Blood group a active difucosyl glycolipid from hog gastric mucosa

135

Biochimica @ Elsevier

et Biophysics

Acta,

Scientific Publishing

388 (1975) 135-145 Company, Amsterdam

- Printed in The Netherlands

BBA 56582

BLOOD GROUP A ACTIVE DIFUCOSYL GASTRIC MUCOSA

GLYCOLIPID

FROM HOG

AMALIA SLOMIANY and BRONISLAW L. SLOMIANY Department

of Biochemistry,

New

York

Medical

College,

Valhalla,

N. Y. 10595

(U.S.A.)

(Received November 18th, 1974)

Summary A new difucosyl from

water-soluble

glycolipid exhibiting blood group A activity was isolated glycolipid fraction of hog gastric mucosa. The structure of

this glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis, as: GalNAc-( 1:3)-Gal-( 124)-GlcNAc-( 15 3)-Gal-( 15 4)-Glc-( l+ l)-Cer 2 3 ta! t 01 1 Fuc Fuc

Introduction Blood-group substances (A,B,H), both of glycolipid and glycoprotein nature are characterized by the presence of a fucose residue linked to C-2 of galactose [l-5]. In Lewis substances, the fucose residue is linked either to C-4 of N-acetylglucosamine (Le”) or to C-2 of galactose and C-4 of N-acetylglucosamine ( Leb ). Fucose may be also linked to C-3 of N-acetylglucosamine in type II chains [6--81. A glycolipid with this type of structure was recently characterized by Yang and Hakomori [7] and was shown to be inactive in both Le” and Leb systems. Blood-group active (A,B,H) difucosyl oligosaccharides of glycoprotein origin with the second fucose residue linked to C-3 of N-acetylglucosamine have been characterized by Lloyd et al., [g-11]. So far the existence of glycolipids of corresponding carbohydrate structures have not been reported. In this report we describe the isolation, purification and structural studies of blood group A-active difucosyl glycolipid from hog gastric mucosa.

136

Experimental

procedures

Materials Frozen hog stomachs used for mucosa preparation were purchased from Pel-Freez Biological Inc., Rogers, Ark. Human red cells A,B,O types, human blood grouping serum anti-A, anti-B, and anti-H (Ulex europeous extract) were from Schering Diagnostics, Port Reading, N.J. Enzymes, fl-N-acetylhexosaminidase, a-galactosidase and /3-galactosidase, were kindly supplied by Dr Y.T. Li, Tulane University, La. Sphingenine and sphinganine were from Miles Laboratories, Inc., Elkhart, Ind. Standard fatty acid methyl esters and 1,2-dipalmitin were from Applied Science Laboratory, State College, Pa. DEAE-Sephadex A-25 was supplied by Ph~macia, Piscataway, N.J. and silicic acid (100-200 mesh) by Bio-Rad Laboratories, Richmond, Ca. Silica gel HR plates 250 nm coating thickness were from Analtech Inc., Wilmington, Dela. Methyl ethers of neutral sugars were kindly provided by Drs II. Choi and K. Meyer, Yeshiva University, N.Y. Partially methylated aminosugar standards were from the same source as reported [ 12,131, Preparation of glycolipid from hoggastric mucosa Hog gastric mucosa scrapings obtained from five fresh hog stomachs were homogenized at top speed in a Waring blendor, with 0.01 M potassium phosphate buffer (pH 6.8). After 5 min, 8 vol. of tetr~ydrofuran [14] was added and mixture was homogenized for an additions 5 min. The homogenate was then centrifuged at 12 000 X g for 10 min at lO”C, supernate collected and the residue was homogenized with the buffer/tetrahydrofuran mixture twice again. The pooled supernates, after addition of 0.3 vol. diethyl ether, were partitioned into organic and aqueous phase [ 141. The aqueous phase was concentrated, dialyzed extensively against ice cold water and lyophilized. The lyophilized crude glycolipids were dissolved in n-propanol/isopropanol/32% NH4 OH (35 : 35 : 30) and chromatographed on silicic acid column [14]. Each fraction was analyzed by thin-layer chromatography on Silica Gel HR plates developed in chloroform/methanol/water/NH, OH (60 : 35 : 1 : 7). The glycolipid fraction, detected on parallel plates with orcinol and resorcinol reagents, [ 151 was dried, dissolved in methanol/chloroform/water (60 : 30 : 8) and applied to a column (2.5 cm X 40 cm) of DEAE-Sephadex A-25 [16]. The neutral glycolipids were eluted from the column with 1000 ml of the above mixture. The gangliosides and other acidic lipids were then eluted with 1300 ml of methanol/chloroform/0.8 M sodium acetate (60 : 30 : 8). Further purification of fucose-containing glycolipid present in neutral glycolipid fraction was accomplished by preparative thin-layer chromatography on Silica Gel HR plates developed in chloroform/methanol/water (65 : 25 : 4) and chloroform/methanol/water (65 : 35 : 8). Lipid homogeneity The homogeneity of the isolated fucose-containing glycolipid was determined on thin-layer plates developed in chloroform/meth~ol/NH~ OH (40 : 80 : 25), and chlorofo~/meth~ol/acetic acid/water (55 : 45 : 5 : 5).

