Bloodgroup a specific glycolipids from human erythrocytes

BiOCHIMICA ET BIOPHYSICA ACTA

313

BBA 4214

BLOODGROUP A SPECIFIC GLYCOLIPIDS FROM HUMAN ERYTHROCYTES j. KO~CIELAK*

Lister Institute of Preventive Medicine, London (Great Britain) and Institute of Haematology, Warsaw (Poland) (Received April 5th, 1963)

SUMMARY

The isolation of two bloodgroup A specific glycolipids from human erythrocytes is described. These glycolipids appear to be homogeneous; both contain residues of galactosamine, glucosamine, galactose, glucose, sialic acid, fucose and sphingosine, but they differ in the composition of their fatty acid moieties. One glycolipid is insoluble in cold methanol and contains lignoceric acid residues, whereas the other is soluble in methanol and contains a high proportion of lower fatty acid residues. The materials are equally active in quantitative precipitation tests. The activity is similar to that of the A substance obtained from ovarian cyst fluid. The haemagglutination-inhibiting activity of the material insoluble in cold methanol is more than ioo times that of the glycolipid which is soluble in methanol, and the haemagglutination-inhibiting activity of the latter material is increased to that of the former substance by combining it with serologicaUy inactive lipid. The activity of the methanol-insoluble glycolipid is decreased to that of the methanol-soluble glycolipid by mixing the two glycolipids. Essentially similar results were obtained when the "Forssman" activity of the two giycolipids was studied by haemolysis inhibition tests. These results and those obtained from examination of the two glycolipids in the ultracentrifuge, suggest that the serological activity of bloodgroup substances from red cells depends largely upon their state of aggregation in aqueous solution.

INTRODUCTION

Since the discovery of the ABO bloodgroup system by LANDSTEINER1, 8, the chemical nature of the material responsible for the bloodgroup activity of the red cell has remained a puzzling problem and many conflicting reports on this subject have appeared. Early work demonstrated that bloodgroup active material could be extracted from red cells with ethanol and it was suggested that the material was lipid in nature 3. The occurrence of bloodgroup substances in a water-soluble form in secretions was k n o w # and the direction of research changed to a study of these materials largely because they were present in the secretions in relatively large amounts. These sub* Present address: Institute of Haematology, U1. Chocimska 5, Warsaw (Poland).

Biochim. Biophys. Acta, 78 (I963) 313-328

314

J. KOSCIELAK

stances were obtained in a highly active and homogeneous form and were shown to be mucopolysaccharides (see refs. 7, 15). On the basis of indirect evidence, it was assumed that the conclusions reached from studies on water-soluble specific substances would also apply to the bloodgroup substances on the red-cell surface and that the latter substances would be mtlcopolysaccharide in nature 7. Some works appeared to confirm this assumption 4, 5,s, but othersg-ll, lS,14 isolated from human red cells a bloodgroup-active substance of glycolipid nature to which a lignoceryl sphingosinegalactosamine trihexoside structure was given 9,1°. Later however, it was reported that this substance contained only a small amount of the active component 12. The bloodgroup activity of the glycolipid-containing material was lower than that of the substances from secretions. Subsequently, it was reported is that the bloodgroup-active glycolipid from red cells was only slightly active in the haemagglutination inhibition test, when freed from major contamination by other lipids co-existing with the preparation and that the activity of the glycolipid could be substantially increased by combining it with a serologically inert carrier lipid. The aim of the present paper was to study this phenomenon using purified bloodgroup-active glycolipid fractions. MATERI:'~LS AND METHODS

Blood Human red cells were obtained as packed, sterile sediments from the M.R.C. Blood Products Unit.

Isolation of stroma Packed cells were haemolysed, as soon as possible after being received, with about 8 volumes of water to which small pieces of solid CO 2 and a few ml of capryl alcohol were added. The haemolysate was allowed to stand for I h, was then centrifuged by passing through a Sharpies centrifuge (22 00o rev./min) and the stroma was collected. About 3 g of freeze-dried stroma was obtained from IO0 ml of packed red cells.

Chemical analyses Total N was estimated colorimetrically, using Nessler reagent, by the micromethod of JOHNSON17. Absorbancy was read at 400 m/, in a Unicam spectrophotometer. Sphingosine N : The method of McKIBBIN ANt) TAYLOR18 was employed. Amino sugars: A modification (PALMER et al. 19) of the method of ELSON AND MORGAN z° w a s used with glucosamine as a standard. The results are expressed as free base. Materials were hydrolyzed in 0. 5 N HC1 in sealed glass ampoules in a boiling water bath for 18 h, cooled, neutralized with an equal volume of 0.5 N NaOH and made up to the required volume. This hydrolysate was also used for reducing-sugar estimations. Reducing sugar was determined by the method of NELSON zl, using galactose as a reference standard. Fucose: GIBBONS'22 modification of the procedure of DISCHE AND SHETTLES2a was employed.

