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Solubilization of human erythrocyte membrane glycoproteins and separation of the MN glycoprotein from a glycoprotein with I, S, and A activity
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BIOCHIMICA ET BIOPHYSICA ACTA BBA
36201
SOLUBILIZATION OF HUMAN E R Y T H R O C Y T E MEMBRANE GLYCOP R O T E I N S AND SEPARATION OF T H E MN GLYCOPROTEIN FROM A GLYCOPROTEIN W I T H I, S, AND A ACTIVITY*
H. H A M A G U C H I AND H. C L E V E
Division of Human Genetics, Department of Medicine, Cornell University Medical College, New York, N.Y. (U.S.A.) (Received March 23rd, 1972)
SUMMARY
Human erythrocyte membrane glycoproteins are recovered in high yields in the aqueous phase after extraction of the membranes with a mixture of chloroformmethanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase reveals the presence of three main glycoprotein components, glycoproteins I, II, and III, and one additional periodic acid-Schiff positive component, without contamination by other membrane proteins. Their apparent molecular weights correspond to 58 ooo, 37 ooo, 24 ooo, and 50 ooo, respectively. The blood group antigens I, Ss, and MN are recovered in the aqueous phase in high yields and with marked increase in their specific activities. The aqueous phase also contains moderate amounts of A and B and small amounts of H antigenic activities. Further separation of the glycoproteins of the aqueous phase was achieved by gel filtration on Sephadex G-ioo columns in the presence of I % dodecyl sulfate. The M and N antigenic activities were found in glycoprotein I. N activity as tested with a rabbit antiserum was also observed in glycoprotein III. The highest activities for A, I, and S antigens were found in glycoprotein III. The chloroform-methanol phase does not contain glycoproteins; only the blood group antigens A, B, and H are found in this phase. The interphase, which contains most of the membrane proteins except the main glycoproteins, shows only weak ABH, I, Ss, and MN activities. The data indicate that the major glycoprotein of the human erythrocyte membrane, glycoprotein I, is the so-called MN glycoprotein, and that the I and S antigenic activities are associated with glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 ooo. Glycoprotein III is also associated with the A antigenic activity.
* Presented in p a r t a t t h e 4th International Congress of H u m a n Genetics Paris, September 4 - I I , I971.
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INTRODUCTION
Recently, three electrophoretically distinguishable glycoprotein components have been demonstrated in the human erythrocyte membrane and all appear to extend from the external cell surface through the cell membrane to the inner surface 1-s. In the course of our studies of the human erythrocyte membrane, we noticed that these three glycoproteins and one additional glycoprotein component can be quantitatively recovered in the aqueous phase after extraction with a mixture, of chloroformmethanol. The preparations were almost completely free from contamination with other membrane proteins and the glycoprotein components contained the blood group specificities ABH, I, Ss, and MN. In this paper, the procedures for solubilization and separation of human erythrocyte membrane glycoproteins and their blood group activities are described. METHODS
Preparation of human erythrocyte membrane glycoproteins H u m a n blood of individual donors in acid-citrate dextrose, less than two weeks old, was used as starting material. Membrane glycoproteins were prepared b y a method adapted from Kornfeld and Kornfeld 4. The blood was centrifuged at 15oo rev./min for 15 min. Plasma and buffy coat were removed by aspiration. Erythrocytes were then suspended in 0.9% NaC1- 5 mM Tris-HC1 buffer at p H 7.4 and washed three times b y centrifugation at 15oo rev./min for IO min. Some erythrocytes were discarded in all wash steps to ensure complete removal of the buffy coat. The packed erythrocytes were hemolyzed in a 9-fold volume of IO mM Tris-o.i mM EDTA-HC1 buffer, p H 7.4. Ghosts were sedimented by centrifugation at 20 ooo × g for 20 min. Ghosts were washed four times by centrifugation at 2o ooo × g for 20 min in 2o-fold volume of the T r i s - E D T A buffer. The washed ghosts were mechanically homogenized with a Dounce homogenizer and diluted to a protein concentration of 2 mg/ml with IO mM Tris-o.I mM EDTA-HC1 buffer, p H 7.4. The entire procedure was performed at temperatures of 0- 4 °C. To one volume of the ghost suspension, nine volumes of a chloroform-methanol mixture (2:1, v/v) were added. The mixture was stirred vigorously at room temperature for 30 rain and centrifuged afterwards at 15oo rev./ rain for IO min at room temperature. The aqueous layer was carefully aspirated and centrifuged again under the same conditions in order to remove contaminating interphase materials. The aqueous phase was concentrated in a rotary evaporator at 37 °C. to between one-tenth and one-forth the volume of the original ghost suspension. The concentrated aqueous phase was clear. Turbidity of the sample at this stage indicated the presence of contaminating interphase components. Ethanol precipitates of the aqueous phase components were prepared by addition of 9 vol. of ethanol, to I vol. of aqueous phase, followed by centrifugation at 3000 rev./min for 20 min at room temperature. The precipitates were air dried and distilled water was added; the obtained solution was clear. The interphase and chloroform-methanol phase were resuspended in distilled water and concentrated in a rotary evaporator. The procedure permits evaporation of organic solvents. Separation of the aqueous phase components, mostly glycoproteins, was carried out at room temperature by gel filtration on Sephadex G-Ioo columns in the presence