137

Thin-layer chromatography Thin-layer chromatography was performed on Silica Gel HR plates activated at 130°C for one hour prior to use. Neutral glycolipids and gangliosides were detected respectively with orcinol and resorcinol reagents. Ninhydrin spray was used for long-chain bases. Visualization with iodine vapors was used for preparative purposes. Methyl esters of fatty acids and methyl glycosides Methyl esters of fatty acids and methyl glycosides were obtained thanolysis of the glycolipid in 1.2 M methanolic HCI at 80°C for 20 h.

by me-

bong-chain bases For long-chain base analyses glycolipid was hydrolyzed in 1.0 M HCl in aqueous methanol [7]. The bases recovered from the hydrolysate [1’7] were examined and thin-layer plates developed in chloroform/methanol/2 M NH, OH (40 : 10 : 1) [IS] and analyzed by gas-liquid chromatography. Partial acid hydrolysis Partial acid hydrolysis of the isolated glycolipid was performed in 1.0 M formic acid at 100” C for one and two hours [ 191. Purified glycolipid was also subjected to partial acid hydrolysis in 0.3 M HCl in chloroform/methanol (2 : 1). After neutr~ization glycolipids recovered from the lower phase of the chloroform~methanol/water p~ition system were chromato~aphed on thinlayer plates developed in chloroform~methanol/water (65 : 25 : 4). The glycolipid fragments were identified by comparison of their migration on the plates with the hexoside ceramides isolated previously [ZO] , as well as by gas-liquid chromatography. Enzyme study Enzymatic hydrolysis of the ceramide tetrasaccharide (obtained by partial acid hydrolysis of native glycolipid) was performed by incubating the substrate at 37” C with a-gaiactosidase, /3-galactosidase [ 211 and /3-N-acetylhexosaminidase [22]. Sodium taurocholate (0.6%) was used as an emulsifying agent in each assay system. After 36 h of incubation, the reaction mixture were shaken with 4 vol. of chloroform/methanol (2 : 1) and centrifuged. Glycolipids in the organic phase after each enzymatic treatment were ~hromato~aphed on thinlayer plates developed in chloroform/methanol/water (65 : 25 : 4) and their carbohydrate composition was determined by gas-liquid chromatography [ $1. The sugars liberated to the aqueous phase were identified in a similar manner. Methylation analysis Native glycolipid and ceramide pentasaccharide obtained by partial acid hydrolysis of native glycolipid (1 M formic acid 100” C, 1 h) were permethylated according to the procedure of Hakomori (231. The permethylated glycolipids recovered from the methylation mixture by extraction with chloroform were purified on thin-layer plates developed in acetone/water/NH~OH 1241. The neutral sugars were (250 : 3 : 1.5) and subjected to hydrolysis separated from hexosamines by filtration through an AG 5OW-X8 (H’) column