Biochim. Biophys. Acta, 78 (1963) 313-328

A SPECIFIC GLYCOLIPIDS

315

Sialic acid: This was estimated b y means of Bial's reagent. I ml of an aqueous solution of the substance was heated with I ml Bial's reagent in a boiling-water b a t h for 15 min. The solution was then extracted with 4 ml of redistilled isoamyl alcohol and the absorbancy was read at 570 m/~. A mucopolysaccharide (No. 350) obtained from the fluid of an ovarian cyst (PoszTAI AND MORGAN~4) which contained 18 % of sialic acid was included as a standard in each set of estimations. Hexose: The phenol-H2SO4 method of DUBOlS et al. 25 was employed. Serological assays

Estimations of the A and B haemagglutination activity of red-cell substances were performed with natural human sera according to MORGAN AND KING~s. The A activity was also tested with rabbit immune anti-A serum and with certain anti-A plant agglutinins 27. Tests for H activity were made by the method of ANNISON AND MORGAN28 using immune rabbit anti-H serum and anti-H reagents of plant origin2L Inhibition of haemolysis of sheep cells by rabbit immune anti-A serum was studied by the technique of MORGAN AND KING~s. Quantitative precipitation tests with rabbit immune anti-A serum were performed according to HEIDELBERGER AND MCPHERSON 29 as modified b y KABAT AND BEZER 3°.

Laboratory standards of purified bloodgroup substances obtained from ovarian cyst fluids were included in each serological determination. EXPERIMENTAL AND RESULTS

Isolation procedure

The method employed involved the following steps: I. Extraction of stroma with ethanol and the recovery of active material precipitated on cooling the extract. 2. Removal of inactive material from the precipitate by extraction with organic solvents. 3. Division of the active material into fractions, based upon their solubility in methanol. 4. Re-fractionation on cellulose columns. 5- Further fractionation on silicic acid columns. 6. Division of the most active subfraction into two distinct glycolipids by utilizing their different solubilities in cold methanol. I. Extraction o f stroma with ethanol

Ethanol was added slowly to the wet stroma paste immediately after collection from the Sharpies' centrifuge. The mixture was vigorously stirred until a final ethanol concentration of 80-85 % (v/v) was reached. The resulting suspension was left for 2-3 days at room temperature, with occasional shaking, and was then filtered. The clear yellow filtrate was cooled to about - - i o °, and the white precipitate which separated, was allowed to settle for 2-3 days at this temperature. The clear supernatant fluid was then removed by decantation. The lower layer, containing the precipitate, was centrifuged at about --IO °, and, the resulting deposit was washed with cold 83 % ethanol and then with acetone. I t was finally dried in vacuo. The yield of this Biochim. Biophys. Acta, 7 8 (1963) 313-328

316

j. KO~CIELAK

"crude" bloodgroup substance which was readily soluble in water, was about o.I % of the dry weight of the stroma extracted. The preparations obtained from each separate batch were pooled, dried and analyzed for reducing sugar, hexosamine and haemagglutination inhibition activity. Yield, 9-7 g from 7 ° 1 of red cells. Reducing sugar I6.O%, hexosamine 5.o%, haemagglutination inhibition activity 6 % , expressed in terms of percentage activity of the laboratory standard A substance obtained from ovarian cyst fluid. 2. Extraction of the "crude substance" with organic solvents The substance (9.6 g) was extracted with acetone for 5 h in a Soxhlet extractor. The acetone-soluble material (1.o4 g) was inactive and was rejected. The acetoneinsoluble residue (8.46 g) was further extracted for 2 h with diethyl ether; 0.78 g of material was recovered from the ether. The material contained 3.8 % of hexosamine, 12.5 % of hexose. Its haemagglutination inhibition activity was 0.4 % of the standard. The material was not studied further. The acetone and ether-insoluble material (7.4 g) was then washed 3 times with portions (IOO ml each) of light petroleum (b.p. 40-60 °) and the soluble material (0.5 g, containing hexosamine 2.I%, hexose 8.1%, haemagglutination inhibition activity, 0.2 %) was discarded. 3. Division of the active material into fractions, based upon their solubility in methanol The residue remaining after extraction with acetone, ether and light petroleum (6.6 g) was extracted with boiling methanol (27 ° ml) and the solution was filtered while hot. The residue which was insoluble in hot methanol (0.47 g, haemagglutination inhibition activity 1%) was discarded. The clear, yellow filtrate was cooled to 4 °, and after 24 h the precipitate which formed was recovered and designated material "M" (yield 4-7 g, hexosamine 6.1%; reducing sugar 2o.5%; haemagglutination inhibition activity 20 % of the A standard). An additional amount of material was recovered from the methanolic solution by cooling it to --8 °. This material (0.41 g, hexosamine 1.4%, hexose 6.2%) has less activity (5 % of the standard substance) than the main fraction. Evaporation in vacuo of the methanolic supernatant solution to dryness yielded a further 0.59 g of material (hexosamine 0.48 %, hexose 2.I%, haemagglutination inhibition activity less than o.oi % of the standard A substance). The last two fractions were, however, not purified further. 4. Fractionation of the material on cellulose columns Cellulose powder (Whatman standard grade) was suspended in the lower layer of a chloroform-ethanol-water (16:4:1, v/v) solvent mixture and poured, in small portions at a time, into a column of 96 × 3.2 cm, and allowed to settle for I day. The material "M", I g at a concentration of IO % (w/v) in the same solvent mixture, was applied to the column and was eluted with the same solvent until the effluent gave no trace of opalescence on the addition of acetone (5 volumes). The effluent, containing acetone-precipitable material, was subsequently evaporated in vacuo at 3 o°. This material is referred to as Fraction CI. The column was then eluted with c h l o r o f o r m - m e t h a n o l - w a t e r solvent mixture (5:15:1, v/v) and the effluent which contained material precipitable b y acetone was evaporated to dryness. This material is referred to as Fraction CII. Biochim. Biophys. Acta, 78 (1963) 313-328