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of sodium dodecyl sulfate. The column, 4 cm × 115 cm, was packed and equilibrated with a 10/0 (w/v) sodium dodecyl sulfate solution in distilled water. Before application, the aqueous phase components were reduced for 4 h b y 1°/0 2-mercaptoethanol in the presence of 3°/0 sodium dodecyl sulfate and subsequently alkylated b y o.i M iodoacetic acid at p H 8.6 for I h at room temperature in the dark. p H was adjusted by addition of Tris-base. The flow rate of the column was 15-3o ml/h. The eluted materials were divided into three fractions as indicated (see below, Fig. 3), and concentrated b y ultrafiltration at room temperature to between one-tenth and one-sixth of the volume of the original eluates. Analytical methods Protein concentrations were measured b y the method of Lowry et al. ~. Three × crystallized bovine pancreatic ribonuclease was used as a standard. Sialic acid was assayed b y the method of Warren 6. Phosphorus was measured b y the method of Bartlett 7 and cholesterol was assayed by the method of Zlatkis et al. s. Sodium dodecyl sulfate-polyacrylamide gel electr0phoresis was performed in 1°/0 sodium dodecyl sulfate-o.I M sodium phosphate buffer at p H 7.1. The acrylamide concentration was either 7.5% or 6.5%. Tubes of 5 m m diameter and 12 cm length were used. Solubilization of samples and preparation of gels were performed according to the method described previously 9. Electrophoresis of the 7.5% gels was carried out at 3 V/cm for 8 h. The 7.5~o gels were fixed overnight b y 12.5% trichloroacetic acid dissolved in 50% ethanol and immersed in distilled water for i h. The gels were stained with 0.25% Coomassie brilliant blue for IO h 1°, or with the periodic acidSchiff reagent 11. In the 6.5% acrylamide gel system, electrophoresis was started with a current of 5 mA per tube. After 30 min the current was raised to IO mA per tube. Electrophoresis was stopped as soon as the B P B reached the 8.0 cm m a r k from the origin. Gels were stained with Coomassie brilliant blue or with the periodic acidSchiff reagent b y the method of Fairbanks et al3. The patterns of the erythrocyte membrane proteins were essentially identical in the two electrophoretic systems described. Blood group activities of glycoproteins were measured by hemagglutination inhibition tests. Two-fold serial dilutions were tested b y the conventional test tube method in the presence of four hemagglutination doses of antisera for A, B, I, S, and s antigens and two hemagglutination doses of antisera for M and N antigens and of anti-H lectin for H antigen. Samples in 0.9% NaC1 and antisera were incubated at 37 °C for I h for A, B. S, s, M, and N activities and at 4 °C for I h and 2 h for I and H activities, respectively. After these incubation periods, erythrocytes in 0.9°/0 NaC1 were added. The total volume of the mixture was 80-200/zl. The mixtures were incubated further at 37 °C for I h for A, B, S, s, M, and N activities and at 4 °C for I h, and 2 h for I and H activities, respectively. Readings were taken after centrifugation at 1500 rev./min for I min. Presence of s antigen was determined by indirect Coombs test. In order to calculate the approximate recoveries of blood group activities, total hemagglutination inhibition units were determined according to Gardas and Kogcielak 12. Removal of sodium dodecyl sulfate and measurement of serological activities of glycoproteins fractionated by gel filtration were performed as follows : To I vol. of concentrated fraction (40-200/zl) in test tubes with I.O cm diameter and 7.5 cm length, 9 vol. of ethanol were added and centrifuged at 2500 rev./min for 30 min at room Biochim. Biophys. Acta, 278 (1972) 271-28o
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temperature. The supernatant was carefully decanted, I ml of 9 o~o ethanol was added, and centrifugation at 2500 rev./min for 30 min was repeated. After decantation of the supernatant, the tubes were put upside-down on filter paper for 3o min. The ethanolwashed precipitates did not cause hemolysis and could be tested for inhibition activities. 5 o - I o o / A of antiserum with eight hemagglutination doses were added to the precipitates in the tubes. The precipitates dissolved in the antiserum. The tubes were shaken overnight at 4 °C to ensure the reaction of antibody and inhibiting components. Equal volumes of a 2% suspension of erythrocytes were added. Hemagglutination inhibition tests were read after appropriate incubation times. Human anti-A, anti-B and anti-S sera, rabbit anti-M, anti-N, and anti-immunoglobulin sera, and anti-H lectin (Ulex europeaus) were purchased from Behring Diagnostics, Inc. Human anti-s serum for indirect Coombs test was purchased from Pfizer Inc. A human cold agglutinin was used as anti-I reagent. RESULTS
