Revue Fransaise de Transfusion et Immuno-hSmatologie Tome XIX - - N ° 1 - - 1976
207
Distribution of the antigens A, D and c on red cell membranes by N.C. H U G H E S - J O N E S and E. ROMANO Medical R e s e a r c h Council's E x p e r i m e n t H a e m a t o l o g y Unit, St Mary's Hospital - LONDON.
Blood group antigens are k n o w n to be p r esen t on the surface of the red cell m e m b r a n e either as glycoproteins (MN, WINZLER, [8]), as glycolipids (ABO, HANFLANDand EGLI, [1]) or as lipoproteins (Rh, LORUSSO and GREEN [4], HUGHES-JONES and coll. [3]). The question arises as to the m o d e of d i s t r i b u t i o n of these antigens on the surface of the red cell. Are they d i s t r i b ute d at r a n d o m , or are they organised in clusters, in pairs o r in }arger groups ? We have a t t e m p t e d to obtain evidence for the relative distribution of A and Rh sites on h u m a n cells using an antiglobulin reagent labelled with colloidal gold which can be readily visualized by electron m i c r o s c o p y (RoMANO and H~JGHES-JONES, [7]). It i s an old o b s e r v a t io n that proteins will stabilize colloidal gold against the p r ec ip it a t in g action of ions. In o u r experience, however, only certain proteins f o r m a sufficiently stable bond to be useful in electron microscopy. H u m a n IgG antibody did not f o r m a stable bond but a h o r s e antiglobulin (which appeared to be a T globulin when e x a m i n e d electrophoretically) has p r o v e d to be suitable. The colloidal gold particles are a p p r o x i m a t e l y 3 n m in d i a m e t e r and are attached to the antiglobulin molecule at an average ratio of 1.6 particles of gold on each antibody molecule. The electron microscopical technique used was a m o d i f i c a t i o n of that of NICOLSON and SINGER [5]. The antibodies (anti-A, anti-c or anti-D) w e r e first a t t a c h e d to the surface of the intact red cell. The cells were t h e n lysed and m o u n t e d on the surface of a carbon-coated grid, wh i ch was then placed in a solution of gdld-labelled antiglobulin (gold-anti-IgG). After r e m o v i n g the excess gold-anti-IgG the grids w e r e dried and e x a m i n e d u n d e r the electron microscope. This technique shows the distribution of blood group antibody molecules p r e s e n t only on one side of the r e d cell (the side away f r o m the grid). The whole surface of a red cell can be vizualized and a two-dimensional p i ct u r e : o f the distribution of the gold-anti-IgG is obtained.
208
H U G H E S - J O N E S N.C.
et R O M A N O
E.
DISTRIBUTION OF D-ANTIGEN SITES,
In the initial experiments, R l r cells w e r e c o m b i n e d w i t h anti-D and labelled w i t h gold-anti-IgG. FIG. 1 shows the result obtained w h e n there w e r e a p p r o x i m a t e l y 3000 separate gold clusters on the surface of t h e cell (corrected for the fact that only half the surface was labelled). We believe that each gold cluster represents one D-antigen site for the following reasons. FIG. 1 ~t 4 . -
Electron microscopy pictures of red cell membranes combined with blood group antibodies and visualized with gold-labelled anti-IgG.
FIG. 1 . -
Rir cells combined with anti-D.
1. - - On average, each cluster contained 2.7 particles of gold. Since each antiglobulin molecule was c o m b i n e d with, on average, 1.6 particles of gold, w e have concluded that there w e r e j u s t u n d e r 2 antiglobulin molecules present at each site. Other studies, using the same anti-IgG labelled w i t h i251, have shown that u n d e r the conditions of this experiment, two antiglobulin molecules w o u l d c o m b i n e w i t h one anti-D molecule. 2 . - Almost all the clusters had a m a x i m u m d i a m e t e r of less than 30 n m and two-thirds w e r e less than 14 nm. Calculations based on the size of IgG molecules indicate that there is only r o o m for one anti-D and 2 anti-IgG molecules at each site,
DISTRIBUTION OF THE A N T I G E N S A, D, C
209
F u r t h e r studies on these cells have shown that the clusters are r a n d o m l y distributed on the red cell surface, and do not differ significantly f r o m that expected of a Poisson distribution. Our conclusion is thus that D sites are single, discrete entities, distributed randomly. Similar results were obtained on cells which w e r e homozygous for the D-antigen. On the other hand, we obtained evidence that the D-antigen is mobile and that aggregation of the antigen can occur. The evidence for this was two-fold. FirsL t r e a t m e n t of the red cells with papain, which is known to bring about aggregation of the intercalated particles (NICOLSON, [6]) resulted in the gold clusters being found in aggregates (FIG. 2) after
FIG. 2 . -
Rir cells treated first with papain and then with anti-D.