A SPECIFIC GLYCOLIPIDS

317

About 88 % (average value) of material "M" was recovered in Fractions CI

and ClI, the individual yields being about 70 % and 18 %, respectively. The corresponding fractions collected in separate runs were pooled and analyzed. Fraction CI : hexosamine 2.7 %, reducing sugar I6.O%, haemagglutination inhibition ~.ctivity not significant. Fraction ClI: hexosamine 12.8 %, reducing sugar 46.o%, haemaggiutination inhibition activity 1 % of the standard. The results of quantitative precipitation experiments are given in Fig. 2. A definite decrease of haemagglutination inhibition activity was observed for both substances obtained after separation of material "M" on the cellulose columns. Fraction CI exhibited no capacity to inhibit in haemagglutination tests and substance Fraction CII no more than 5 % of the activity of the original material. The activity of Fraction CI, measured by the quantitative precipitation test was negligible. Substance Fraction ClI, however, was about 4 times more active than the starting material. Earlier work 16 showed that the whole of the bloodgroup activity resides in Fraction cII, whereas material Fraction CI, has so-called "carrier" properties. The two fractions form a complex in chloroform-methanol CI :I, v/v) mixture and, after combination, these materials develop a haemagglutination inhibition activity similar to that of the original. 5. Chromatography on silicic acid columns

Fraction ClI was further fractionated on columns of silicic acid (Mallinckrodt) which had been activated by heating at IiO ° for I h. The proportion of the adsorbent to the material to be separated was in all experiments IOO:i (w/w). The columns which were employed had a length/width ratio of 6:1. Silicic acid was mixed with chloroform to form a thin slurry, poured into the column and allowed to settle for I day. Fraction ClI, which was insoluble in chloroform, was dissolved in the minimum volume of warm pyridine, diluted with lO-2O volumes of chloroform and applied to the top of the column. The sides of the column were then washed down twice with chloroform, the washings allowed to pass into the column filling and the column was eluted successively with chloroform, with methanol-chloroform mixtures containing successively 2o, 4 o, 60, 80 % methanol, and finally with pure methanol. The progress of the fractionation was followed by evaporating suitable aliquots (0.050.3 ml) to dryness at 6o °, dissolving the residues obtained in i ml of water and analyzing each for hexose by the phenol-H~S04 test. The results are presented in Table I, and Figs. I and 2. The highest activity, as measured by quantitative precipitation tests, was exhibited b y Fractions SIV and SV, whereas the main glycolipid peak, SIII, was almost inactive. Fractions SV and SVI were less active than Fraction SIV. When the activities were measured by the haemagglutination inhibition test, fractions SV and SVI were found to be more active than SIV. The recombination TM of these fractions (SIV, SV, SVI) with the carrier material gave preparations, the activities of which were closely similar, and it was concluded that Fractions SV and SVI still contained some substance which functioned as a carrier material. The small yields of Fractions SV and SVI did not permit any detailed study of the materials and further investigation was limited to Fraction SIV. Fraction SIV (167 mg) was obtained from 740 mg of Fraction CII. Biochim. Biophys. Acta, 78 (I963) 313-328

318

I. KOSCIELAK TABLE I PROPERTIES

OF

FRACTIONS

Fraction I

W e i g h t % of t h e original Fraction C II

FROM

Fraction II

A SILICIC

ACID

Fraction [ l i

COLUMN

Fraction IV

Fraction V

Fraclion V1

3.6

4 .8

.55.5

23.0

0.2

I. 7

~C 0. 3

1.7

l i.,~

14,8

13. 4

8. 4

R e d u c i n g s u g a r (%)

5.o

9.o

5o.o

51.9

38.2

26.6

tlaemagglutination inhibition a c t i v i t y as % of the s t a n d a r d A substance

o

o. 3

o i

o. 5

5.o

5.o

Haemagglutination inhibition a c t i v i t y of fractions c o m b i n e d w i t h 3 p a r t s (w/w) of t h e carrier lipid*

o

0. 3

io.o

8o.o

80.0

80.0

H e x o s a m i n e (%)

* % of t h e a c t i v i t y of the s t a n d a r d A s u b s t a n c e as c a l c u l a t e d for t h e p u r e f r a c t i o n p r e s e n t in the m i x t u r e .

i3°I ~2o E

/

i 10

%

45O 9OO Solvent (ml)

Fig. 1. F r a c t i o n a t i o n of C I I A m a t e r i a l on a silicic acid column. Arrows i n d i c a t e s o l v e n t c h a n g e . O r d i n a t e : lO, 2o, 3 ° m g of hexose per fraction. Abscissa: 45o, 9oo ml of s ol ve nt .

