Anomeric Structures of Globoside and Ceramide Trihexoside of Human Erythrocytes and Hamster...

transcript

THE JOURNAL OF I~IOLOGICAL CHEMIBTRY Vol. 246, No. 7, Issue of April 10, pp. 2271-2277, 1971

Printed in U.S.A.

Anomeric Structures of Globoside and Ceramide Trihexoside

of Human Erythrocytes and Hamster Fibroblasts*

(Received for publication, November 27, 1970)

SEN-ITIROH HAKOMORI AND BADER SIDDIQUI

From the Department of Pathobiology, School of Public Health and Community Medicine, University of Washing- ton, Seattle, Washington 98105

Yu-TEH LI AND SUCHEN LI

From the Department of Biochemistry, Tulane University, Delta Regional Primate Research Center, Covington, Louisiana 70433

CARL GUSTA F HELLERQVIST!:

From the Department of Organic Chemistry, Stockholm University, Stockholm, Sweden

SUMMARY

The terminal galactosyl residue of ceramide trihexoside of human erythrocytes and BHK fibroblasts and that of the ceramide trihexoside derived from “globoside” of human erythrocytes were hydrolyzed and totally converted to ceramide lactoside by an oc-galactosidase isolated from fig. The enzyme showed a strict substrate specificity for a- galactosides and was completely freed from ,&galactosidase activity. With the sequential application of glycosidases, the carbohydrate sequence and anomeric linkages of glyco- sphingolipids have been simultaneously determined. The presence of cr-anomeric (equatorial) proton in theseglycolipids was supported by a doublet at 6 4.6 ppm (J = 2 Hz) in the nuclear magnetic resonance spectrum of a trimethylsilyl derivative and by an absorption at 850 cm-l in the infrared spectra. From these results and the results of methylation study, the structures of ceramide trihexoside and of globoside were determined, respectively, galactopyranosyl-cr-(1 + 4). galactopyranosyl$(l ---t 4)glucopyranosyl-(1 + l)ceramide, and 2-acetamido-Z-deoxy-galactopyranosyl-P-(1 + 3)galactopyranosyl-ac-(1 + 4)galactopyranosyl-P-(1 --+ 4)glucopyranosyl(1 + 1)ceramide.

Globoside,l the major glycolipid of the human erythrocyte membran;, has been characterized (1, 2) and has the structure

* This investigation was supported by Research Grants T-475 from American Cancer Society and CA 10909 from the National Cancer Institute, United States Public Health Services (to S. H.) and by Grant GB 18019 from the National Science Foundation and Grant RR OOlG4 from the National Institute of Health, United States Public Health Services (to Y.-T. L.).

$ Supported by Hierta Retzius Stipendiefond. 1 Globoside is a trivial name used as an abbreviation for a

crystalline glycolipid having a carbohydrate sequence and structure as stated in the text.

2-acetamido-2.deoxygalactosyl(1 + 3)galactosyl(l -+ 4)galactosyl(1 -+ 4)glucosylceramide (3, 4). It was shown to be a membrane antigen of some cell species (5-S). The terminal 2-acetamido-2-deoxygalactopyranosyl linkage was hydrolyzed by P-N-acetylhexosaminidase (9-11) in contrast to that of Forssman antigen glycolipids (9, 12); however, anomeric linkages of the two int,ernal galactoses still remain to be elucidated. Globoside contains an internal carbohydrate chain, very similar to that of ceramide trihexoside; therefore, these compounds appear meta- bolically related.

The composition of CTH2 of erythrocytes has been shown to have carbohydrate moiety identical with that which accumu- lates in tissues of patients with Fabry’s disease (13) or in Naka- hara sarcoma of mice (14); however, the detailed structure of CTH of erythrocytes has not been studied. The structures of these CTH present in pathological tissues have been studied by Miyatake (15), by Kawanami (14), and, in greater detail, by Sweeley, Sinder, and Griffin (16). These studies have shown a common structure of galactopyranosyl(1 + 4)galactopyranosyl (1 -+ 4)glucopyranosylceramide. Kawanami (14) suggested the presence of an oc-anomeric linkage at the terminal galactosyl residue of CTH of mice sarcoma based on the infrared and nuclear magnetic resonance spectra, whereas Sweeley et al. (16) have stated that the terminal galactosyl residue of Fabry’s CTH could be a p-configuration upon a detailed study by NMR spectra, although deficiency of an a-galactosidase in the leukocytes of patients with Fabry’s disease was pointed out by Kint (17).

The anomeric specificity of an enzyme or enzymes capable of splitting the terminal galactosyl residue of CTH found in tissue (17) and in serum (18, 19) is obscure.

