Studies on B-antigenic sites of human erythrocytes by use of coffee bean α-galactosidase

ARCHIVES

OF

BIOCHEMISTRY

Studies

AND

BIOPHYSICS

on B-Antigenic of Coffee

NOAM Department

HARPAZ,

170,676-663

(1975)

Sites of Human Erythrocytes Bean a-Galactosidasel

H. M. FLOWERS

of Biophysics,

The Weizmann Received

April

AND

Institute

NATHAN of Science,

by Use

SHARON2 Rehovoth,

Israel

29, 1975

Treatment of human type B erythrocytes, erythrocyte membranes and membrane fractions with highly purified coffee bean a-galactosidase resulted in the disappearance of B activity and enhancement of H activity with concomitant release of galactose. In the case of B erythrocytes, e&me treatment led to the release of 3.0 x lo6 molecules of galactose per cell, about 75% of which were found in the aqueous phase of butanollwater extracts of cell membranes. AB and 0 cells released 1.9 x 10s and 0.8 x 10’ molecules per cell, respectively. All the galactose released from 0 cells came from glycolipid material lacking B activity. Application of a correction for the enzymically labile, non-B-active ol-galactosides presumably also present in B cells would suggest that such B cells have approximately 2.2 x 106 B-antigenic sites. No release of galactose was observed when membranes and cell fractions, prepared from B erythrocytes which had been previously incubated with o-galactosidase were treated with the enzyme, showing that all the a-galactosidase-labile galactose present in B-active macromolecules in intact red cells is located at sites available to the enzyme and can be readily removed. We did not find any significant differences in the number and distribution of the Bantigenic sites present in erythrocytes from secretors and from a nonsecretor B individual.

The detailed carbohydrate structures of secreted, water-soluble glycoproteins with ABO blood group activity have been elucidated in recent years (for reviews, see Refs. l-3). However, our knowledge of the structures of these determinants in erythrocytes is limited. Although serologically active glycosphingolipids have been isolated from human erythrocyte membranes and chemically characterized (31, the yields of the purified compounds were extremely low. Recently, significant amounts of ABO blood group-active materials have been shown to enter the aqueous phase upon extraction of erythrocyte membranes with solvent systems

such as butanol/water (41, chloroform/ methanol/water (51, and phenol/water (6). The nature of the water-soluble active component is still unresolved, various authors having identified it as glycoprotein (4-7) or glycolipid (8, 9). The use of exoglycosidases, capable of removing immunodominant sugar moieties from ABO antigenic determinants and thereby altering their specificities, was an early and valuable means of elucidating the structural and immunochemical relationships among these antigens. Treatment of secreted, B-specific substances with crude a-galactosidase from Trichomonas foetus (10) or from coffee beans (11) caused the loss of B specificity and appearance of H specificity, and this was one of the earliest indications that the difference between the two antigens is the presence of the cr-n-galactosyl moiety in the B antigen. Similarly, the conversion of human

’ This study was supported by a grant from the Israel Ministry of Health and by a contribution from a friend of the Weizmann Institute in Buenos Aires, Argentina. * Established Investigator of the Chief Scientist’s Bureau of the Israel Ministry of Health. 676 Copyright All rights

0 1975 by Academic Press. of reproduction in any form

Inc. reserved.

B-ANTIGENIC

SITES

OF

erythrocytes from type B to type 0, first by a crude preparation of a clostridial cY-galactosidase (12) and subsequently with the purified enzyme (13), has been reported. Whereas in the earlier paper (12) data on the sugars released were presented, none were given in the later report (13). Experiments in our laboratory have recently demonstrated the destruction of the B specificity of the water-soluble material obtained by butanollwater extraction of erythrocyte membranes, with concomitant release of n-galactose, by a partially purified a-galactosidase from coffee beans (14). In the present paper we describe the serological effects of treating intact B erythrocytes, isolated erythrocyte ghosts, and butanol- and water-soluble membrane fractions, with highly purified coffee bean CYgalactosidase isolated by affinity chromatography (15). The enzyme was used to determine the quantitative distribution of B-antigenic sites among erythrocyte membrane fractions and, in addition, to probe the orientation of the B-active components in the membrane of the intact cell. MATERIALS