-~ 5 0

I

o~-

5'0

,;o

,;o

2;0

Added antigen (~g)

Fig. 2. Precipitation curves of various bloodgroup-active fractions from red ceils and of the standard A substance. O - - 0 , standard A substance; O - - r n , Fraction SIV; O - - e , Fraction SV; I - - I I , F r a c t i o n C I I ; /~--LX, F r a c t i o n SVI; A - - A , m e t h a n o l i c p r e c i p i t a t e . O r d i n a t e : 25, 50, 75 tzg of a n t i b o d y N p r e c i p i t a t e d . Abscissa: 5o, IOO, 15o, 200/ *g of a n t i g e n a dde d.

Biochim. Biophys. Acta, 78 (1963) 313-3x8

A SPECIFIC GLYCOLIPIDS

319

6. Fractionation of material SIV by methanol The chromatographic behaviour (Fig. I) of the Fraction SIV indicated the presence of more than one component which, however, might have arisen from "trailing". It was therefore decided to investigate the homogeneity of the Fraction SIV by fractional solubilitv tests, as applied earlier. A solution (6.0 mg/ml) in methanol of Fraction SIV was prepared at room temperature and a series of the appropriate dilutions, contained in tightly stoppered centrifuge tubes, were cooled to o °. After 24 h the precipitates formed were removed by centrifuging at o °, and determinations were made on the supernatant methanol solutions for hexosamine, hexose and serological activity, as measured by quantitative precipitation tests. After the basic shape of the curve was established, the additional estimations in the region of the point of inflection on the curve were made. The results are given in Fig, 3. 1.st A

=~ ,.o

OV

I

0

0.5

L

I

1

1.0

1.5

2.0

215

Hexose (mg/ml sample)

=as1 B g "--0

E ¢~0.3

°~a2 o.1 o

, 0.2

, OA

, (16

Hexosomine (rag/m!

~.oo

,.0 8

sample)

C

OC c~

2.0

~ ,.o 1~)

2~)

3D

4~)

5.0

5ubstance (mg/ml sample) Fig. 3. Fractional solubility test of Fraction SIV in methanol. For explanation see text. A: As m e a s u r e d b y hexose estimation. Ordinate: 0. 5, i.o, i. 5 mg of hexose per ml of s u p e r n a t a n t . Abscissa: o.5, i.o, 1.5, 2.0, 2.5 m g of hexose per ml of sample. B: As measured b y hexosamine estimation. Ordinate: o.i, 0.2, o.3, 0.4, 0.5 mg of hexosamine per ml of s u p e r n a t a n t . Abscissa: o.2, o.4, o.6, 0.8 m g of hexosamine per ml of sample. C: As measured by the estimation of antib o d y N precipitated. Ordinate: i.o, 2.0, 3.0 mg of substance per ml of s u p e r n a t a n t as estimated from the precipitation curve of Fraction SiV. Abscissa: i.o, 2.0, 3.0, 4.0, 5.0 mg of substance per ml of sample.

Biochim. Biophys. Acta, 78 (1963) 313-328

320

j. KO~CIELAK

It is evident that Fraction SIV contains at least two components, both of which have the same ratios of activity to hexosamine and hexose. Unfortunately, the saturation point of the second component, or components, could not be reached because of its high solubility in cold methanol. However, from the shape of the curve, it was believed that the less soluble component precipitating beyond the concentration of I.I mg/ml was comparatively homogeneous. Therefore, it was decided to separate these components by cold methanol-fractionation based upon their solubility properties, as obtained from the solubility curve. A portion of Fraction SIV (IOl.8 rag) was dissolved in methanol (18 ml) at room temperature, and the solution was left at o ° for 24 h. The precipitate which formed was collected by centrifugation at o °, and was washed three times with cold methanol. The washings were added to the main methanol supernatant fluid and the soluble material was recovered; it was dissolved in 8 ml of methanol and the solubility test was repeated. The precipitate obtained at o ° was added to that recovered from the first experiment. The material remaining soluble in methanol was recovered, dissolved again in methanol (6 ml) and the precipitate which formed at o ° was washed with cold methanol and the washings discarded. The material soluble in methanol (36 nag) and that less soluble and precipitated (45 rag) were freed from methanol and dialyzed separately against distilled water. These materials were subsequently examined in the ultracentrifuge and were found to give single peaks. The substances were also studied by means of thin-plate chromatography 32, using silicic acid-CaS04 as the adsorbent and chloroform-methanol-water (4:6:0.4, v/v), as the solvent system. The materials migrated at about the same rate and were free from major contaminants. Traces of Fractions SIII and SV were, however, still present. Spot tests for phosphorus on the two materials were negative 31.