138

[ 121. The acetylated partially methylated sugar alcohols were analyzed by gas-liquid chromatography. The peaks were identified by comparing their retention times with that of standard compounds and with the reported [24,25] retention values. The 1,2-dipalmitin was permethylated by refluxing with, methyl iodide in the presence of Ag, 0 [ 261. The permethylated diglyceride was purified on thin-layer plates, developed in petroleum ether/diethyl ether/acetic acid (70 : 20 : 4) and deacylated with methanolic NaOH [16], After neutralization, the methyl esters of fatty acids were extracted with petroleum ether. The partially methylated glycerol present in the methanolic layer was acetylated [27] and analyzed by gas-liquid chromatography. Periodate oxidation of partially methylated hexosamines A part of hexosamine fraction obtained from hydrolyzate of permethylated native glycolipid was subjected to oxidation with periodate [28] . The oxidation mixture was treated with NaBH, to stop reaction and reduce the products. The reduction mixture was acidified with glacial acetic acid, and boric acid was removed as methyl borate. After acetylation [27], the products were identified by gas-liquid chromatography. Gas-liquid chromatographic analysis Gas-liquid chromatography analyses were performed with Perkin-Elmer Model 801 apparatus. Glass columns (6 feet X l/8 inch) packed with 3% SE-30 on chrom-W-DMCS, 100-120 mesh or packed with 1% ECNSS-M on Gas Chrom-Q were used. Trimethylsilyl derivatives of methyl glycosides and longchain bases prepared from the dry residues by addition of silylating reagents [29] were analyzed on SE-30 columns [30] . The alditol acetates obtained from the glycolipid according to procedure of Yang and Hakomori [7] were analyzed on ECNSS-M columns programmed at 2” C/min from 150 to 210°C. The methylated sugars were analyzed as the partially methylated alditol acetates on the same columns at 140 and 150°C for the neutral sugars, and at 190°C for hexosamines. The methyl esters of the fatty acids were analyzed on SE-30 columns programmed at 4”C/min from 120 to 220°C [31]. The peaks were identified by comparing their retention times with the known standard mixtures of saturated, unsaturated and cw-hydroxy fatty acid methyl esters. The acetate derivatives of glycerol, and partially methylated glycerol were analyzed on ECNSS-M columns at 90” C. Hemagglutination and hemagglutination-inhibition assay Hemagglutination and hemagglutination-inhibition assays were performed with the Takatsy microtitrator using 0.025 ml loops and 2% suspension of A-positive human cells. The anti-A serum was used diluted to contain 4 units/O.025 ml. Calorimetric procedures Sialic acid was determined by the method kainen [32] and long-chain bases by the method

of Miettinen and Takki-Laukof Lauter and Trams [33].

139

Results Extraction of lipids from 340 g of wet mucosa scrapings with tetrahydrofuran followed by partitioning with diethyl ether yielded 0.79 g of lipids from the aqueous phase and 8.24 g from the organic phase. The glycolipids present in the aqueous phase were freed of phospholipid contaminants by silicic acid column chromatography, and fractionated on DEAE-Sephadex column into two fractions. The fucose-containing glycolipid fraction was eluted from DEAE-Sephadex with methanol/chloroform/water and the acidic glycolipids (gangliosides, sulfatides) with methanol/chloroform/O.8 M sodium acetate. Thin-layer chromatography was used as a final step in the purification of fucose-containing glycolipid. The purified glycolipid, obtained in a yield of 28.2 mg, was homogeneous on thin-layer chromatography with chloroform/ methanol/water (Fig. l), chloroform/methanol/NH, OH (R, 0.32) and chloroform/methanol/water/acetic acid (Rf 0.26). The extent of A activity of the isolated glycolipid was 1.2-2.5 pg/O.l ml.

Fig. 1. Thin-layer chromatography of purified difucosyl glycolipid from hog gastric mucosa and its degradation products. (1) Native difucosyl glycolipid: (2) Ceramide pentahexoside (derived from native glycolipid after 1 h hydrolysis); (3) Ceramide tetrahexoside (derived from native glycolipid after 2 h hydrolysis): (4) Ceramide tetrahexoside following incubation with P-galactosidase: (5) Ceramide tetrahexoside following incubation with &Ilactosidase and P-N-acetylhexosaminidase; (6) Ceramide tetrahexoside following sequential incubation with fl-galactosidase, P-N-acetylhexosaminidase and P-galactosidase: (7) Standard glycolipids: a, ceramide monohexoside; b, ceramide dihexoside; c. ceramide trihexoside; d. ceramide tetrahexoside. Conditions: Silica gel HR-250 nm developed in chloroform/methanol/water (65 : 25 : 4). Visualization: orcinol spray.

140

-l

II

29

25

Fig.