AND

METHODS

a-Galactosidase (a-n-galactoside galactohydrolase, EC 3.2.1.22; specific activity, 94.5 units/mg of protein) from coffee beans was purified by affinity chromatography (15). It was free of the following contaminating glycosidase activities when tested with p-nitrophenyl glycosides (Koch-Light): D-DGalactosidase, a- and P-n-glucosidase, a-L&mosidase, a-D-mannosidase, N -acetyl-P-D-glucosaminidase and N-acetyl-/3-n-galactosaminidase. The purified enzyme migrated as a single band when 100 pg of protein was subjected to electrophoresis on 7.5% polyacrylamide gel in the presence of 1% sodium dodecyl sulfate. Freshly drawn or recently outdated bank blood was obtained from healthy donors. Erythrocytes were prepared for treatment with a-galactosidase by removing the plasma and buffy coat and washing four times with phosphate-buffered saline, pH 7.4, to prevent their agglomeration in glycerol solution (16). After determination of the packed cell volume, by use of an International Equipment Company microcapillary centrifuge, 10.0 ml of packed erythrocytes were washed three times with four volumes of 0.1 M citric acid/O.2 M Na*HPO, buffer, pH 5.0, containing 3% (by weight) of glycerol (B. D. H.) To onehalf of the packed cells was added 5 ml of a-galactosidase dissolved in the buffered glycerol solution, and to the other half, buffered glycerol without en-

HUMAN

ERYTHROCYTES

677

zyme. To facilitate the recovery and quantitation of the liberated galactose, l-2 x lo4 cpm of D 13H]galactose (Amersham Radiochemical, 100 mCi/mmol) was added to each suspension. Incubation was at 37°C with occasional gentle agitation. At varying periods of time, aliquots (10 ~1) were taken and tested for B activity by adding 0.1 ml of anti-B blood-grouping antiserum (Hylandl, incubating for 10 min at room temperature and observing the cells under a microscope. From the time when agglutination failed to occur in the test sample, the cells were incubated with enzyme for an additional hour, centrifuged, and washed three times with four volumes of buffered glycerol solution. Under the above conditions no hemolysis of erythrocytes was observed even after 9 h of incubation, either with or without enzyme. The supernatant fluid and combined washings were deionized by passage through columns (0.5 x 5.0 cm) of Dowex 50 (H+ form) and Amberlite IR-45 (OH- form), and lyophilized. Glycerol was removed by an adaptation of the method of Adachi and Sugawara (17): The sample was dissolved in 50 ml of npropanol and passed through a column (2.0 x 5.0 cm) of Dowex l-X4 (HSO,form) equilibrated with the same solvent. The column was washed with 100-150 ml of n-propanol, which removed glycerol, and then 50 ml of distilled water was passed through. The propanol eluate did not contain any radioactivity and was discarded; the aqueous eluate was freed of bisulfite with Amberlite IR-45 (OHform), and lyophilized. Ghosts were prepared from 20 ml of salinewashed erythrocytes by the procedure of Dodge et al. (181, modified by including 0.1 mM EDTA (disodium salt) in the hemolysis buffer. One-half of the resulting ghost preparation was retained for a-galactosidase treatment (see below), and the other half used for extraction with n-butanol/water by the procedure of Rega et al. (19). Prior to extraction, the ghosts were dialyzed overnight against cold, distilled water containing sufficient ammonium hydroxide to give pH 8-9. The dialyzed ghost suspension was brought to 20 ml with ice-cold water in a centrifuge tube, 10 ml of cold a-butanol was added and the suspension shaken vigorously for 30 s. The mixture was kept for 15 min at 0°C and then centrifuged for 15 min at 30,OOOg in a Sorvall refrigerated centrifuge equipped with an SS-34 angle rotor. The lower, aqueous phase was carefully removed, and the upper, butanol phase, as well as any interphase layer, were reextracted with 20 ml of cold water as above. After centrifugation, no interphase layer was observed, and the butanol phase was removed. The pooled aqueous phases were reextracted with 20 ml of cold a-butanol, concentrated to ca. 5 mg/ml of protein by rotary evaporation or by ultrafiltration with a Minicon B-15 apparatus, and dialyzed against distilled water at 4°C. The pooled butanol