Properties of the bloodgroup active materials (a) insoluble, and (b) soluble in methanol Gas chrornatograpt~v of fatty acids: The materials were dissolved in anhydrous methanol containing 2 N HC1 and heated in sealed glass ampoules at I00 ° for I0 h. The fatty acid esters formed, were recovered 33 and were chromatographed on columns of 2 % Apiezon L on 80-100 mesh celite at 200 °, using argon as the mobile phase (see Table II). The retention volumes of individual peaks were calculated relative to that of palmitic acid and plotted against the number of C atoms. A straight line was obtained which thus made possible the identification of all saturated acids present. In the case of the methanol-soluble material, additional runs were made on the column loaded with 15 % ethyleneglycolisophthalate on 8O-lOO mesh eelite at 19°0 to identify the unsaturated fatty acids by the method of JAMES31. The material insoluble in methanol contained virtually only one fatty acid, i.e. lignoceric acid, whereas the methanol-soluble material contained a range of fatty acids having between 16 and 24 C atoms. The presence of one fatty acid in the material insoluble in methanol can be regarded as additional evidence of its homogeneity. Sphingosine: Sphingosine was identified in the hydrolysis products of both materials by paper chromatography in butanol-acetic acid-water (4:I:5, v/v) solvent. An authentic sample of sphingosine was included in the runs. The quantitative determination of sphingosine 18 provided further evidence that this substance is a Biochim. Biophys. ,4eta, 78 (1963) 313-328

A

SPECIFIC

321

GLYCOLIPIDS

constituent of each bloodgroup-active material. Sphingosine is the only base in the materials which can be extracted from acid solution by chloroformTM. Sugars: The identification of individual sugars was achieved by means of descending paper chromatography in three separate solvent systems, (a' pyridineethyl acetate-water (4: IO:3, v/v), (b) p y r i d i n e - b u t a n o l - w a t e r (9:6:3, v/v,) and (c) butanol-acetic acid-water (4:1:5, v/v). The presence of galactose, glucose, galactosamine, glucosamine and a small amount of fucose was established in both bloodgroup-active materials. The ratio of the two hexosamines was determined by GARDELL'Sa5 procedure, and the galactose/glucose ratio by paper chronnatography in pyridine-ethyl acetatewater (4:1o:?~ v/v). .: ~ The results of the analysis of the materials which were soluble or insoluble respectively i~ met1',anol under the conditions described, are given in Table III, and lead to the c{mdusion that they are both giycolipids and are similar in chemical compositicn. The differe:2ce in solubility of these materials can be attributed to the TABLE

~I

FA'r'rY ACID COMPOSII'ION OF BLOODGROUP GLYCOLIPIDS R e s u l t s a r e e x p r e s s e d a s a p e r c e n t a g e of t h e t o t a l f a t t y a c i d c o n t e n t . NumbcT qf C atom~ present( --: indicates a do~¢l}l~ ;,O~;dl

MctJmnvl*LzPoluble fraction

Methanol-soluble fraction

,c, 18 :,, 2{',

o o o o

19. 7 lO. 7 2. 3 3.8

21 ~

O

0.6

22 ,2-23 2,~= 24

8.1 o z-4 o 89.5

22.9 I.I 2.3 7.8 26.8 2.o not identified

TABLE ANALYSIS

OF

THE

III

BLOODGROUP

ACTIVE

GLYCOLIPIDS

Solubility of glycolipid in methanol

Hexosamine (%) Reducing sugar (%) Sialic acid (%) Fucose (%)

Insoluble

Soluble

15.8 43.0 lO.4 1.2

12.1 41.4 IO.9 2.3

N (%)

2.46

2.48

Sphingosine N (%) Glucosamine[ galactosamine ratio Galactose/glucose ratio

o.81

o.91

3.o : I 3-I : i

3. I : I

2.8: i

Biochim. Biophys. Acre, 78 ( I 9 6 3 ) 3 1 3 - 3 2 8

322

j. KOSCIELAK

differences in their fatty acid content. In the case of the glycolipid insoluble in methanol, the sum of the hexosamine, sialic acid and sphingosine N accounts for the total N content of the substance, whereas about 93 % of N could be accounted for in the methanol-soluble glycolipid. Neither fraction contains any amino acids detectable by paper chromatography in p r o p a n o l - w a t e r (8:2, v/v) solvent of the 6-N hydrolysates of the glycolipids. Immunological: The activities of the glyeolipids which are soluble and insoluble respectively in methanol are similar, when measured by quantitative serological precipitation (Fig. 4). There is, however, a striking difference in the capacity of each material to inhibit the haemagglutination of red cells by various anti-A reagents, and also in the capacity of each to inhibit the haemolysis of sheep red cells (Table IV). The activity of methanol-insoluble glycolipid approached that of the laboratory standard A substance which was obtained from ovarian cyst fluid. This result was somewhat surprising as both the methanol-insoluble and methanol-soluble glycolipids, when present in the mixture (Fraction SIV, Table IV), exhibited considerably less activity when tested with human anti-A serum. Moreover, on mixing these fractions together in an organic solvent (chloroform-methanol), evaporating to dryness and reconstituting in aqueous solution, the serological activity of the mixture falls to a much lower level. It would appear that the glycolipid insoluble in methanol is no longer activated by combining with the "carrier lipid", whereas the soluble material retains this property. The haemagglutination activity of the methanol-soluble material, combined with the carrier is exactly the same (calculated for the glycolipid soluble in methanol present in the reaction mixture) as that of the methanol-insoluble glycolipid (see Fig. 5). The reactivation of the methanol-soluble material is only slight when measured by plant-seed agglutinins. The methanol-soluble and methanol-insoluble glycolipids do not exhibit Hactivity with any of the reagents tested, which confirms an earlier observation is. The materials do not precipitate with anti-type X I V pneumococcus serum, but do so after treatment with Trichomonas foetus enzymes is. The glycolipids were not tested for other bloodgroup activities. The crude material isolated from the whole red cells

v75[

z

o

25

<

o

10o 150 Added antigen (jug)