Electrophoretic and chemical analysis of erythrocyte membrane glycoproteins Fig. I presents the 1% sodium dodecyl sulfate-7.5~/o polyacrylamide gel electro-
Fig. I. E l e c t r o p h o r e t i c p a t t e r n s of h u m a n e r y t h r o c y t e m e m b r a n e s on I ~o s o d i u m dodecyl s u l f a t e 7.5~o p o l y a c r y l a m i d e gels. (i) C o o m a s s i e brilliant blue stain, (2) Periodic a c i d - S c h i f f stain. G P = glycoprotein.
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phoresis patterns of human erythrocyte membranes. Staining with Coomassie brilliant blue reveals the presence of at least 15 polypeptide components in red cell ghosts. Components I-VI are designated in accordance with the notation used by Fairbanks et al. 1. In order to show the periodic acid-Schiff stain positive components clearly, 15o pg of the membrane proteins were applied to the gel. The periodic acid-Schiff positive proteins are named glycoproteins I, II, and III. The less intensely stained zones and glycolipid components are named Components A, B, and C. Glycoproteins I, II, and III correspond to the components periodic acid-Schiff-I, periodic acid-Schiff-2, and periodic acid-Schiff-3, respectively, described as by Fairbanks et al. 1. Component III corresponds to the component a reported by BretscherS, 13. Component A reacts only weakly with the periodic acid-Schiff reagent. Electrophoretically, Components III and A show complete correspondance. It may be assumed that they are identical and that their carbohydrate content is relatively low. On sodium dodecyl sulfate gel electrophoresis, Component B is not clearly separated from glycoprotein I. By gel filtration, however, the two components were more distinctly separated (see below, Fig. 3). The periodic acid-Schiff positive Component C is supposed to be a glycolipid 14. Coomassie blue stain of this component is positive. Apparent molecular weights of the glycoproteins were determined from the migration rates of the proteins in i%
A
Fig. 2. ElectrophoreUc p a t t e r n s of the three phases obtained by chloroform-methanol extraction of h u m a n erythrocyte membranes, on I ~o sodium dodecyl sulfate-6.5 ~o polyacrylamide gels. (A) Coomassie brilliant blue stain, (B) Periodic acid-Schiff stain. Samples and amounts of protein applied are: (I) h u m a n erythrocyte membrane 6o pg, (2) aqueous phase 25/~g, (3) ethanol precipitates of the aqueous phase which contained 25/~g proteins, (4) interphase 60 t'g, and (5) chlorof o r m - m e t h a n o l phase.
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sodium dodecyl sulfate-7.5% polyacrylamide gels according to Weber and Osborn 15. The molecular weights for glycoprotein I, Component B, glycoprotein I I and glycoprotein III were found to be 58 ooo, 50 ooo, 37 ooo, and 24 ooo, respectively. Fig. 2 presents the electrophoretic analysis of the chloroform-methanol extracts. I°/0 sodium dodecyl sulfate~6.5°/0 polyacrylamide gels were used and the gels were stained with Coomassie brilliant blue for protein (Fig. 2A) and with the periodic acidSchiff reagent for carbohydrate (Fig. 2B). Glycoproteins I, II, and III are recovered in the aqueous phase of chloroform-methanol extracts almost quantitatively. The patterns also reveal that glycoprotein I, glycoprotein II, and glycoprotein III are precipitable by 90% ethanol at room temperature. In order to obtain clearly visible protein bands, 25/zg aqueous phase proteins were applied to the gels. Application of relatively large protein quantities leads to a retardation of the electrophoretic migration of glycoprotein III and two bands appear in the glycoprotein III region. Component A was present only in the interphase and Component B was present in two phases, aqueous phase and interphase. Component C is present in the chloroformmethanol phase only. Apart from the glycoproteins, the aqueous phase does not contain any additional protein components. The electrophoretic patterns of the aqueous phase components show complete correspondance in mobilities on periodic acid-Schiff stain and Coomassie brilliant blue stain. The recovery of ghost proteins in the aqueous phase of chloroform-methanol extracts was on the average 3.7~o. The range observed in seven preparations was from 2.6-4.90/0 . The recovery of sialic acid was 83% with a range from 79-90% in three preparations. Cholesterol was not recovered in detectable quantities; the sensitivity of the assays excluded the presence of more than o.15% of ghost cholesterol. Phosphorus recovery was on the average 3.3%- In the ethanol precipitates of the aqueous phase, however, phosphorus could not be detected; the sensitivity of the phosphorus assay indicated a molar N/P ratio of more than 300.