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FLOWERS

phases were evaporated to dryness, the residue extracted for 1 min with 12 ml of boiling 83% ethanol (20), and the extract kept at -20°C for 48 h. The precipitate formed, designated lipid fraction I, was collected by centrifugation, and the mother liquor, designated lipid fraction II, was evaporated to dryness. Both lipid fractions were washed by stirring for 30 min with cold, anhydrous acetone and dispersed in 10 ml of boiling distilled water to give stable suspensions. Ghosts, aqueous fraction and lipid fractions I and II were each divided into two equal portions: To one portion was added a-galactosidase (50 units) in 0.1 M sodium acetate buffer, pH 5.0, and to the other portion buffer alone, to give final volumes of 10 ml. Radioactive gala&se was introduced to each sample, as described for enzyme treatment of erythrocytes, a drop of toluene added to prevent bacterial contamination, and the samples incubated at 37°C for 72 h. Aliquots (0.1 ml) were taken after 36 h of incubation and tested for hemagglutination inhibition activity with anti-B and anti-H reagent. After removal of sedimentable material by centrifugation, the samples were passed through a column (2.0 x 50 cm) of Sephadex G-10 equilibrated with pyridine acetate buffer, pH 6.5 (50 ml of pyridine and 2 ml of acetic acid per liter), and 6-ml fractions collected. An aliquot (60 ~1) of each fraction was dissolved in 10 ml of Bray’s scintillation fluid and counted in a scintillation counter. The radioactive .fractions were pooled, concentrated by rotary evaporation, and passed through a column (0.5 x 5.0 cm) of Dowex 50 (H+ form). The eluate was lyophilized and the residue dissolved in distilled water in a final volume of 1.00 ml. Aliquots (0.1 ml) were assayed for galactose by the coupled galactose dehydrogenase-NAD+ method (21), with the final assay mixture scaled down to 0.3 ml. The reaction was followed at 340 nm with a Gilford Model 2400-S recording spectrophotometer in parallel with assay mixtures containing known quantities of n-galactose. The minimum quantity of n-gala&se detectable was 0.1 erg per assay mixture. The radioactivity in O.l-ml aliquots was determined and a correction applied for loss of galactose incurred in the isolation procedure. Identification and semiquantitative estimation of galactose were performed by descending paper chromatography on Whatman No. 1 paper in ethyl acetate/pyridine/water (8:3:1, by volume, solvent A) and n-butanol/acetic acid/water (25:6:25, by volume, solvent B). Staining was with the silver nitrate reagent (22). Serological tests were performed with Cooke microtiter plates. Anti-A and anti-B blood-grouping sera were obtained from Hyland Corporation. H activity of erythrocytes was tested with a 1:l (by weight) saline extract of Ulex europeus seeds (F. W. Schumacher). For hemagglutination inhibition

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SHARON

tests, serial dilutions of the inhibitor (50 ~1) in phosphate-buffered saline were incubated with 4 hemagglutinating units of the appropriate agglutinating reagent (50 ~1) at 37°C. After 30 min, 0.05 ml of a 2% erythrocyte suspension was added, and agglutination patterns were scored after 3 h at room temperature. One hemagglutinating unit is defined as the minimum concentration of agglutinating reagent giving complete agglutination in the absence of inhibitor. The secretor status of blood donors was determined by hemagglutination inhibition tests on boiled saliva specimens, by use of both U. europeus lectin and the appropriate blood-grouping antiserum. Protein was determined by the method of Lowry et al. (23) with crystalline bovine serum albumin (Sigma) as standard. Total hexose was determined by the phenol/sulfuric acid method (24) with Dgalactose as standard. For identification of component sugars, samples hydrolyzed for 4 h in 2 N HCl at 100°C were examined by paper chromatography in solvents A and B. Erythrocyte suspensions were analyzed for their sialic acid content by the method of Warren (25) after hydrolysis in 0.1 N HsSO, for 1 h at 80°C and precipitation of protein by the method of Tischer and Peters (26). After preparation of ghosts, the combined hemolysate and washings were lyophilized and similarly assayed for sialic acid. In calculating the number of antigenic sites per erythrocyte, 1 ml of erythrocytes, packed under conditions of cell volume determination, was taken to contain 11.5 x lo9 cells (27). All chemicals used were of the highest purity available commercially. RESULTS

Incubation of a 50% suspension of human B erythrocytes with purified coffee bean a-galactosidase, at a final enzyme concentration of 170 units/ml of incubation medium, led to a gradual decrease in their anti-B agglutination titer. After 2.5 h, undiluted antiserum failed to agglutinate the erythrocytes, as observed by macro- and microscopic inspection (Fig. 1). The agglutination titer against U. europeus extract increased Is-fold within 0.5 h of incubation, equalling the titer of type 0 erythrocytes, and did not. increase on further incubation. The time required for the disappearance of B activity varied inversely with enzyme concentration. Less than 20 min were required when the concentration was 400 units/ml of incubation medium or higher, while 9 h were required when 70 units/ml were employed.