2.

Gas-liquid

sate

of purified

samine.

.

” I5

TIME (min

chromatogram glycolipid.

Conditions:

1%

1.

of Fucose;

ECNSS-M

)

alditol

acetate

2 galactose;

columns,

derivatives

of

3.

4. N-acetylglucosamine;

temperature

glucose;

monosaccharides

programmed

at 2OC/min

found

in the

hydroly-

5. N-acetylgalactofrom

150

to 210°C.

Gas-liquid chromatography of the alditol acetates, formed from the carbohydrate portion of the glycolipid (Fig. 2, Table I), established the presence of fucose, galactose, glucose, N-acetylglucosamine and N-acetylgalactosamine in amolarratioof2:2:1:1:1. Partial acid hydrolysis in formic acid for 1 h removed all of the fucose from the glycolipid (Fig. 1: Table I). Prolongation of the hydrolysis for 2 h resulted in the formation of tetra- and dihexoside ceramides in addition to penta- and trihexoside ceramides (minor products). Partial acid methanolysis of the isolated glycolipid gave mono-, di-, tri- (major product) and tetrahexoside ceramides (Table I). These results suggest that the sequential arrangement of TABLE

I

THE

COMPOSITION

AND

ITS

Hexoside

AND

PARTIAL

MOLAR

HYDROLYSIS

RATIOS

OF

CARBOHYDRATES

IN

THE

NATIVE

GLYCOLIPID

PRODUCTS

ceramide

Molar

Native

ratios*

FUC

Gal

1.92

2.10

Glc-Cer

Glc

GalNAc

GlcNAc

1.0

0.97

0.94

(1.0)

Gal-Glc-Cer

(0.96)

(1.0)

GlcNAc-Gal-Glc-Cer

1.98

1.0

Gal-GlcNAc-Gal-Glc-Cer

1.97

1.0

GalNAc-Gal-GlcNAc-Gal-Glc-Cer

2.03

1.0

0.93 0.95 0.96

0.98

______ * Determined by

by gas-liquid

the analysis

chromatography

of trimethylsilyl

derivatives

of alditol [30]

acetates

(71.

Values

in brackets

were

obtained

141

the sugar units in the pentasaccharide chain of this glycolipid is GalNac -+ Gal -+ GlcNAc -+ Gal -+ Glc + Cer. The results of sequential enzymatic de~adation of the saccharide chain of the ceramide tetrahexoside (Gal + GlcNAc -+ Gal + Glc + Cer), derived from native glycolipid by partial hydrolysis, are shown in Fig. 1. The ceramide tetrahexoside was resistant to the action of a-galactosidase, but was hydrolyzed to ceramide trihexoside (GlcNAc + Gal -+ Glc -+ Cer) by fl-galactosidase. The ceramide trihexoside was, in turn, converted to ceramide dihexoside (Gal -+ Glc + Cer) after incubation with ~-~-acetylhexosaminid~e. The ceramide dihexoside was further degraded by ,&galactosidase to a glucosylceramide. The results of enzymatic degradation presented above indicate that the ceramide tetrasaccharide has the structure: Gal (0) + GlcNAc (0) -+ Gal (0) -+ Glc --f Cer. The permethylated native glycolipid after hydrolysis, reduction and acetylation gave, on gas-liquid chromato~aphy, peaks corresponding to the acetates of 2,3,4-tri-0-methylfucositol, 2,4,6-tri-0-methylg~actitol, 2,3,6-tri-Omethylglucitol, 4,6-di-0-methylgalactitol (Fig. 3A), 3,4,6-tri-O-methyl-Nmethylacetamidogalactitol and 6-0-methyl-N-methylacetamidoglucitol (Fig. 4A). The ceramide pentasaccharide (GalNAc -+ Gal -+ GlcNAc + Gal -+ Glc -+ Cer), derived from native glycolipid by partial hydrolysis, gave rise to partially methylated alditol acetates of 2,4,6-tri-O-methylg~actitol, 2,3,6-tri-O-methylglu~ito1 (Fig. 3B), 3,4,6-TV-0-methyl-~-methylacetamidogalactitol and 3,6-di-O-methyl-Wmethylacetamidoglucitol (Fig. 4B). Periodate oxidation of the partially methylated hexosamines (obtained from permethylated native glycolipid) followed by reduction and acetylation, gave on gas-liquid chromatography (at 90°C) two peaks (Fig. 5B), one of which corresponded to the acetate derivative of l-O-methylglycerol (Fig. 5A). The second peak (Fig. 5B) which was not identified, presumably arose from the