2

Fig. 4. Precipitation curves of the methanol-insoluble and methanol-soluble glycolipids. O - - O , glycolipid insoluble in methanol; [ 3 - - O , glycolipid soluble in methanol. Ordinate: 25, 5o, 75 /2g of a n t i b o d y N precipitated. Abscissa: 50, ioo, i5o, 200/~g of antigen added.

Biochim. Biophys. Acta, 78 (1963) 313-328

90

-~~D

• ~' eV

P~

eV

A substance

H substance

A substance*

* The standard

Standard

Standard

with

with

with

and

was A 4Ol exhibiting

i :i

reconstituted

glycolipid

glycolipid

3

glycolipid

glycolipid

the methanol-sol,

Methanol-insol.

thecarrieri:

Methanol-sol.

Methanol-sol.

reconstituted

reconstituted

glycolipid

Methanol-insol.

I: 3

glycolipid

Methanol-insol.

thecarrier

the methanol-sol•

glycolipids

IV S containing

methanol-insol,

Fraction

IV

about

--

--

3.2. Io 5

.

6.4.1o 4

3 . 2 " lO 4

--

2 . 5 " lO 5

.

.

.

--

8. lO 4

potent

.

4.1o 4

8. Io 3

__

4" lO4

.

Dolichos biflorus extract(anti-A) and A t cells

of the most

Viccia cracca extract(anti-A) and A t cells

2 5 % of activity

5" I o 6

3.2. io 4

2.5.1o 8

I. lO 4

2.5-IO e

2.5" lO 6

1 . 2 5 . lO 4

Natural human anti-A serum and At cells

--

.

.

preparations

6.4' lO 6

.

2

5" lO2

io 2

5" IOa

<5.1o

<

<5.

<

.

Ulex europa*as extract(anti H) and 0 cells

2

obtainable

3.2" lO 5

--

2

5" lO2

<5-1o

<

<5•1o

< 5" lO2

Cystisus sessilifolius extract(anti-H) and 0 cells

2

2

from secretions.

6 . lO 5

--

<5.1o

"< 5" lO2

<5•1o

< 5" lO2

I" 107

5" 106

5" 107

5" 106

I" I O 8

I ' 108

Immune rabbit anti-A hacmolytic serum and serum and 0 cells sheep cells Immune rabbit (anti-O cells)

H A E M A G G L U T I N A T I O N A N D HAEMOLYSIS I N H I B I T I O N E N D P O I N T S OF BLOODGROUP-ACTIVE GLYCOLIPIDS FROM RED CELLS

TABLE

~o ba

©

c~

"O

>

324

j. KOSCIELAK

does not exhibit Le a, Rh, MN, P activity, and does not inhibit the haemagglutination brought about b y influenza virus13,14. 7c O

g 6c ol

O1

5c

>o 4c 3c D "0

1C

o o

2'5

io

7'5 --

Currier present (%)

,;o

Fig. 5. Effect of addition of carrier u p o n the serological activity of the methanol-insoluble and methanol-soluble glycolipids as m e a s u r e d b y the h a e m a g g l u t i n a t i o n inhibition test. Ordinate: i.o, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, log of dilution of the active substance giving complete inhibition of haemagglutination. Abscissa: 25, 5 o, 75, lOO% of carrier present.

Physical methods

The glycolipids were examined in a Spinco Model E ultracentrifuge. I n all cases the substances were dissolved directly in the buffer solution (o.157 M NaC1, 0.0054 M NaH2POa, o.o125 M N a 2 H P O ~, p H 7.2, I 0.20). The methanol-soluble glycolipid gave a single symmetrical peak which remained fairly sharp over the duration of the experiment which was 65 min (Fig. 6a). No material with appreciably higher, or lower, sedimentation coefficient could be seen. The sedimentation coefficient at a concentration of 0.62 g per IOO ml was S2o,w = 11. 9 S; at o.15 g per IOO ml, s20,w = 12.4 S. The methanol-insoluble glycolipid gave a single, rather skewed, peak which both m o v e d and spread rapidly (Fig. 6 b and c). The sedimentation coefficient at a concentration of 0.90 g per IOO ml was s20,w = 64 S ; at 0.2 g per IOO ml S2o,w = 79 S.

Fig. o. Ultracentrifuge p a t t e r n s obtained with glycolipids. Sedimentation is from right to left; t e m p e r a t u r e 2o ° t h r o u g h o u t , a: Methanol-soluble glycolipid, after 44 rain at 42 o4o rev./min, phase-plate angle 5 o°. b, c: Methanol-insoluble glycolipid after 28 min (b) and 44 min (c) at 21 74 ° rev./rnin, phase-plate angle 6o °. d, e: Mixed glycolipids (see text) after t4 min (d) and 42 min (e) at 42 o4o rev./min. Phase-plate angles 55 ° and 45 °, respectively.