the addition of anti-D. This change suggests that the D antigen may be related to the intercalated particles. Secondly, w h e n ailti-D was c o m b i n e d with ceils of the r a r e phenotype -D-, the gold clusters were also present as aggregates, w i t h o u t enzyme t r e a t m e n t (FIG. 3). It was thought that the anti-D had b r o u g h t about the aggregation of the antigen sites on these cells, which have about 100,000-200,000 D sites on each cell, c o m p a r e d to 10,000- 30,000 sites on the c o m m o n phenotypes (HuGI~IES-JONES, GARDNER and LINCOLN, [2]). RELATIONSHIP BETWEEN C AND D SITES OF THE RH SYSTEM. In order to study the relationship between c and D antigen sites, red cells of the phenotype ccDEE were c o m b i n e d with anti-c or ailti-D separately or with b o t h antibodies at the same t i m e and it was found that the average n u m b e r of gold particles in each cluster was 2.3, 2.7 and
14
210
H U G H E S - J O N E S N.C.
et R O M A N O
FIG. 3. - - D-red c e l l s c o m b i n e d w i t h anti-D,
FIG. 4. - - G r o u p a r e d c e l l s c o m b i n e d w i t h a n t i - A ; m o r e anti-A m o l e c u l e s o n e a c h r e d cell.
t h a n 10,000
E.
DISTRIBUTION OF THE ANTIGENS A, D, C
211
4.0 respectively. Not only were the n u m b e r of gold particles in each cluster greater when anti-c a n d anti-D was p r e s e n t at the same time, but also the clusters were larger in size. The i n t e r p r e t a t i o n was made that c and D antigen sites are close together, either p r e s e n t on the same protein, or as 2 proteins in close association, possibly as dimers. DISTRIBUTION OF A SITES. Similar experiments were carried out using A1 cells a n d an IgG anti-A. A somewhat different d i s t r i b u t i o n p a t t e r n was found. If only small a m o u n t s of anti-A were c o m b i n e d with the red cell surface, so that where less t h a n 10,000 gold clusters on each red cell, the clusters were for the most part discrete b u t there was also evidence that the A sites were aggregating together. An increase in the a m o u n t of anti-A bound to the cells rapidly altered the picture a n d there was obvious evidence of aggregation of A-antigen into a reticular n e t w o r k (Fro. 4). I t was also n o t e d that agglutination of the A cells by the IgG anti-A did not take place when there were only a small n u m b e r of anti-A molecules on the cell, b u t agglutination did occur when sufficient anti-A was combined with the red cells to b r i n g about aggregation of A antigen on the red cell surface, This gives support to the theory of Nicolson [6] that agglutination is potentiated by clustering of antigen sites on the cell surface. CONCLUSION The use of a gold-labelled anti-IgG d e t e r m i n i n g the d i s t r i b u t i o n of blood group antigens has revealed that 1) D antigen sites are n o r m a l l y discrete b u t can b e made to aggregate by proteolytic enzyme t r e a t m e n t ; 2) that D sites on -D- cells are aggregated by anti-D; 3) that the c a n d D antigen sites are close together on the red cell surface; 4) that A antigen sites are readily aggregated by anti-A once the n u m b e r of anti-A molecules on each red cell exceeds a b o u t 10200.
RESUME Une anti-IgG m a r q u d e ~ l'or colloidal a 6t6 utilisde pour visualiser en microscopie 61ectronique la d i s t r i b u t i o n des antig~nes de groupe sanguin A,D et c sur la m e m b r a n e des globules rouges h u m a i n s . Les sites antigdniques D c o n s t i t u e n t des entitds distinctes, disposdes au hasard g la surface de l'hdmatie. Aprbs t r a i t e m e n t g la papaine on observe une d i s t r i b u t i o n en grappes des sites D. Sur les hdmaties de phdnotype -D-, n o n traitdes p a r la papa'ine, cette m6me d i s t r i b u t i o n est observde. Les sites antigdniques c et D semblent 6troitement associds sur les hdmaties ccDEE. La d i s t r i b u t i o n des sites antigdniques A d6pend de la
212
HUGHES-JONES N.C. et ROMANO E.
quantit6 d'IgG anti-A lide par hdmatie. Quand le n o m b r e d'IgG anti-A est supdrieur ~t 10.000 p a r globule rouge une agr6gation des sites A est observ4e sur la surface cellulaire. Doctor N.C. HUGHES-JONES, Medical Research Council's E x p e r i m e n t a l Haematology Unit St Mary's Hospital, Paddington, LONDON.