13

5

IO TIME (mln

2

)

Fig. 3. Gas-liquid chromatograms of partially methylated hexitol acetates present in the hydrolysates of permethylated native glycolipid (A) and ceramide pentasaccharide (B) obtained by partial acid hydrolysis of native glyeolipid. 1. 2,3.4-tri-0-methylfucositol; 2. 2,4,6-tri-0-methylgalactitol: 3. 2.3.6”t&O-methylglucitol; 4. 4,6-di-O-methylgalactitol. Conditions: 1% ECNSS-M columns. temperature 140°C.

142

12

IO

5 TIME

Fig.

4.

Gas-liquid

hydrolysates partial

of

acid

(mln

chromatograms

of partially

permethylated

hydrolysis

native

of native

columns.

temperature

3.

methylated.

glycolipid

glycolipid.

methyl-N-methylacetamidoglucitol:

2

1

(A)

N-methylated. and

ceramide

hexosaminitol

acetates.

pentasaccharide

(B),

1. 3.4,6-tri-0-methyl-N-methylacetamidogalactitol;

6-0-methyl-N-methylacetamidoglucitol.

found

in

obtained

by

2. 3,6-di-O-

Conditions:

1%

ECNSS-M

190°c.

portion of 6-0-methyl-N-methylacetamidoglucose containing N-methylacetamido group. Analysis of the same mixture for acetate derivatives of partially methylated hexosaminitols (at 190” C), gave only a peak corresponding to the acetate derivative of 3,4,6-tri-0-methyl-N-methylacetamidogalactitol.

I IO Fig.

5.

Gas-liquid

reduced

and

samines

found

Conditions:

products.

in the permethylated ECNSS-M

1

(min

cbromatograms

acetylated 1%

2

5 TIME

columns.

of

acetylated

(B)

resulting

native

standards; from

glycolipid.

temperature

9O’C.

(A)

periodate

of

glycerol,

oxidation

1. l-0-methylglycerol:

l-0-methylglycerol of

partially 2. not

and

methylated

identified:

the

hexo-

3. glycerol.

143

TABLE

II

FATTY

ACID

Fatty

AND

LONG-CHAIN

acid

BASE

COMPOSITION

Long-chain

OF

THE

ISOLATED

GLYCOLIPID

base

(%) 14:

0

15

:0

16

: 0

(%)

4.7 2.5 22.1

18:

0

18.6

18:

18:

1

4.1

18

3.0

Unidentified

18 20 22

: OCY-OH :0 :0

22:

1 : 0

1.6 0.7

:

: 0

24

: :

24

1 1

6.9

5.8

23 23

1.7 91.4

0.3

:

24

1

4.8

22

O&-OH

0

:

1.7

1.3 17.6

Oa-OH

3.1

Unidentified

8.1

The fatty acids and long-chain base composition of the studied glycolipid is given in Table II. Hexadecanoate, octadecanoate and tetracosenoate were the principal fatty acids, whereas octadecasphingenine was the major long-chain base of the glycolipid. Discussion The fucose-containing glycolipids were found along with gangliosides and sulfatides in the aqueous phase resulting from extraction of hog gastric mucosa with buffered tetrahydrofuran and partition with diethyl ether. The fuco-lipids were separated from acidic glycolipids on DEAE-Sephadex and purified further by thin-layer chromatography. Purification of the fucose-containing glycolipids gave, in addition to the previously described A-active glycolipids [3,4,13], a new glycolipid which was composed of fucose, glucose, galactose, N-acetylglucosamine and N-acetylgalactosamine in molar ratios of 2 : 1 : 2 : 1 : 1. The isolated glycolipid exhibited blood-group A-activity and differed from the previously characterized A-active glycolipids from the same source [ 3,4] primarily by the presence of additional fucose residue. From the results of analysis of sugar composition, partial acid hydrolysis, sequential hydrolysis of saccharide chain with specific glycosidases and permethylation studies, the structure of the glycolipid is proposed to be: GalNAc-( 123)Gal-( l~4)-GlcNAc-(l~3)-Gal-(l~4)-Glc-(l-+ 2 3 t CY f 01 1 Fuc Fuc