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A mixture of the two fractions, containing o.5 g of each per IOO ml gave one sharp peak and a faster, rapidly spreading peak (Fig. 6 d and e). Analysis of the patterns gave the apparen t proportions of the components (corrected for radial dilution) as about 7 ° % slow and 30 % fast. The accuracy of the area analysis is limited, because of the difficulties of comparing sharp and diffuse peaks, but confirmation was obtained by estimating the absolute concentration of the slow component from the later exposures of the experiments, when its resolution from the fast component is complete. The value obtained was 0.73 g per IOO ml; this figure, of course, does not represent the original concentration of the slow component in the mixture, due to the JOHNSTON-OGSTOlq ~ effect. The sedimentation coefficients were 13.6 S for the slow component and 28.2 S for the fast. Insufficient material was available for a range of concentrations to be investigated. No quantitative estimate of the degree of heterogeneity of the t w o materials can be made from the evidence so far obtained; however, the general behaviour of the methanol-soluble fraction in the ultracentrifuge is similar to that of purified proteins, and it is therefore likely to be of the same order of homogeneity. The methanol-insoluble fraction is certainly much more heterogeneous, but a more or less continuous distribution of molecular weight is indicated. The result of the experiment on a mixture of the two materials is most easily interpreted on the assumption that complex formation occurs between the two components. The difference between the apparent proportions and the known original composition, and the increase in absolute concentration of the slow component, must be due in part to the operation of the JOHNSTON--OGSTON effect; nevertheless the changes are larger than would generally be expected at such low concentrations (cf. ref. 37). Moreover the fact that the sedimentation coefficient of the slow component (13.6 S at 0.73 g per IOO ml) was greater than the value to be expected at this concentration when examined alone (11.7 S), is strong evidence in favour of complex formation. The decrease in the sedimentation coefficient of the faster component from its value when examined alone must be due in part to direct concentrationdependence effects, but the decrease is unusually large, and therefore suggests that disaggregation of the original fast component has occurred as a consequence of the interaction. A rapidly reversible equilibrium reaction of the type considered by GILBERT AND JENKINSa8 is ruled out in view of the apparently complete resolution of the two components. DISCUSSION The data presented relate the bloodgroup-A activity of human erythrocytes to the glycolipid components of the red-cell membrane. However, the major hexosaminecontaining glycolipid of red cells (Fraction SlII) was devoid of bloodgroup activity when measured by haemaggiutination inhibition and by quantitative precipitation tests. This glycolipid contained residues of sphingosine, lignoceric acid, galactose, glucose and galactosamine and it was most probably identical with the lignocerylsphingosinegalactosamine trihexoside described by YAMAKAWA et al.lL The glycolipid fractions which possessed the bloodgroup activity were relatively more soluble and less soluble in methanol and they differed from those described by YAMAKAWAet al. 1~ in that they had a higher content of sialic acid and glucosamine. Evidence for the homogeneity of their preparations was not given byYAMAKAWA Biochim. Biophys. Acta, 78 (1963) 313-328

326

J. KO~CIELAK

and it is therefore possible that their materials were contaminated with a substance having a high galactosamine and a low sialic acid content. The analytical figures of the two glycolipids indicate that they cannot be simple comFonnds containing one sphingosine residue per molecule, since the molar ratios of either glucose or sialic acid residues to sphingosine are from 0.57 to 0.58. Two or more sphingosine residues are therefore most probably present in the molecule and, in this respect, the bloodgroup active glycolipids are similar to the gangliosides 39. The occurrence of several different bloodgroup active glycolipids in the materials isolated from a pool of human red cells obtained from many donors raises the question as to whether these materials are present in the cells of each individual, or whether they represent a genetic variation in glycolipid composition and structure associated with different members of the population. This question is not readily answered as it would be difficult to obtain sufficient blood from one person to carry out the required investigation. It is known, however, that the immunological specificity of polysaccharide antigens resides in small oligosaccharide chains present as surface structures in the molecules (see refs. 7, 15). The directing action of the bloodgroup genes, mediated presumably by specific enzymes, would be to incorporate simple sugar residues into a preformed substance, thereby converting it into an antigen of definite specificity. Thus, assuming in the case of bloodgroup substance from red cells that the acceptor portion is cerebroside or ceramide, many bloodgroup-active glycolipids, differing only in fatty acid composition, could be present in the red cells of a single individual. The unusual behaviour of the bloodgroup activity of the substances from red cells as measured by the inhibition of haemagglutination may be best explained by assuming that the activity depends on the degree of aggregation of individual glycolipid molecules in aqueous solution. Some of the active fractions (CII, SIV and the fraction soluble in methanol) developed the full activity only when in combination with the carrier lipid from which they had been separated. Since the glycolipid would probably exist in aqueous solution as a molecular aggregate or micelle, the addition of a waterinsoluble "carrier" lipid could be expected to increase the hydrophobic portion of the complex, and hence give rise to larger micelles which in turn would contain more active carbohydrate structures and be a part of a more rigid structure. The similar reasoning could be applied to the methanol-insoluble glycolipid which probably existed in aqueous media as aggregates of sufficient size to display high activity, whereas the soluble glycolipid did not. Upon recombination of the two glycolipids, the necessary inicelle structure was modified, probably through disaggregation, and gave rise to a loss in activity. The results of the ultracentrifugal analysis of the two glycolipids alone and after mixing, support this assumption. The fact that the methanol-insoluble substance contains the C,4 lignoceric acid whereas the methanolsoluble substance possesses lower fatty acids, might account for the observed differences in solubility and in ability to form aggregates in aqueous solution. However, all lhe glycolipid fractions developed full activity in the quantitative precipitation test without any carrier substance added.This apparent discrepancy can be explained on the basis of qualitative difference between the precipitation and haemagglutination inhibition test. The latter is essentially a competitive one in which the inhibitor comFetes for antil:ody molecules with the native antigen on the red-cell surface. Thus the forces which bind the inhibitor-antibody complex must be strong enough et al. l~