A CTH was also found recently in the original clone of BHK C13/21 (20). The quantity of the CTH increased in the con- fluent state of cells and decreased or disappeared after extensive

2 The abbreviations used are: CTH, ceramide trihexoside, Gal- Gal-Glu-Cer; NMR, nuclear magnetic resonance; IR, infrared spectra; globo-CTH, CTH derived from globosidk by. chemical or enzvmatic degradation of terminal GalNAc: CDH. ceramide dihexoside or ceramide lactoside; TMS, trimethylsilyl.’

by guest, on July 17, 2011w

.jbc.orgD

ownloaded from

2272 Anomeric Structure of Globoside Vol. 246, No. 7

passages or after viral transformation (21, 22). The cell density- dependent responses of CTH may be due to the addition of a terminal galactosyl residue to the ceramide dihexoside when cells are contact-inhibited (21, 22). Therefore, the terminal gnlactosyl linkage might be different from that of the penultimate galactosyl residue. This paper reports the anomeric configuration of galactose residues in the CTH of erythrocytes and fibroblasts as well as that of globoside of erythrocyte membrane.

From NMR and IR spectra, and by using cy- and ,8-galactosidases, it was concluded that terminal galactose in the ceramide trihexoside and the galactose residue internally next to the N-acetylgalactosamine in globoside have an a! configuration. Among various ar-galactosidases examined, only a-galactosidase isolated from ficin was capable of hydrolyzing these galectosidic linkages.

MATERIALS AND METHODS

Sph&gogZycoZipids-Ceramide trihexoside and globoside were prepared from human erythrocyte membrane. One part membrane suspension in water was mixed with 9 parts absolute ethanol, extracted at 80” for 15 min, and filtered. The precipitate which occurred upon cooling of the filt’rate at -20” was purified by solvent fractionation and by column chromatography on Biosil A column as previously described (23). Finally, crys-

E ; a, cu 20 30 40 50 60 70 80

E 0.2L .c

0.05M NaCl

lo 20 30 40 50 60 70

Fraction Numbers FIG. 1. A, Bio-Gel P-60 filtration of crude ol-galactosidase prep-

aration obtained by acetone precipitation. The enzyme solution (4 ml) containing 0.7 g of protein was applied to a Bio-Gel P-60 column (2 X 120 cm) which had been previously equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. The column was eluted with the same buffer at a flow rate of 6 ml per hour controlled by a peristaltic pump. o , absorption at 280 nm; 0, or-galactosidase activity; 3 ml per fraction were collected. B, DEAE-Sephadex A-50 chromatography of ol-galactosidase fraction obtained by Bio-Gel P-60 filtration. Protein solution (7 ml) containing 167 mg of protein was applied to a DEAE-Sephadex A-50 column (2.5 X 35 cm) which had been equilibrated with 0.06 M sodium phosphate buffer, pH 7.0. l , absorption at 280 nm; 0, cu-galactosidase activity; 6 ml per fraction were collected.

talline precipitates of CTH and globoside were obtained by centrifugation from methanol solution at -20”. The preparations were homogeneous as to the carbohydrate moiety liberated upon the cleavage of glycosidic-lipid bond (24). The total glycolipids of hamster BHK cells (20) were prepared by the acetylation procedure (25). Globoside was degraded to a ceramide trisaccha- ride by the controlled periodate oxidation (0.5% glycolipid in 0.015 M aqueous sodium periodate solution containing 0.02 M

acetate buffer, pH 4.5, 4” for 18 hours) followed by Smith degradation in aqueous methanol solution. Because of a strong ag- gregation of glycolipid micelles in aqueous salt solution, the terminal sugar was preferentially oxidized and eliminated under such conditions. The yield of globo-CTH was 30% of the theo- retical. Globoside was also degraded to ceramide trihexoside (globo-CTH) by fl-N-acetylhexosaminidase (P-2.acetamido-2- deoxy-n-hexoside acetamidodeoxyhexose hydrolase, EC 3.2.1.30) of jack bean (11).

Incubation of Xphingoglycolipids with Various Glycosidases- Enzymic hydrolysis of CTH, globo-CTH, CDH, CDH derived from CTH, and CDH derived from globe-CTH was performed by incubating the substrate separately with the following enzymes: fl-galactosidase (P-u-galactoside galactohydrolase, EC 3.2.1.23) from jack bean (26) ; cr-galactosidase (cr-u-galactoside galactohydrolase, EC 3.2.1.22) from J1oritereZla vinacea (27) ; ar-galactosidase prepared from Aspergillus niger (28); and Lu-galactosidase isolated from ficin. The conditions for hydrolyzing glycolipids by these enzymes were similar to those described earlier (11) and are given in Table I. After the incubations, the reaction mixtures were shaken with 4 volumes of chloroform- methanol, 2:1, and then centrifuged. The lower layer was removed by capillary pipette, evaporated to a dryness, and analyzed by thin layer chromatography on Silica Gel JI plates. The solvent systems used were: chloroform-methanol-water, 65:30:8 (lower phase) or 65 :25 :4. The spots on the plate were developed by heating at 120” after spraying with 2 ~5 sulfuric acid containing 0.2% orcinol. The intensities of the spots given by individual glycolipids were compared with that of the known quantities (3 to 30 pg.) of the standard glycolipids. The conversion rates of a given glycolipid to its derivatives with one less sugar moiety were determined both by the dcgrec of disappear- ance of that original glycolipid and by the appearance of the new glycolipid as a degradation product.