Blood group activities of erythrocyte membrane glycoproteins Table I presents the results of hemagglutination inhibition assays performed with ghosts and the aqueous phase of chloroform-methanol extracts. In the aqueous phase the blood group specificities A, B, H, I, S, s, M, and N were found. High recoveries in the aqueous phase were observed for the I, S, s, M, and N activities. Specific activities for A, B, I, S, s, M, and N antigens were increased in the aqueous phase. Recovery and specific activity of A blood group antigens in the aqueous phase were not significantly different when A-secretor and A-non-secretor were compared. Ethanol precipitates of the aqueous phase also contained A, B, H, I, S, s, M, and N activities. The chloroform-methanol phase is rich in membrane lipids. This fraction did not show any activities for I, S, s, M, and N, but contained inhibitory activities for ABH. Weak A, B, H, I, S, s, M, and N activities were observed in the interphase fraction which contained most of the membrane proteins except the main glycoproteins. The ethanol soluble fraction of the aqueous phase, the supernatant after precipitation with 90% ethanol, contained inhibitory activities for A and B, suggesting that at least parts of the A and B activities of the aqueous phase are attributable to contaminating A and B glycolipids. The aqueous phase of chloroform-methanol extracts also showed weak activity for the P antigen. Rh (D), k, and Fy a antigenic activities were not detected in the aqueous phase. Biochim. Biophys. Acta, 278 (1972) 271-28o
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SOLUBILIZATION OF ERYTHROCYTE GLYCOPROTEINS TABLE I
ACTIVITIES AND RECOVERIES OF BLOOD GROUP ANTIGENS IN THE AQUEOUS PHASE OF CHLOROFORM-METHANOL EXTRACTS Figures for activities a n d recoveries are m e a n values f r o m three p r e p a r a t i o n s (A-secretor, B, H, I, S, a n d M) or f r o m t w o p r e p a r a t i o n s (A-non-secretor, s a n d 1~). The activity is expressed as the m i n i m u m a m o u n t of p r o t e i n ( i n / , g ) which inhibits h e m a g g l u t i n a t i o n b y four h e m a g g l u t i n a t i n g doses of a n t i s e r a for A, B, I, S, and s antigens and b y t w o h e m a g g l u t i n a t i n g doses of antisera for M, N, a n d H antigens. Total i n h i b i t o r y activities are s h o w n as total h e m a g g l u t i n a t i o n u n i t s of g h o s t p r e p a r a t i o n s or a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l extracts, respectively, which were o b t a i n e d f r o m IO ml a c i d - c i t r a t e - d e x t r o s e blood. The recoveries of blood g r o u p s u b s t a n c e s in the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s are expressed as percentage of h e m a g g l u t i n a t i o n inhibition units of the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s against h e m a g g l u t i n a t i o n inhibition u n i t s of ghosts.
Antigen
A, secretor A, non-secretor B H I S s M N
Aqueous phase ofchloroform-methanol extracts
Ghosts
Activity (l~g/ml)
Total activity (hemagglutination units)
Recovery (%)
Activity (t~g/ml)
13 16 34 53 53 62 56 35 53
380 240 250 90 lO5 ioo 83 177 131
4° 32 29 9 75 77 75 93 73
i2o 14o 19o 12o iooo 12oo 99 ° 670 64o
Total activity (hemagglutination units) 960 74 ° 870 97 ° 14o 13o IiO I9o 18o
Separation of the glycoproteins with blood group activities The aqueous phases of chloroform-methanol extracts of erythrocyte ghosts from several persons with the phenotype A, MN, S were fractionated by gel filtration on Sephadex G-Ioo columns in the presence of 1% sodium dodecyl sulfate. A representative diagram is shown in Fig. 3. 1% sodium dodecyl sulfate- 7.5 % polyacrylamide gel electrophoresis revealed the presence ofglycoprotein I in Fraction I, glycoprotein II and Component B in Fraction II, and glycoprotein n I in Fraction I n (Fig. 3). Table n shows the results of hemagglutination inhibition assays performed with the fractionated materials from a representative experiment. In several experiments the highest activity for A antigen was always found in Fraction n I . Fraction n and, to a lesser degree, Fraction I also showed some A activity. The I antigenic activity was also associated with Fraction III. The M blood group antigen was associated with Fraction I. Peculiarly, in several experiments it was found that all three fractions strongly inhibited rabbit N-antisera. It is further noteworthy that S antigen was not associated with Fraction I. The strongest S antigenic activity was associated with Fraction III.