REFERENCES [1] HANFLANDP. a n d EGLI H, - - Quantitative isolation and purification of blood group-active glycosphingolipids from h u m a n B erythrocytes. Vox Sang., 68, 438, 1975. [2] HUGHES-JONES N.C., GARNDER B. and LINCOLN P.J. - - Observations on the n u m b e r of available c, D and E antigen sites on red cells. Vox Sang., 21, 210, 1971. [ 3 ] H U G H E S - J O N E S N . C . , GREEN E.J. and HUNT V.A.M. - - Loss of Rh antigen activity following the action of phospholipase A2 on red cell stroma. V o x Sang., 29, 184, 1975. [4] LORUSSO D.J. a n d GREEN F.A. - - Reconstitution of Rh (D) antigen activity f r o m h u m a n erythrocyte m e m b r a n e s solubilized from deoxycholate. Science, 188, 66, 1975. [5] NICOLSON G.L. a n d SINGER S.J. - - Ferritin-conjugated p l a n t agglutinins as specific saccharide stains for electron m i c r o s c o p y : application to saccharide b o u n d to cell m e m b r a n e s . Pro& Nat. Acad. Sci. (U.S.A.), 68, 942, 1971. [6] NICOLSON G.L. - - The relationship of a fluid m e m b r a n e structure to cell agglutination a n d surface-topography. Ser. Haemat., 6, 275, 1973. [7] ROMANO E.J. and HUGHES-JONES N.C. - - Distribution and mobility of the A, D and c antigens on h u m a n red cell m e m b r a n e s ; studies with a gold-labelled antiglobulin reagent. Brit. J. Haematol., 30, 507, 1975. [8] W~NZLERR.J. - - A glycoprotein in h u m a n erythrocyte m e m b r a n e s . I n : Red Cell M e m b r a n e Ed. b y JAMIESON G.A. a n d GREENWALTT.J., Lippincott, Philadelphia, 1969.
207
Distribution of the antigens A, D and c on red cell membranes by N.C. H U G H E S - J O N E S and E. ROMANO Medical R e s e a r c h Council's E x p e r i m e n t H a e m a t o l o g y Unit, St Mary's Hospital - LONDON.
Blood group antigens are k n o w n to be p r esen t on the surface of the red cell m e m b r a n e either as glycoproteins (MN, WINZLER, [8]), as glycolipids (ABO, HANFLANDand EGLI, [1]) or as lipoproteins (Rh, LORUSSO and GREEN [4], HUGHES-JONES and coll. [3]). The question arises as to the m o d e of d i s t r i b u t i o n of these antigens on the surface of the red cell. Are they d i s t r i b ute d at r a n d o m , or are they organised in clusters, in pairs o r in }arger groups ? We have a t t e m p t e d to obtain evidence for the relative distribution of A and Rh sites on h u m a n cells using an antiglobulin reagent labelled with colloidal gold which can be readily visualized by electron m i c r o s c o p y (RoMANO and H~JGHES-JONES, [7]). It i s an old o b s e r v a t io n that proteins will stabilize colloidal gold against the p r ec ip it a t in g action of ions. In o u r experience, however, only certain proteins f o r m a sufficiently stable bond to be useful in electron microscopy. H u m a n IgG antibody did not f o r m a stable bond but a h o r s e antiglobulin (which appeared to be a T globulin when e x a m i n e d electrophoretically) has p r o v e d to be suitable. The colloidal gold particles are a p p r o x i m a t e l y 3 n m in d i a m e t e r and are attached to the antiglobulin molecule at an average ratio of 1.6 particles of gold on each antibody molecule. The electron microscopical technique used was a m o d i f i c a t i o n of that of NICOLSON and SINGER [5]. The antibodies (anti-A, anti-c or anti-D) w e r e first a t t a c h e d to the surface of the intact red cell. The cells were t h e n lysed and m o u n t e d on the surface of a carbon-coated grid, wh i ch was then placed in a solution of gdld-labelled antiglobulin (gold-anti-IgG). After r e m o v i n g the excess gold-anti-IgG the grids w e r e dried and e x a m i n e d u n d e r the electron microscope. This technique shows the distribution of blood group antibody molecules p r e s e n t only on one side of the r e d cell (the side away f r o m the grid). The whole surface of a red cell can be vizualized and a two-dimensional p i ct u r e : o f the distribution of the gold-anti-IgG is obtained.