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to withstand the addition of the native antigen. In the precipitation test only one antigen and antibody are present in the reaction mixture and no competition occurs. In the field of lipid haptens, observations relevant to the subject discussed are numerous. LANDSTEINER AND LEVENE4° reported that purification of their heterogenetic antigen decreased its activity but that the activity could be largely restored by the addition of an ox-brain extract which consisted chiefly of sphingomyelin. More recently, it was shown that Cytolipin-H is inactive in the complement fixation test unless some auxiliary lipids, or lecithin, are added to the system 41. Finally, the total absence of H-activity in bloodgroup substances from red cells must be considered. The bloodgroup substances obtained from secretions contain fucose, and there is strong evidence that fucose is a component of the active determinant group of the H-antigen (see ref. 15). Bloodgroup substances from red cells contain only small amounts of fucose and it is not surprising, therefore, that they are not H-active. However, a plausible scheme for the biosynthesis of bloodgroup active mucoids from secretions has been developed42 and this requires that the precursor molecule is transformed into an H-active substance before the conversion into A- and B-active substances can take place. It has been suggested 4~ that the genetically controlled biosynthesis of the bloodgroup substances from red cells follows a different path. In most instances, the erythrocyte surface undoubtedly has H-activity, and it may be that two types of bloodgroup substances, namely both glycolipid and mucopolysaccharide, exist on the red-cell surface. Some preliminary evidence in support of this view has appeared 44. However, .further studies are indicated. ACKNOWLEDGEMENTS

The author wishes to thank Professor W. T. J, MORGAN and Dr. W. M. WATKINS for their interest and advice throughout this work. He is indebted to Dr. J. M. CREETH for performing and interpreting the ultracentrifuge experiments and to Dr. G. M. GRAY for carrying out the fatty acid analyses. The author is also grateful to Dr. T. J. PAINTER for his help in preparing the manuscript. The work has been supported by a Grant from the International Atomic Agency. REFERENCES LANDSTEINER, Zentr. Bahteriol. Parasitenk. Abt. i, 27 (19oo) 357. LANDSTEINER, Wien. I4tin. Wochschr., 14 (19Ol) 1132, LANDSTEINER, J. VAN DER SCHEER AND D. H. WITT, Proc. Soc. Exptl. Biol. Med., 22 (1924) 289. HALLAUER, Z. Immunitaetsforsch., 83 (1934) I I 4 . V. STEPANOV, A. KUSlN, Z. MAKAJEVA AND P. N, KOSSJAKOW, Biokhimiva, 5 (194 o) 547. LEHRS, Z. Immunitaetsforsch., 66 (193 o) 175. A. KABAT, Blood Group Substances, Academic Press, New York, 1956. H. RASCH, Z. Immunitaetsforsch., i i o (1953) 243. YAMAKAWA AND S. SUZUKI, J. Biochem. (Tokyo), 39 (1952) 393. KLENK AND K. LAUENSTEIN, Z. Physiol. Chem., 291 (1952) 249. I. HAKOMORI AND R. W. JEANLOZ, J. Biol. Chem., 236 (1961) 2827. YAMAKAWA, R. IRm AND M. IWANAGA,J. Biochem. (Tokyo), 48 (196o) 49. I~OSCIELAK AND I~. ZAKRZE'~VSKI, in E. MIKULAGZEK AND W. T. DOBRZANSKI, Proc. Intern. Syrup. Biologically Active Mucoids, Warsaw 1959, Polish Academy of Science, W a r s a w , 1959, p. 2i. 14 j. KOSCIELAK AND K. ZAKRZEWSKI, Nature, 182 (196o) 516. 15 W. T. J. MORGAN, Proc. Roy. Soc. (London), Set. B, 151 (1959-196o) 308. le j . KOgCIELAK, Nature, 194 (1962) 751. 1~ M. J. JOHNSON, J. Biol. Chem., 137 (1941) 575. 1 K. K. 3 K. * C. 5 A. H. 7 E. 8 L. 9 T. 10 E. 11 S. 12 T. 13 j.

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