Infrared and Nuclear Magnetic Resonance Spectra-Infrared spectra were determined by using KBr tablets in a I’crkin- Elmer apparatus. NMR spectra were determined as trimethyl- silylated derivatives according to Van der Veen (29), Sweeley et al. (16), and Hellerqvist, BjBrndal, and Lindberg (30) in car-

TABLE I

PuriJication of cr-galacto.sidase from 10 g of &in

Crude extract 9.7 Dialysis against 0.05 M sodium

phosphate buffer, pH 7.0.. 9.4 Acetone precipitate. . 8.6 Bio-Gel P-60.. 8.4 DEAE-Sephadex A-50.. 5.7

Total Specific protein activity

w u?ds/mg

x 10-s

8380 0.11

3530 0.26 1363 0.62

167 5.04 21 27.14

:eco”ery

96.8 88.8 87.0 59.1

.jbc.orgD

ownloaded from

issue of April 10, 1971 Hakomori, Xiddiqui, Li, Li, and Hellerqvist 2273

hou tetrachloride by using an HA 100 Varian apparatus with time-averaging computer Cl024 (Varian) .

Methylation Analysis and Optical Rotation--The positions of linkages between monosaccharides were analyzed by methylation (31) followed by formolysis, hydrolysis, reduction, and acetylation (32, 33). The partially methylated alditol acetates in the hydrolysates of permethylated glycolipids were identified by gas chromatography (on a B-foot, 3% ECNSS column at 150°C) and the peaks were analyzed in the Hitachi-Perkin-Elmer mass spectrometer. Optical rotation was determined in a Perkin- Elmer photoelectric polarimeter by using a IO-cm cell.

Isolation of oc-Galactosidase from Ficin-All of the operations were carried out between 0” and 4”. Ten grams of ficin (Nu- tritional Biochemicals, Lot, 6380) were dissolved in 100 ml of water and centrifuged to obtain a clear extract. It was then dialyzed exhaustively against 0.05 M sodium phosphate buffer, pH 7.0, overnight with several changes of buffer. Inactiv: proteins precil)itated during dialysis were removed by centrifugation to obtain 120 ml of clear enzyme solution. To this solution 48 ml of acetone were added dropwise with vigorous stirring. After 10 min the precipitate was removed by centrifugation and an additional 48 ml of acetone were added to the supernatant fluid to precipitate the a-galactosidase. The precipitate was separated by centrifugation, dissolved in 8 ml of 0.1 M sodium phosphate buffer, pII 7.0, and designated crude a-galactosidase. Four milliliters of the crude a-galactosidase were applied to a Bio-Gel P-60 column (2 X 110 cm) previously equilibrated with 0.1 M sodium phosphate buffer, pH 7.0. The column was eluted with the same buffer at the rate of 6 ml per hour (Fig. IA). The active fractions indicated by the horizontal bar were pooled, and the oc-galactosidase was precipitated by adding ammonium sulfate to 0.8 saturation. Three hours after the addition of ammonium sulfate to the pooled fractions the precipitate was centrifuged at 12,000 x g for 15 min and dissolved in 2 ml of 0.05 M sodium phosphate buffer, pI-I 7.0. Pooled fractions from two such gel filtrations were dialyzed exhaustively against 0.05 M

sodium phosphate buffer, pH 7.0, and applied to a DEAE- Sephadex A-50 column (2 X 30 cm) already equilibrated with the same buffer. The column was eluted with the same buffer to wash off the unadsorbed proteins and then eluted with 0.05 M

sodium citrate buffer, pH 6.0, containing 0.05 M NaCl. The elution profile is shown in Fig. 1B. a-Galactosidase fractions indicated by the horizontal bar were pooled and precipitated by adding ammonium sulfate to 0.8 saturation. The precipitate was dissolved in 1 ml of 0.05 M sodium phosphate buffer, pH 7.0, and designated purified ar-galactosidase. The specific activity and the yield achieved in various purification steps, based on 10 g of ficin, are summarized in Table I. a-Gala&o- sidase activity wits assayed by incubating 0.05 ml of the enzyme solution with 1 ml of 2 mM p-nitrophenyl-or-n-galactoside (dissolved in 0.05 M sodium citrate buffer, pII 4.5) as described previously (27). One unit of enzyme was defined as that amount of enzyme which hydrolyzed 1 pmole of p-nitrophenyl-a-n-galactoside at. 25” per min. The specific activity of the enzyme was expressed as units per mg of protein. Protein was determined by the method of Lowry et al. (34), with crystalline bovine albu- min as the standard.