208
H U G H E S - J O N E S N.C.
et R O M A N O
E.
DISTRIBUTION OF D-ANTIGEN SITES,
In the initial experiments, R l r cells w e r e c o m b i n e d w i t h anti-D and labelled w i t h gold-anti-IgG. FIG. 1 shows the result obtained w h e n there w e r e a p p r o x i m a t e l y 3000 separate gold clusters on the surface of t h e cell (corrected for the fact that only half the surface was labelled). We believe that each gold cluster represents one D-antigen site for the following reasons. FIG. 1 ~t 4 . -
Electron microscopy pictures of red cell membranes combined with blood group antibodies and visualized with gold-labelled anti-IgG.
FIG. 1 . -
Rir cells combined with anti-D.
1. - - On average, each cluster contained 2.7 particles of gold. Since each antiglobulin molecule was c o m b i n e d with, on average, 1.6 particles of gold, w e have concluded that there w e r e j u s t u n d e r 2 antiglobulin molecules present at each site. Other studies, using the same anti-IgG labelled w i t h i251, have shown that u n d e r the conditions of this experiment, two antiglobulin molecules w o u l d c o m b i n e w i t h one anti-D molecule. 2 . - Almost all the clusters had a m a x i m u m d i a m e t e r of less than 30 n m and two-thirds w e r e less than 14 nm. Calculations based on the size of IgG molecules indicate that there is only r o o m for one anti-D and 2 anti-IgG molecules at each site,
DISTRIBUTION OF THE A N T I G E N S A, D, C
209
F u r t h e r studies on these cells have shown that the clusters are r a n d o m l y distributed on the red cell surface, and do not differ significantly f r o m that expected of a Poisson distribution. Our conclusion is thus that D sites are single, discrete entities, distributed randomly. Similar results were obtained on cells which w e r e homozygous for the D-antigen. On the other hand, we obtained evidence that the D-antigen is mobile and that aggregation of the antigen can occur. The evidence for this was two-fold. FirsL t r e a t m e n t of the red cells with papain, which is known to bring about aggregation of the intercalated particles (NICOLSON, [6]) resulted in the gold clusters being found in aggregates (FIG. 2) after
FIG. 2 . -
Rir cells treated first with papain and then with anti-D.
the addition of anti-D. This change suggests that the D antigen may be related to the intercalated particles. Secondly, w h e n ailti-D was c o m b i n e d with ceils of the r a r e phenotype -D-, the gold clusters were also present as aggregates, w i t h o u t enzyme t r e a t m e n t (FIG. 3). It was thought that the anti-D had b r o u g h t about the aggregation of the antigen sites on these cells, which have about 100,000-200,000 D sites on each cell, c o m p a r e d to 10,000- 30,000 sites on the c o m m o n phenotypes (HuGI~IES-JONES, GARDNER and LINCOLN, [2]). RELATIONSHIP BETWEEN C AND D SITES OF THE RH SYSTEM. In order to study the relationship between c and D antigen sites, red cells of the phenotype ccDEE were c o m b i n e d with anti-c or ailti-D separately or with b o t h antibodies at the same t i m e and it was found that the average n u m b e r of gold particles in each cluster was 2.3, 2.7 and
14
210
H U G H E S - J O N E S N.C.
et R O M A N O
FIG. 3. - - D-red c e l l s c o m b i n e d w i t h anti-D,
FIG. 4. - - G r o u p a r e d c e l l s c o m b i n e d w i t h a n t i - A ; m o r e anti-A m o l e c u l e s o n e a c h r e d cell.
t h a n 10,000
E.