1)-Cer

144

The sequential arrangement of sugar units in the ceramide pentahexoside chain of the isolated glycolipid (GalNAc + Gal + GlcNAc --f Gal + Glc + Cer) was established on the basis of the results obtained from partial acid hydrolysis. The ability of the isolated glycolipid to inhibit hemagglutination in A- anti-A system indicated that the terminal N-acetylgalactosamine is attached by a(1 -+ 3) glycosidic linkage to subterminal galactose and that one of the two fucose residues is attached to this galactose by e( 1 + 2) glycosidic bond. The ceramide tetrahexoside (Gal + GlcNAc + Gal + Glc + Cer) fragment obtained from native glycolipid was degraded to glucosylceramide by sequential treatment with ,0-galactosidase, /3-N-acetylhexosaminidase and P-galactosidase, thus indicating that the anomeric configuration of the glycosidic bonds in ceramide tetrasaccharide is /3. The attachment of the second fucose residue to N-acetylglucosamine as well as the type of linkages between the sugars in studied glycolipid were established by permethylation analysis. Identification of 4,6-di-o-methylgalactitol and 2,4,6-tri-0-methylgalactitol among the products of permethylated native glycolipid and only 2,4,6-tri-0-methylgalactitol from permethylated ceramide pentahexoside fragment, indicated that the subterminal galactose was substituted on C-2 with fucose. Tentative identification of 6-O-methyl-N-methylacetamidoglucitol among the products of permethylated native glycolipid and presence of 3,6-di-0-methyl-N-methylacetamidoglucitol in permethylated ceramide pentahexoside fragment, indicated that the subterminal galactose is linked to N-acetylglucosamine by (1 + 4)bond and that the second fucose residue of the studied glycolipid is attached to N-acetylglucosamine by (1 -+ 3)linkage. The supporting evidence for the presence of 6-0-methylglucosamine among the products of permethylated native glycolipid was obtained from the results of periodate oxidation of partially methylated hexosamines of this glycolipid. The analysis of the reduced products of oxidation have shown the presence of intact 3,4,6-tri-0-methyl-JJmethylacetamidogalactitol, formation of l-0-methylglycerol and disappearance of partially methylated glucosamine. The oligosaccharide(lacto-ZV-fucopentaose III) containing fucose residue linked by (1 + 3) bond to N-acetylglucosamine has been found in human milk [ 61 . More recently, the monofucosyl glycolipid of identical carbohydrate structure was also characterized [7] . Oligosaccharides with the similar structures were isolated from blood group H, Lea and Leb active glycoproteins [34] . Although these substances resemble Le” haptens, they do not inhibit Lea-antiLe” hemagglutionation. According to Kobata and Ginsburg [6] and Vicari and Kabat [35] the determinants with fucose residue on C-3 of N-acetylglucosamine are formed by specific fucosyltransferase different from the one responsible for Lea activity. It has been also suggested [ll] that fucosyltransferase produced by Lea gene has a wide specificity and will add fucose to N-acetylglucosamine in both types of chains [36] . The fucose attached to N-acetylglucosamine in type II chain may form products with other genes such as H and A and/or B [6]. This is supported by the finding [g-11] of blood group A,B,H active glycoproteins containing difucosyl oligosaccharides with type II chains. These difucosyl oligosaccharides were shown to be considerably poorer inhibitors of precipitation and hemagglutination as compared with the corresponding monofucosyl oligosaccharides. The carbohydrate moiety and

145

antigenic properties of the glycolipid described here appears to be similar to difucosyl oligosaccharides characterized by Lloyd et al., f9-111. The existence of difucosyl blood group A-active glycolipid with the carbohydrate structure, identical to the A-active difueosyl oligosaccharides of glycoprotein origin, further indicates that the same antigenic determinant may be linked to a lipid or protein core. Acknowledgments This investigation was supported tional Institute of Arthritis, Metabolism

by Grant AM-15565-03 and Digestive Disease.

from

the Na-

References 1 2 3 4 5

15 16 17 18 19 20 21 22 23 24 25 26 27 28

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6 7 8 9 10 11 12 13 14