The purified ol-galactosidase preparation was free from any contaminating glycosidases including a- and @-galactosidase, 0-N.acetylhexosaminidase, LY- and P-L-fucosidase, P-xylosidase, and or-mannosidase when assayed with p-nitrophenylglycosides

WAVENUMBER (cd 1

1000 900 800 720

WAVELENGTH IN MICRONS

FIG. 2. Infrared spectra (between 720 and 1000 cm-l) of ceramide lactoside (A), ceramide trihexoside (B), globoside (C), and ceramide trihexoside derived from globoside by the controlled Smith degradation (D), as determined in KBr pellet. Concentra- tion: approximately 1% for A to C; 0.2y0 for D (because of short- age of the material and impurity). Chart D was recorded with an expanded scale.

as substrates. In addition to cr-galactosidase, no other glycosidases were originally present in ficin. Incubation of 2 mg of melibose and raffinose, respectively, with 0.05 unit of fig a-galactosidase in 0.2 ml of 0.05 RI sodium citrate buffer, pH 4.5, resulted in complete liberation of all terminal nonreducing cu-galactosidic linkages from these two oligosaccharides in 18 hours at 37” according to the method described previously (27). By the same procedure lactose was not hydrolyzed, even with 2 units of enzyme. The optimum pH for enzyme activity was between 3.5 and 4.5.

Blood Group B Actitity-This was determined by inhibition of hemagglutination, with or without “auxiliary lipid” (6, 35) by using anti-B serum (3 hemagglutination units). Microtiter plates and pipettes were used. Blood group B-active glycolipid isolated from rabbit erythrocytes (36) was used as a positive control and globoside was used as a negative control.

RESULTS AND DISCUSSION

The structures of ceramide trihexoside and globoside have been determined as oc-galactopyranosyl(1 --t 4)-P-galactopyranosyl- (1 + 4)glucopyranosylceramide and P-(2-acetamido-2-deoxygalactopyranosyl)(l + 3).oc-galactopyranosyl(1 -+ 4)-fl-galactopyranosyl(1 --f 4)glucopyranosylceramide, respectively, on five experimental findings :

The methylated CTH gave, by the described procedure (33), approximately 1 mole each of acetates of 1,3,4,5-tetramethyl galactitol, 1,4,5-trimethyl galactitol, 1,4,5-trimethyl glucitol; methylated globoside gave approximately 1 mole each of acetates of 1,3,5-trimethyl galactitol, 1,4,5-trimethyl galactitol, 1,4,5-trimethyl glucitol.3

3 IUPAC nomenclature; carbon number of the substitution of hexitols is reversed from that of hexose.

.jbc.orgD

ownloaded from

2274 Anomeric Structure of Globoside Vol. 246, No. 7

FIG. 3. Nuclear magnetic resonance spectra of trimethylsilyl derivative of cerebroside (A), ceramide trihexoside (B), and globoside (C). Note a small doublet in Chart B at 4.61 6 ppm (5.39 7 ppm) with a very low spin-spin coupling constant (J = 1 N 2 Hs), which is characteristic for cu-anomeric proton. Note also a doublet at 4.1 8 ppm and 4.28 6 ppm with much larger coupling constant (J = 7 Hz).

Infrared spectra of CTH and globo-CTH showed an absorption at 840 to 850 cm-1 which is characteristic of the presence of a! anomeric (G-equatorial) proton in addition to an intensive absorption at 890 to 900 cm-1 which is due to the deformation vibration of axial protons (Fig. 2).

NMR of trimethylsilyl derivative of CTH (Fig. 3) showed a signal at 4.6 6 ppm with a low coupling constant (J = 2 Hz), at 4.1 6 ppm (J = 7 Hz), and at 4.28 6 ppm (J = 7 Hz). By comparison with the spectra of glucosylceramide (4.05 6 ppm; J = 7 Hz) and galactosylceramide (4.0 6 ppm; J = 7 Hz), the peak at 4.1 6 ppm (J = 7 Hz) could represent the anomeric proton of &glucosyl residue. The spectrum at 4.28 6 ppm (J = 7 Hz) should be due to a anomeric proton of P-galactoside of galactopyranosyl(1 + 4)glucose in comparison with the spectrum of TMS derivative of lactose gave a signal at 4.35 d ppm (J = 7 Hz); TMS-&galactopyranosyl(l + 6)galactose gave 4.47 6