B-ANTIGENIC

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absent at pH 7.0. Between pH 4.5 and 5.5, similar enzyme activities were observed whether the incubations were carried out in 0.1 M sodium acetate or in McIlvaine citrate/phosphate buffers. When ghosts from B erythrocytes were treated with a-galactosidase in the presence of various sugars, only D-galactose and methyl a-n-galactopyranoside inhibited the reaction (Fig. 4). As shown in

Tlmo (h)

I-,

1.5

2.5 Type 0 Erythrocyis 0

2

8 32 Aggluiinqtion

128 Titer

512

FIG. 1. Change with time of B and H activities of type B erythrocytes incubated with a-galactosidase. Intact B erythrocytes, packed volume 1.0 ml, were incubated with 240 unit8 of Cx-galacto8idaNe in 0.1 M citric acid/O.2 M NazPOa buffer, pH 5.0, containing 3% glycerol (by weight), in a final volume of 2.4 ml, at 37°C. Aliquots of 0.1 ml were taken at different times and suspended in 2.4 ml of phosphate-buffered saline. Aliquots of the resulting suspension (50 ~1) were tested for agglutination with equal volumes of serially-diluted anti-B antiserum and U. europeus anti-H lectin. Black bars designate B activity and white bar8 H activity.

When type AB erythrocytes were treated with a-galactosidase at a concentration of 100 units/ml, their B activity was lost within 6 h, while their A activity remained unchanged throughout the incubation period. Similarly, 0 erythrocytes, incubated under identical conditions, maintained the same anti-H titer as untreated control cells. The results of cy-galactosidase treatment of ghosts prepared from B erythrocytes and of extracts of such ghosts are shown in Fig. 2. At an enzyme concentration of 5 units/ml, the B activities of these fractions were destroyed within 36 h, as determined by hemagglutination inhibition test. The enzyme-treated ghosts and the aqueous extract showed enhanced H activity. However, in lipid fraction I, H activity was slight or altogether absent even after enzyme treatment. Lipid fraction II displayed neither B nor H activity. The pH dependence of cr-galactosidase activity on ghosts prepared from B erythrocytes is shown in Fig. 3. Activity was highest between pH 4.5 and 6.0, dropped sharply between pH 6.0 and 6.5, and was

B Ghosts

0 Ghosts

B Aqueous 6 LIpid

Phase Froct~on

I 0

2

8

32

0

Hcmagglut~nat~on

2

8

32

lnhnbition Titer

FIG. 2. Effect8 of cr-galactosidase on B and H activities of ghosts and of butanohwater ghost extracts. Ghosts, aqueous phase or lipid fraction I were prepared from 5.0 ml of packed B erythrocytes as described in the text and incubated with 50 units of a-galactosidase in 0.1 M sodium acetate buffer, pH 5.0, in a final volume of 10 ml, at 37°C for 36 h. Enzyme was omitted from controls. Hemagglutination inhibition tests were performed as described in the text. Black bar8 designate B activity and white bar8 H activity. II! 4.5 5.0 5.5 6.0 6.5 70 Enzyme

Absent 2

0

32

Hemogglutmation

128 lnhibitlon

Titer

FIG. 3. Effect of pH on destruction of B activity in ghosts by a-galactosidase. Ghosts from type B erythrocytes, 25 pg of protein, were incubated at 37°C with 0.7 unit8 of a-galactosidase in 0.1 M citric acid/O.2 M Na2P0, buffer, of different pH values, in a final volume of 75 ~1. After 24 h, 25 ~1 of isotonic sodium phosphate buffer, pH 7.4, was added and serial dilution8 of the mixture tested for hemagglutination inhibition as described in the text.

680

HARPAZ,

FLOWERS

agar Added NOW

FIG. 4. Effects of various sugars on destruction of B activity in ghosts by cx-galactosidase. Ghosts from type B erythrocytes, containing 25 pg of prctein, were incubated at 37°C with 2 units of a-galactosidase and 10 pmol of the saccharide to be tested in 0.13 M sodium acetate buffer, pH 5.0, in a final volume of 75 ~1. After 24 h, 25 ~1 of isotonic sodium phosphate buffer, pH 7.4, was added and serial dilutions of the mixture tested for hemagglutination inhibition as described in the text.