DISTRIBUTION OF THE ANTIGENS A, D, C
211
4.0 respectively. Not only were the n u m b e r of gold particles in each cluster greater when anti-c a n d anti-D was p r e s e n t at the same time, but also the clusters were larger in size. The i n t e r p r e t a t i o n was made that c and D antigen sites are close together, either p r e s e n t on the same protein, or as 2 proteins in close association, possibly as dimers. DISTRIBUTION OF A SITES. Similar experiments were carried out using A1 cells a n d an IgG anti-A. A somewhat different d i s t r i b u t i o n p a t t e r n was found. If only small a m o u n t s of anti-A were c o m b i n e d with the red cell surface, so that where less t h a n 10,000 gold clusters on each red cell, the clusters were for the most part discrete b u t there was also evidence that the A sites were aggregating together. An increase in the a m o u n t of anti-A bound to the cells rapidly altered the picture a n d there was obvious evidence of aggregation of A-antigen into a reticular n e t w o r k (Fro. 4). I t was also n o t e d that agglutination of the A cells by the IgG anti-A did not take place when there were only a small n u m b e r of anti-A molecules on the cell, b u t agglutination did occur when sufficient anti-A was combined with the red cells to b r i n g about aggregation of A antigen on the red cell surface, This gives support to the theory of Nicolson [6] that agglutination is potentiated by clustering of antigen sites on the cell surface. CONCLUSION The use of a gold-labelled anti-IgG d e t e r m i n i n g the d i s t r i b u t i o n of blood group antigens has revealed that 1) D antigen sites are n o r m a l l y discrete b u t can b e made to aggregate by proteolytic enzyme t r e a t m e n t ; 2) that D sites on -D- cells are aggregated by anti-D; 3) that the c a n d D antigen sites are close together on the red cell surface; 4) that A antigen sites are readily aggregated by anti-A once the n u m b e r of anti-A molecules on each red cell exceeds a b o u t 10200.
RESUME Une anti-IgG m a r q u d e ~ l'or colloidal a 6t6 utilisde pour visualiser en microscopie 61ectronique la d i s t r i b u t i o n des antig~nes de groupe sanguin A,D et c sur la m e m b r a n e des globules rouges h u m a i n s . Les sites antigdniques D c o n s t i t u e n t des entitds distinctes, disposdes au hasard g la surface de l'hdmatie. Aprbs t r a i t e m e n t g la papaine on observe une d i s t r i b u t i o n en grappes des sites D. Sur les hdmaties de phdnotype -D-, n o n traitdes p a r la papa'ine, cette m6me d i s t r i b u t i o n est observde. Les sites antigdniques c et D semblent 6troitement associds sur les hdmaties ccDEE. La d i s t r i b u t i o n des sites antigdniques A d6pend de la
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HUGHES-JONES N.C. et ROMANO E.
quantit6 d'IgG anti-A lide par hdmatie. Quand le n o m b r e d'IgG anti-A est supdrieur ~t 10.000 p a r globule rouge une agr6gation des sites A est observ4e sur la surface cellulaire. Doctor N.C. HUGHES-JONES, Medical Research Council's E x p e r i m e n t a l Haematology Unit St Mary's Hospital, Paddington, LONDON.
REFERENCES [1] HANFLANDP. a n d EGLI H, - - Quantitative isolation and purification of blood group-active glycosphingolipids from h u m a n B erythrocytes. Vox Sang., 68, 438, 1975. [2] HUGHES-JONES N.C., GARNDER B. and LINCOLN P.J. - - Observations on the n u m b e r of available c, D and E antigen sites on red cells. Vox Sang., 21, 210, 1971. [ 3 ] H U G H E S - J O N E S N . C . , GREEN E.J. and HUNT V.A.M. - - Loss of Rh antigen activity following the action of phospholipase A2 on red cell stroma. V o x Sang., 29, 184, 1975. [4] LORUSSO D.J. a n d GREEN F.A. - - Reconstitution of Rh (D) antigen activity f r o m h u m a n erythrocyte m e m b r a n e s solubilized from deoxycholate. Science, 188, 66, 1975. [5] NICOLSON G.L. a n d SINGER S.J. - - Ferritin-conjugated p l a n t agglutinins as specific saccharide stains for electron m i c r o s c o p y : application to saccharide b o u n d to cell m e m b r a n e s . Pro& Nat. Acad. Sci. (U.S.A.), 68, 942, 1971. [6] NICOLSON G.L. - - The relationship of a fluid m e m b r a n e structure to cell agglutination a n d surface-topography. Ser. Haemat., 6, 275, 1973. [7] ROMANO E.J. and HUGHES-JONES N.C. - - Distribution and mobility of the A, D and c antigens on h u m a n red cell m e m b r a n e s ; studies with a gold-labelled antiglobulin reagent. Brit. J. Haematol., 30, 507, 1975. [8] W~NZLERR.J. - - A glycoprotein in h u m a n erythrocyte m e m b r a n e s . I n : Red Cell M e m b r a n e Ed. b y JAMIESON G.A. a n d GREENWALTT.J., Lippincott, Philadelphia, 1969.