FIG. 4. Thin layer chromatography of glycolipids and that which was hydrolyzed by various glycosidases, and some examples of sequential degradation of globoside and ceramide trihexoside. t, standard neutral glycolipids: a, ceramide monohexoside; b, two spots of CDH; c, CTH; d, globoside. 8, hydrolysate of globoside with p-N-acetylhexosaminidase showing globo-CTH. 8, hydrolysate of globo-CTH with fig a-galactosidase showing globo-CDH. 4, incubation mixture of globo-CTH with jack bean p-galactosidase, no reaction and showing globo-CTH. 6, hydrolysate of globo-CDH with p-galactosidase, showing ceramide monohexoside. 6, incubation mixture of CTH of erythrocytes with jack bean fi-galactosidase, showing no reaction and recol ered original CTH. 7, the same mixture of CTH of erythrocytes with fig a-galactosidase showing a hydrolysis product containing CDH. 8, CDH derived from CTH and incubated with jack bean fi-galactosidase, showing the hydrolysate contains ceramide monohexoside. 9, standard rabbit ceramide pentasaccharide having blood group B activity (42). 10, the rabbit blood-group B glycolipid incubated with fig cu-galactosidsse, showing conversion to a ceramide tetrasaccharide (lacto -N-tetraosylce - ramide). ii, the rabbit blood group B glycolipid incubated with jack bean p-galactosidase, showing no reaction. 19, standard asialo-GM-l (gangliotetraosylceramide; galactosyl-b-1 -+ a-N-acetylgalactosaminosyl-8-1 3 4-galactosyl+l -+ 4-glucosylceramide) . IS, asialo GM-l incubated with jack bean fl-galactosidase, showing triosylceramide (N-acetylgalactosaminosyl-P-1 ---f 4 galactosyl-D-1 --f 4-glucosylceramide). 14, asialo GM-l incubated with fig cu-galactosidase, showing no reaction. 16, triosylceramide derived from “asialo GMI,” incubated with ,9- (N-acetyl)-hexosamini- dase, showing the presence of CDH.

ppm (J = 6.5 Hz)~ (30). The spectrum at 4.6 6 ppm (J = 2 Hz) is most probably due to the anomeric proton of a-galactoside of cu-galactopyranosyl(1 -+ 4)galactosyl in comparison to the 6 4.5 (J = 2.0 Hz) for cz-methyl galactoside (16) and to 6 4.9 (J = 1 Hz) for the TMS derivative of c-y-D-galaCtOpyrsnOSyl-

(1 -+ 6)n-glucopyranose (melibiose), although 6 = 5.31 ppm (J = 3.2 Hz) has been reported for the TMS derivative of cr-glucosyl(1 -+ 4)glucose (maltose) (16, 29). The signal at 4.6 6 ppm

of TMS-CTH was with a low spin-spin coupling constant and agreed with the signs by other o-protons which were all charac-

terized by a much weaker coupling constant (J = 1 N 2 Hz) due to the axial-equatorial arrangement of a-linkages4 (16, 29, 30). The TMS derivative of globoside showed a signal at 4.5 6 ppm (J = 8 Hz) and a broad signal at 4.2 to 4.3 6 ppm and at

4 C. G. Hellerqvist and B. Lindberg, unpublished observation.

.jbc.orgD

ownloaded from

Issue of April 10, 1971 Halcomori, Siddiqui, Li, Li, and Helleqvist 2275

TABLE II

Conversion 0-f glycolipicls by hydrolysis of terminal nonreducing sugars with specijic glycoside hydrolases

One hundred micrograms of glycolipids were dissolved in 100 pl of 0.05 M sodium citrate buffer containing 100 pg of sodium taurocho- late in small conical test. tubes. With warming and followed by agitating with Vortex mixer (Scientific Products, Evanston, Illinois) and immersing in an ultrasonic cleaning bath (Balsonic, Bausch and Lomb, supplied by Van Waters, Seattle, Washington), glycolipids were readily dissolved. The pH of the citrate buffers was4.5 for a-galactosidase, 4.0 for ,“l-galactosidase, and 5.0 for P-N-acetylhexosaminidase. The enzyme was then added to the solution and incubated at 37” for 18 hours. The activities of enzymes used were: jack beanp-galactosidase, 0.5 unit/50 ~1; jack bean &Vactylhexosaminidase, 2 units per PI; Mortierella vinacea or-galactosidase, 5.5 units/5 ~1; fig a-galactosidase, 0.G unit/GO ~1.

Substrates (glycolipids)

CT11 from human red cells CTH from human red cells CTH from human red cells CTH from human red cells CTH from RHK cells CTH from BHK cells Globoside” Globo-CTH Globo-CTH Red cell CDH Ited cell CDH CD11 derived from red cell CTH CD11 derived from Globo-CTH Asialoganglioside Asialoganglioside

Rabbit blood group I&aciive glycolipid

Rabbit blood group B-active glycolipid

EnZyltXS

a-Galactosidase (fig) a-Galactosidase (fig) p-Galactosidase (jack bean) a-Galactosidase (M. vinacea) a-Galactosidase (fig) p-Galactosidase (jack bean) p-Hexosaminidnse” a-Galactosidase (fig) p-Galactosidasc (jack bean) a-Galactosidasc (figj p-Galactosidase (jack bean) p-Galactosidase (jack bean) &Galactosidase (jack bean) a-Galactosidase (fig) &Galactosidase (jack bean)

a-Galactosidase (fig)

@-Galactosidasc (jack bean)

a Confirmation of previous results by Li and Li (11). b C&III, ceramide monohexoside; Cer, ceramide.

4.0 6 ppm (J = 7 Hz). Although the chemical shift value at 4.5 6 ppni is within a range of that given by nnomeric proton of cy-galactosides (see above), the spectrum has a relat.ively large coul)ling constant (J = 8 Hz) for a single anomcric proton of cr.glycosides, suggesting that the spectrum could be superimposed wit,h another of unidentified origin. The spectrum at 4.0 6 ppm (J = 7 112) should be due to the nnomeric proton of P-glycoside of glucosylceramide as mentioned above. A broad peak at 4.2 to 4.3 6 ppm has not been identified.