?:+“I .

> I

0

2

8

32

I

12s 0

Hemagglutinatmn

2

Inhibitkm

8

32

128

I

Titer

FIG. 5. B and H activities of ghosts and butanobwater extracts derived from a-galactosidasetreated erythrocytes. Type B erythrocytes, packed volume 5.0 ml, were treated with 500 units of (Ygalactosidase for 9 h, as described in the text. The resulting cells were not agglutinated by anti-B antiserum. Ghosts were prepared from them and from erythrocytes incubated in parallel but without enzyme, and one-half of each of the resulting ghost preparations was extracted with butanobwater. Ghosts and ghost extracts were brought to 10 ml with phosphate-buffered saline and hemagglutination inhibition tests performed as described in the text. Black bars designate B activity and white bars H activity.

Fig. 5, ghosts and butanollwater extracts prepared from B erythrocytes whose B activity had been abolished with cr-galactosidase showed no residual B activity and, except for the lipid fractions, showed enhanced H activity.

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SHARON

Paper chromatographic analysis of the low molecular weight products released from B erythrocytes, ghosts and butanol/water extracts upon enzyme treatment showed that galactose was the major sugar released. Traces of other monosaccharides were often observed, in equal quantities, both in enzyme-treated samples and in parallel incubation controls, but the amounts of these products were less than 10% of the total silver nitratestaining material as estimated visually. The quantities of galactose released from erythrocytes and ghosts of the various ABO phenotypes, in terms of galactose molecules released per cell, are given in Table I. Corrections for losses of galactose during its isolation and for nonspecific release of galactose in incubation controls were applied as described in Materials and Methods. The average recovery of [3H]galactose was about 90%. Galactose in incubation controls was routinely below the assay sensitivity (0.02 pg/ml of packed erythrocytes), but occasionally higher values (up to 1.0 pglml) were obtained, particularly when samples were incubated for 57 days. The quantity of galactose liberated from ghosts and buerythrocytes, tanol/water extracts is tabulated in Table II. The distribution of a-galactosidase-labile residues among the various fractions was determined separately for each of the blood donors. The galactose released enzymatically from B ghosts represented about 4% of their total galactose content, as determined by assay of their acid hydrolysates, and represented less than 1% of TABLE

I

CT-GALACTOSE LIBERATED PER ERYTHROCVTE GHOST BY LX-GALACTOSIDASE”

Specimen Type Type Type Type Type

B erythocytes B ghosts AB ghosts 0 ghosts A ghosts

OR

MoleculeslcelP (X 10-G) 3.0 2.8 1.9 0.8

+ 0.3 + 0.6 + 0.8 f 0.3 0.4 (1)

(3) (6) (2) (31

D For experimental details, see text. b Results are expressed as median values * standard deviations. The number of donors is given in parentheses.

B-ANTIGENIC

SITES OF HUMAN TABLE

DGALACTOSE

Sample

LIBERATED

Erythrocytes Ghosts Aqueous phase Lipid fraction I Lipid fraction II

II

FROM CELLS AND CELL FRACTIONS

10.4*0.9(3) 9.5’?1.9(6) 7.5?2.3(4) 1.6?0.3(4) 0.8?0.1(2)

BY TREATMENT

WITH

WGALACTOSIDASE”

Type 0

Type AB

Type Bb r+Galactose liberated’ w

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ERYTHROCYTES

Percentd

101*7 100 77%10 19k-3 7+-l

DGalactose liberated’ (PP) n.t.’ 6.4’t3.1(2) 6.30) 1.0(l) n.t.

Percentd

nt. 100 73 12 nt.

n-Galactose liberated’ (M)

Percentd

nt. 2.7*1.5(3) 0.2?0.3(2) 1.520.2(2) 1.7?0.5(21

n.t. 100 8?10 48%6 5321

a For experimental details, see text. All blood donors were of the secretor phenotype except where noted otherwise. b Includes one individual of nonsecretor phenotype. Values of ngalactose released from fractions of this individual were: Erythrocytes, 11.1; ghosts, 11.0; aqueous phase, 8.6; lipid fraction I, 1.7; and lipid fraction II, 0.7 fig/ml. (- Results are normalized to 1.0 ml of packed erythrocytes from which the fractions were prepared and are expressed as median values + standard deviations. The number of donors is given in parentheses. d n-Galactose liberated from cell or cell fraction as percentage of ngalactose liberated from ghosts prepared from the same blood donor. Results are expressed as median percentage values 2 standard deviations. The number of donors is the same as the number given in the previous column. ’ nt., Not tested. ’ Includes one individual whose secretor phenotype was not determined.

