purify (32). The native lipid was analyzed for homogeneity on thin layer chromatography in acidic (Fig. 3) and basic systems (16), in its acetylated form (32), and by osmium-catalyzed periodate oxidation (21). In each solvent system there was found a single band on thin layer plates for L and U, respectively, and in their acetylated form they possessed the same RF value (Fig. 4).

The oligosaccharides released from L and U upon osmium- catalyzed periodate oxidation were indistinguishable from one another by paper chromatography. Both migrated 4.8 cm from the spotting line after 48 hours of descending chromatography in System A, and 0.5 cm after 18 hours of development in System B. Spectrophotometric determination of the components of blood group substances (methanolysis in 2 N HCl, 18 hours at 80”) gave a molar ratio of neutral sugars to long chain base of 4 to 1. Gas-liquid chromatography of the aldit,ol acetate derivatives of the sugars (Fig. 5) showed that these sugars were galactose and glucose in a molar ratio of 3 to 1. This analysis also revealed the presence of fucose, N-aoetylglucosamine and N-acetyl- galactosamine and their molar proportion with respect to glucose was 1: 1: 1: 1. From these results, it was concluded that the A active compound is a heptahexoside ceramide.

Gas-liquid chromatography of the aldehydes (Fig. 6) derived

12 6 4 TIME (MIN.)

FIG. 6. Gas-liquid chromatogram of the aldehydes derived from long chain bases of A active glycolipid. Condition: glass column (180 X 0.4 cm, inner diameter) packed with 15% diethyl- eneglycol-succinate on CHROM-W-HMDS, temperature 140”. Helium was used as the carrier gas at 50 p.s.i. The numbers indi- cate chain lengths of the aldehydes and degree of unsatnration.

6237

from periodate oxidation of long chain bases isolated from blood group A active glycolipids showed the presence of almost the same saturated and unsaturated bases in L and U preparations (Table V). The major bases were sphingenine and heptadec- asphinganine. The sole difference between L and U glycolipids was revealed in fatty acid composition. C&C18 were the major fatty acids found in L sample, while a-hydroxy CZZ-CZ4 were the major fatty acids in U glycolipid (Table VI). Changes in fatty acid patterns have been observed with development (33), with the long chain fatty acids C&-C26 and their hydroxy forms be- coming prominent with age. It is possible that the pooled scrapings of hog stomach were obtained from a heterogeneous age population, and this may, conceivably, have contributed to the variety of fatty acids found in the isolated L and U blood group A substances.

Partial acid hydrolysis was used to aid in the elucidation of the sugar sequence in this heptahexoside ceramide. The most satisfactory results were obtained when the glycolipid was hydrolyzed for 50 min (0.3 PJ HCl in chloroform-methanol 2: 1 at 60”). Examination of the organic layer of the hydrolyzate by thin layer chromatography (chloroform-methanol-water 65 : 25 :4) showed the presence of mono-, di-, tri-, tetra-, and hexahesoside ceramides. The composition of those hexoside ceramides and the molar ratios of the sugars with respect to glucose were established by gas-liquid chromatography of the alditol acetate derivatives, and the following sequence was deduced: GalNAc- Gal-GlcNAc-Gal-Gal-Glc-Cer. Presence of the terminal N- acetylgalactosamine a-glycosidically bound to the subterminal galactose was established by the enzyme study. The N-acetyl- galactosamine was cleaved by the action of a-hi-acetylgalactos- aminidase. The high A activity of the lipids isolated from the hog stomach strongly suggests the presence of the same immuno- determinant group, i.e. GalNAc-Gal, as found in A positive hog

I Fuc

stomach glycoprotein (34) and also in the A active glycolipids of the human red cell stroma (35). It is then quite likely that in hog A active glycolipids fucose is also attached to the subtermi- nal galactose moiety.

Studies of the reduced oligosaccharides recovered from the aqueous layer of the partial hydrolyzate gave supporting evidence for the proposed sequence of sugars in A active glycolipids.

The only difference in oligosaccharide between that proposed by Iseki (36), Hakomori (Compound Aa) (35), and by us resides in the presence of the additional galactose in the internal se- quence i.e. -GlcNAc-Gal-Gal-Glc-Cer of the hog stomach glyco- lipid. The additional galactosyl residue present in our prepara- tion may be a result of polymorphism of blood group substance, which has been observed first by Hakomori et al. (32) on red blood cell determinants. It appears that the blood group A active glycolipids may contain hexa- (32, 36), hepta- as shown here, or even longer and branched oligosaccharides (35). It will be interesting to see if glycolipids are found which contain oligo- saccharides as long (14 to 18 sugars) as those postulated to be present in blood group A active glycoproteins of hog stomach mucosa (8).

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Amalia Slomiany and Martin I. HorowitzPARTIAL CHARACTERIZATION

Blood Group A Active Glycolipids of Hog Gastric Mucosa: ISOLATION AND

1973, 248:6232-6238.J. Biol. Chem. 

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