CTH of crythrocytes and BHK fibroblasts which were not affected l)y P-galactosidase from jack bean were completely hydrolyzed by fig a-galactosidase and totally convcrt,ed to C’DII (Fig. 4, Table II). The CDH of erythrocytes and the CDH derived from the CTH by hydrolysis of CTH with fig oc-galact’o- sidase ncre completely hydrolyzed by jack bean &galactosidase under the same conditions (Fig. 4, Table II). The or-galactosidases from either A. niger or Jf. winacea were not able to hydrolyze ceramide trihexoside.

Removal of terminal 2-acetamido-2-deoxygalactose from globoside either by Smith degradation under controlled conditions or by jack bean /3-N-acetylhexosaminidase produced a compound which was indistinguishable from CTH of erythrocytes or fibroblasts on thin layer chromatography (globo-CTH). The globo-CTH was not hydrolyzed by jack bean P-galactosidase but was easily hydrolyzed by fig ar-galactosidasc and was

Conversion reaction

CTH + Cl)H CTH ---f CDH No reaction No reaction CTH ---f CDH No reaction Globoside --f CTHa CTH + CDH No reaction No reaction CDH ---f CMHb CDH --f CMH CDH ---f CMH No reaction

Gal-GalNAc-Gal-Glu-Cer6 -+ GalNAc-Gal-Glu-Cer

Gal-Gal-GluNAc-Gal-Glu- Cer+ + Gal-GluNAc-Gal- Glu-Cer

No reaction

Amount of Amount of substrate .%ZyEl~

ia 250

75 100 100

250 80 20

100 100 30 30

100 100

ZW~ilS

0.G 0.6

10.0 11.0

1.0 0 .5

10.0 2.5 0.5 0.4 1.0 0.5 0.5 0.4 2.0

totally converted to CDH (Fig. 4, Table II). The CDH obtained from globe-CTH can be hydrolyzed by jack bean P-galactosidase and is converted to ceramidc monohexoside (Table I).

The present study confirmed the sequence of monosaccharide and the positions of glycosidic linkages as previously reported by Yamakawa (3, 4). The terminal galactosyl linkage of ceramide trihexosides from either erythrocytes or BHK fibroblasts and the penultimate galactosyl linkage of globoside of human erythrocytes were proven to be oc linkage in this study in contrast to the other sugar residues which have been demonstrated as fl linkage. The structure of CTH from Nakahara sarcoma may also have LY anomeric linkage based on IR spectra and NMR determined in DzO (14). It is known that infrared patterns of complex carbohydrates arc greatly affected by the conformations

and intramolecular interactions of anomeric proton with neigh- boring groups as well as wit,h any interacting contaminants; thus

the absorption assignment for anomeric proton is not always reliable. The Forssman antigen glycolipid has an oc-galactos- aminosyl linkage but the infrared spectra has no assignment for (Y anomeric (axial) proton at 840 to 850 cm-’ (12). Globoside, which now appears to have an ar-galactosyl linkage at penultimate position, did not show any absorption at 840 to 850 cm-’ (see Fig. 2c), in agreement with the published results (36, 37). The CTH and globo-CTH showed an absorption at 850 cm+ in

.jbc.orgD

ownloaded from

2276 Anomeric Struciure of Globoside

a A r \

Vol. 246, No. 7

FIG. 6. Structures of globoside, ceramide trihexoside of human erythrocyte membrane, and BHK fibroblasts. A, globoside; B, ceramide trihexoside of erythrocytes and BHK fibroblasts; C, showing a ceramide trihexoside with the terminal fl-galactosyl residue; n, N-lignocerylsphingosine.

these preparations whereas the published spectrum of Fabry’s CTH by Miyat)ake (15) had no absorption in this area.

For the same reason, as described above for IR spectra, NMR spectra for the nnomeric proton of the complex carbohydrates is not always reliable, although analysis of anomeric proton as trimethylsilyl derivative has been recommended because the anomeric proton is more separated from other protons than in free carbohydrates (29). Variation of the chemical shift for o( or p anomeric proton is quite large, depending on the structure of carbohydrates. The interpretation of the spectrum as described above should be considered as tentative unless a pure synthetic compound with the same structure is available.

The specific optical rotation ([c&~ value) of the CTH and globoside of human erythrocyte was found to be +20.5” (in pyridine; c, l), and +X5” (in pyridine; c, l), respectively, in close agreement with the published values (15, 36, 37). The dextrorotatory properties of these glycolipids were in striking contrast to the levorotatory properties of ceramide lactoside, hematoside, and gangliosides (3638).