their total hexose, as determined by the phenol/sulfuric acid method. When butanol/water extracts from 5 ml of erythrocytes whose B activity had been abolished by enzyme treatment (500 units, 9 h) were treated again with a-galactosidase (5 units/ml, 72 h), no further gala&se was released. Comparison of the sialic acid content of erythrocytes and of hemolysates and washes separated from ghost suspensions showed that the pooled supernatant fluids contained less than 0.3% of the sialic acid present in the cells, i.e., negligible amounts of membranous material were lost during preparation of the ghosts. In performing the butanol/water extractions of ghosts, 87-90% of the ghost protein was routinely recovered in the aqueous phase, and little or no interphase material was obtained if the original ghost suspension was made mildly alkaline (pH 8-9). Butanollwater extracts of ghosts derived from a type B nonsecretor individual showed anti-B hemagglutination inhibition titers similar to those of secretors. CYGalactosidase treatment of erythrocytes, ghosts and butanol/water extracts from this individual abolished their B activities, and, as shown in Table II, the quanti-

ties and distribution of liberated gala&se were similar to those obtained for secretors. DISCUSSION

We describe in this paper the enzymatic modification of the B specificity of intact human erythrocytes, of isolated erythroand of butanol/water cyte membranes, membrane extracts, by highly purified coffee bean a-galactosidase. Losses of B activity were accompanied by the liberation of galactose and the development of H specificity. Enzyme-treated lipid fractions, however, exhibited little or no appearance of H activity despite complete loss of B specificity. Since it is known that the H activity of glycolipids extracted from type 0 erythrocytes is inhibited by contaminant lipids (20), similar inhibition might well have occurred in the case of cw-galatosidasetreated B lipids. The conversion of B specificity to H specificity by the cleavage of the a-n-galactosyl moiety has been observed in the past with human secreted blood-group glycoproteins (10, 11) and erythrocytes (12, 13) and is in accordance with the immunochemical relationship defined for the B and H antigens. The time required for the loss of B speci-

682

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FLOWERS

ficity of intact erythrocytes varied inversely with the a-galactosidase concentration and was reduced to several minutes by employing concentrations as high as 400 units/ml (about 4 mg/ml) of the enzyme. Full development of H activity occurred well before the loss of B activity, presumably because a relatively small number of H sites are sufficient for a strong agglutination reaction with anti-H reagent to take place. a-Galactosidase treatment of B erythrocytes and erythrocyte ghosts released 3.0 x 10” and 2.8 x lo6 galactose molecules per cell, respectively, while ghosts from 0 erythrocytes yielded 0.8 x lo6 galactose molecules per cell (Table I). Ghosts from AB erythrocytes yielded 1.9 x lo6 molecules of gala&se per cell, intermediate between the values for B and for 0 cells and in agreement with the theory that the synthesis of A and B antigens in AB erythrocytes involves competition for the same precursor molecules. Assuming that the presence of a-n-galactosyl moieties in 0 cells is independent of ABO phenotype, a similar number of sites might exist in B cells in structures other than those that are B antigenic. Hence, correcting for these sites, the number of B-antigenic sites per B cell is about 2.2 x 106. Fujisawa et al. (12) treated B erythrocytes with a crude, B-decomposing enzyme preparation from Clostridium maebashi and reported 14 x lo6 molecules of galactose liberated per cell. Since this enzyme preparation also liberated large quantities of other carbohydrates, including oligosaccharides, a large proportion of the galactose may not necessarily have originated solely from B antigens. Economidou et al. (28) used lz51-labeled anti-B antibody to estimate the number of antigenic sites per erythrocyte and obtained a value of about 0.75 x 106, which is much lower than ours. Their assumption that each antibody molecule binds one rather than two gala&se residues might not be correct. Moreover, possible interference in the binding of antibody molecules to adjacent antigen sites was overlooked. Schenkel-Brunner and Tuppy (29) transferred enzymatically N[3Hlacetylgalactosamine from its UDP de-

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SHARON

rivative to 0 and to B erythrocytes, and the difference in incorporation of lable to the two cell types corresponded to 2.1-2.5 x lo6 molecules/cell, in agreement with our results. Examination of the distribution of agalactosidase-labile residues among the butanol/water membrane extracts (Table II) showed that with B and AB erythrocytes, about three-fourths of these residues were in the aqueous fraction and most of the remainder in lipid fraction I. The distribution among the fractions from 0 erythrocytes was markedly different, with nearly all the enzymically labile galactose being present in equal quantities in lipid fractions I and II and only a small percentage in the material found in the aqueous phase. Since the quantities of gala&se liberated from the butanol fractions of B, AB and 0 cells were similar, we conclude that the overwhelming proportion of Bantigen sites are associated with the bloodgroup substances of the aqueous phase. This conclusion is in agreement with the observation that only extremely small quantities of blood-group glycolipids can be isolated from erythrocyte membranes (4). The presence of a-n-galactosyl moieties in erythrocyte membrane lipids which are not B-specific has been previously reported. A trihexosyl ceramide with the structure a-n-galactosyl-(1+4)-Pn-galactosyl-( 1-*4)+glucosyl ceramide, which accumulates in tissues of individuals with Fabry’s syndrome, is normally present in erythrocytes in quantities of 15 pglg of wet cells (30). Coffee bean a-galactosidase readily hydrolyzes cr-n-galactosyl-(1+4)-n-galactose (31), and the galactose enzymically released from the above trihexosyl ceramide could account for the amount of galactose that we found to be liberated from A and 0 cells. The antigenic determinant of P1 specificity occurs in human erythrocyte glycosphingolipid extracts (32) and apparently also contains a terminal a-n-galactosyl residue, since treatment of cross-reacting P, glycoprotein from sheep hydatid cyst fluid with a-galactosidases abolishes its antigenic activity and liberates n-galactose (33, 34).

B-ANTIGENIC

SITES

OF HUMAN

Assuming that cY-galactosidase may modify only molecules that protrude from the outer membrane surface of intact erythrocytes, our observation that the extracts obtained from enzyme-treated cells contained no residual B specificity nor ogalactosidase-labile residues suggests that all the B-antigen sites are oriented towards the extracellular medium. Nevertheless,. %ryptic” a-n-galactosyl sites in quantities below 0.02 pg/ml of packed erythrocytes (6 x lo4 per erythrocyte) might not have been detected with our assay, and verification of the absence of such sites would require more sensitive techniques, such as radioimmunoassay. Finally, the B activity observed in the aqueous extract derived from erythrocytes of a type B nonsecretor and the normal quantity of galactose released from this material by a-galactosidase corroborate recent evidence on the similarity of secretors and nonsecretors with respect to the presence of B-active material in the aqueous phases prepared from their erythrocyte membranes (35). REFERENCES 1. WATKINS, W. M. (1972) in Glycoproteins: Their Composition, Structure and Function (Gottschalk, A., ed.), 2nd ed., pp. 830-891, Elsevier, Amsterdam. 2. GINSBURG, V. (1972) Aduan. Ensymol. 36, 131147. 3. HAKAMORI, S. I., AND KOBATA, A. (1974) in The Antigens (Sela, M., ed.), Vol. 2, pp. 79-140, Academic Press, New York. 4. WHITTEMORE, N. B., TRABOLD, N. C., REED, C. F., AND WEED, R. I. (1969) VOX Sang. 17,289299. 5. HAMAGUCHI, H., AND CLEVE, H. (1972)Biochim. Biophys. Acta 278, 271-280. 6. MARCHESI, V. T., TILLACK, T. W., JACKSON, R. L., SEGREST, J. P., AND Scow, R. E. (1972) Proc. Nat. Acad. Sci. USA 69, 1445-1449. 7. FUKUDA, M. AND OSAWA, T. (1972) J. Biol. Chem. 248, 5100-5105. 8. GARDAS, A., AND KOSCIELAK, J. (1973) Eur. J. Biochem. 32, 178-187. 9. BRENNESSEL, B. A., AND GOLDSTEIN, J. (1974) VOX Sang. 26,405-414. 10. WATKINS, W. M. (1956) Biochem. J. 64, 21P22P. 11. ZARNITZ, M. L., AND KABAT, E. A. (1960) J. Amer. Chem. Sot. 82, 3953-3957. 12. FUJISAWA, K., FURUKAWA, K., AND ISEKI, S.

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