Only suggestive data for the presence of an cu-galactosyl linkage in CTH or globoside have been obtained by IR, NMR, or optical rotation. In contrast, the discovery of a specific a-galactosidase, which is capable of hydrolyzing the a-galactoside of glycolipids, has enabled the terminal and the penultimate structure of CTH and globoside to be established conclusively. In addition, it has been shown in this study that the sequence of carbohydrates and the anomeric properties of glycosides in globoside and CTH have been determined by sequential application of glycosidases of plant origin. Such a method is much more useful and specific as compared with analysis of physical spectra.

Although the presence of an enzyme which can hydrolyze the terminal galactosyl residue of CTH has been found in tissues (18) and in serum (19, 39), the anomeric specificity of this enzyme action has been obscure. It is of great interest that CTH is easily hydrolyzed by a-galactosidase isolated from ficin but not by other a-galactosidases from many other sources. Further studies on the isolation and properties of a-galactosidase from ficin will be published elsewhere.5

It has been well established that cr-galactosyl(1 + 3)galactosyl residue is a determinant structure of blood group B activity (40, 41), although the presence of n-a-fucosyl(1 -+ 2) residue at the penultimate galactosyl residue greatly enhances the activity through stabilizing the configuration of the terminal cy-galactosyl- (1 --f 3) residue (41). It is known that a glycolipid isolated from rabbit erythrocytes having ol-galactosyl (1 -+ 3)galactosyl residue, without fucosyl residue, showed a B activity (42). Since the structure of CTH is now established to contain a-galactosyl- (1 + 4)galactosyl residue at the terminus, the B activity of CTH was compared with blood group B activity of the B-active rabbit glycolipid that contains a-galactosyl(1 ---f 3)galactosyl structure at the terminus. Even 200 pg of CTH did not inhibit B hemagglutination; by contrast 12.5 pg of the rabbit glycolipid inhibited B hemagglutination. The determinant structure of blood group

B activity is, therefore, not replaceable by a-galactosyl(1 + 4)- galactose where two galactoses are perpendicularly linked

through axial to axial hydroxyl groups, and the configuration of this structure is quite unique (see Fig. 5).

The penultimate galactosyl residue of globoside is now estab-

5 T.-Y. Li and S.-C. Li, manuscript in preparation.

.jbc.orgD

ownloaded from

Issue of April 10, 1971 Hakomori, Xiddiqui, Li, Li, and Hellerqvist 2277

lished to be a: linkage; this is, to the best of our knowledge, the first clear demonstration of the presence of ol-galactoside in the middle of a carbohydrat’e chain of glycolipids or glycoproteins.

This structure of globoside is quite unique in the way that the terminal disaccharide (Fig. 5~) and the internal disaccharide (Fig. 5b) are linked perpendicularly, and the two parts, a and b, are in two different planar dimensions. Gangliotetraosyl- ceramide [galactosyl /3(1 + 3)-N-acetylgalactosaminosyl-/3-

(1 + 4)-galactosyl-P-(1 -+ 4)glucosyl-p-(1 + l)ceramide] (38) and lacto-N-tetraosylceramide or Iacto-N-neotetraosylceramide

(galactosyl-P-(1 + 3) or (1 + 4).N-acetylglucosaminosyl-~- (1 + 3)-galactosyl-P-(1 + 4)gluco&3-(1 + 1)ceramide (or both)) (43, 44) have no axial to axial linkage within the carbohydrate chain; therefore, all of the pyranosidic rings should be arranged on one planar dimension.

Globoside was recently found to be a membrane antigen of fetal erythrocytes (5), in striking contrast to that of the same glycolipid of adult erythrocytes which was in a cryptic state, being masked by a protein cover (5-7). Globoside was also found as a major glycolipid in the plasma membrane of bovine kidney cells as well as the envelop membrane of parainfluenza

virus budding from the same cells, suggesting that it could be a viral antigen (8). Undoubtedly, the mentioned morphology of globosidic carbohydrate chain is important in defining the im- munochemical specificity of this membrane antigen (6, 35, 45).

Acknowledgments-The authors are extremely grateful to Pro- fessor William S. Chilton and Mr. B. J. Nist, Department of Chemistry, University of Washington, for their kind courtesy in determination of NMR spectra.

REFERENCES

1. KLENIC, E., AND LAUENSTEIN, K., Hoppe-Seyler’s Z. Physiol. Chem., 291, 249 (1952).

2. YAMAKAWA, T., AND SUZUICI, S., J. Biochem. (Tokyo), 39, 393 (1952).

3. YAMAKAW~, T., YOICOYAMA, S., BND KISO, N., J. Biochem. (Tokyo), 52, 228 (1962).

4. YAMAKAWA, T., NISHIMURA, S., AND KAMIMURA, M., Jap. J. Exp. Med., 35, 201 (1965).

5. HAI~OMORI, S., VOX Sang, 16, 478 (1969). 6. KOSCIELAK, J., HAI~OMORI, S., AND JE~NLOZ, R. W., Zmmuno-

chemistry, 6, 441 (1968). 7. UHLENURUCR, G., REIFENBERG, V., AND HEGGEN, M., 2. Zm-

munitaetsjorsch. Allergic Klin. Zmmunol., 139, 486 (1970). 8. KLENIC, H.-D., AND CHOPPIN, P. W., Proc. IVat. Acad. Sci.

U. S. A., 66, 57 (1970).

9. ABE, T., HANDA, S., AND YAMAKAWA, T., Seikagaku, 39, 555 (1967).

10. MIYATAICE, T., HANDA, S., AND YAMAICAWA, T., Jap. J. Exp. Med., 38, 135 (1968).

11. 12.

LI, S.-C., AND LI, Y.-T., J. Biol. Chem., 246, 5153 (1970). MAKITA, A., SUZUICI, C., AND YOSIZ~WA, Z., J. Uiochem.

(Tokyo), 60, 502 (1966). 13. SWEELEY, C. C., AND KLIONSKY, B., J. Biol. Chem., 238,

PC3148 (1963). 14. 15. 16.

KAWANAMI, J., J. Biochem. (Tokyo), 62, 105 (1967). MIYATAKE, T., Jap. J. Exp. Med., 39, 35 (1969). SWEELEY, C. C., SINDER, P. D., JR., AND GRIFFIN, C. E., Chem.

Phys. Lipid, 4, 409 (1970). 17. 18.

20. 21. 22. 23.

KINT, J. A., Science, 167, 1268 (1970). BRADY, R. O., GAL, A. E., BRADLEY, R. M., AND M~P~TENSSON,

E., J. Biol. Chem., 242, 1021 (1967). MAPES, C. A., ANDERSON, R. L., AND SWEELEY, C. C., Fed.

Eur. Biochem. Sot. Lett., 7, 180 (1970). STOKER, M., AND MACPHERSON, I., Virology, 14, 359 (1961). HAKOMORI, S., Proc. Nat. Acad. Sci. U. S. A., 67, 1741 (1970). ROBBINS, P. W., AND MACPHERSON, I., Nature, 229, 569 (1971). HAKOMORI, S., AND STRYCHARZ, G. D., Biochemistry, 7, 1279

(1968). 24. 25. 26. 27.

HAKOMORI, S., J. Lipid Res., 7, 789 (1966). SAITO, T., AND HAICOMORI, S., J. Lipid Res., 12, in press (1971). LI, Y.-T., AND LI, S.-C., J. Biol. Chem., 243, 3994 (1968). SUZUKI, H., LI, S.-C., AND LI, Y.-T., J. Biol. Chem., 246, 781

(1970). 28. LEE, Y. C., AND WACEK, V., Arch. Biochem. Biophys., 138, 264

(1970). 29. 30.

31. 32.

VAN DER VEEN, J. M., J. Org. Chem., 28, 564 (1963). HELLERQVIST, C. G., LINI~BERG, B., L~~NNGREN, J., AND J,IND-

BERG, A., Carbohyd. Res., 16, in press (1971). HAKOMORI, S., J. Biochem. (Tokyo), 66, 205 (1964). BJ~RNDAL, H., LINDBERG, B., AND SVENSSON, S., Carbohyd.

Res., 6, 433 (1967). 33.

BJ~RNDAL, II.; HELLERQVIST, C. G., LIXDBERG, B., AND SVENSSON, S., Angew. Chem., 82, 643 (1970).

LOWRY, 0. II., ROSEBROUGH, N. J., FARII, A. L., AND RANDALL, R. J.. J. Biol. Chem.. 193. 265 (1951).

35. 36. 37.

RAPPORT, M. M., AND ~RAF;L., Progr.‘Allergy, 13, 273 (1969). MAKITA, A., J. Biochem. (Tokyo), 56, 269 (1964). YAMAICAWA, T., IRIE, R., AND IWANAGA, R/I., J. Biochem.

(Tokyo), 48, 490 (1960). 38. 39.

KUHN, R., AND WIEGANDT, H., Chem. Rer., 96, 866 (1963). MAPES: C. A., ANDERSON, R. L., SWEELE~, C. C.,‘I)&NICK,

R. J., AND KRIVIT, W.. Science, 169. 987 (19701. 40. 41.

WATKINS, W. M., Science, 152, i72 (iBG6). LLOYD, K. W., AND K~II~T, E. A., Proc. Nat. Acad. Sci.

U. S. A., 61. 1470 (1968). 42.

43. 44.

ETO, T., I~HIKA~A, y., N~HIMURA, II.: ANJ~O, S., ANU YAMA-

ICAWA, T.: J. Biochem. (Tokyo), 64, 205 (1968). KUHN, Ii.; AND WIEGANDT, II., 2. Naturjorsch., 19b, 80 (1964). YANG, H.-J., BND HAKOMORI, S., J. ZZioZ. Chem., 246, 1192

(1971). 45. MARCUS, D. M., AND JAXIS, Il., J. Zmmunol., 104, 1530 (1970).

.jbc.orgD

ownloaded from

Anomeric Structures of Globoside and Ceramide Trihexoside of Human Erythrocytes and Hamster...

Documents