DISCUSSION
Human erythrocyte membranes have at least three electrophoretically distinguishable glycoprotein components in which most of the erythrocyte membrane sialic Biochim. Biophys. Acta, 278 (1972) 271-28o
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GPI GP 1[ GPlU
030
0.08
I
0.06
I
I CME
I
II
Ill
0.04
0.02
0.00
//.__j
250
A___~r
300
I
I
I
I
I
350
400
4 50
500
550
"~-
L
_____
600
Effluent Volume (ml) Fig. 3. E l u t i o n d i a g r a m of the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s (CME) fractionated b y gel filtration on a S e p h a d e x G - i o o c o l u m n in the presence of 1% s o d i u m dodecyl sulfate. 2.6 m g reduced a n d alkylated p r o t e i n s in 4.5 ml of 2% s o d i u m dodecyl sulfate were applied. Sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e gel electrophoresis p a t t e r n s stained b y periodic acid-Schiff are s h o w n in the u p p e r right. G P = glycoprotein. TABLE II DISTRIBUTION OF BLOOD GROUP SPECIFICITIES IN ERYTHROCYTE GLYCOPROTEINS FRACTIONATED BY GEL FILTRATION ON SODIUM DODECYL SULFATE SEPHADEX G-Ioo COLUMNS. RESULTS OF FIEMAGGLUTINATION INHIBITION TESTS*.
Tests were carried o u t at four h e m a g g l u t i n a t i n g doses of antisera.
Fraction I
(glycoprotein I) F r a c t i o n I I (glycoprotein II, B) F r a c t i o n I I I (glycoprotein I I I ) Control (saline) Protein c o n c e n t r a t i o n (/2g/ml) used for inhibition tests
A
I
M
N
S
+ +
+ + + +
--
--
+ + + +
+ -++++
+ + + -++++
+ + + + + + ++++
--++++
+ + -++++
5°
5°
ioo
IOO
60
acid is present 1,16. In this paper, the three main glycoproteins and one additional periodic acid-Schiff stain positive component are shown to be extractable without detectable contamination with other membrane proteins by a rather simple procedure. Electrophoretic patterns indicate a high degree of purity and sialic acid analysis as well as the comparison of the electrophoretic patterns show recovery of the glycoproteins in high yields. The procedure is also applicable for the solubilization and separation of other mammalian erythrocyte membrane glycoproteins 9. The M and N antigens of the human erythrocyte reside in an oligosaccharide side chain of a membrane glycoprotein 16-19. The data presented in this paper demonBiochim. Biophys. Acta, 278 (1972) 271-28o
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strate that the Ss and I antigens of the human erythrocyte membrane are glycoproteins also. The antigenic activities of Ss and I are recovered in high yields in the aqueous phase of chloroform-methanol extracts, similar to the MN activities. High recoveries of these antigens are associated with an increase in their specific activities. The interphase of chloroform-methanol extracts which contains most of the other membrane proteins with the exception of the main glycoproteins, possesses only weak activities for these antigens; the activities might be explained by contamination of the interphase with low concentrations of these glycoproteins. The lack of Ss and I activities in the chloroform-methanol phase indicates that these antigens are not lipid or glycolipid in nature. Recently, several laboratories have shown that the ABH blood group antigen of the erythrocyte membrane may be present, in part, as a glycoprotein12,2°,~1. After chloroform-methanol extraction, moderate activities of A and B and low activities of H are recovered in the aqueous phase. There are no significant differences in recovered A activities between A-secretor and A-non-secretor. Part of the A and B activities of the aqueous phase can be extracted by 9O~o ethanol. This observation suggests that the aqueous phase of chloroform-methanol extracts contains a certain amount of AB glycolipids. The precipitate after 90% ethanol extraction of the aqueous phase, however, reveals ABH activities also, including the precipitate of the aqueous phase obtained from A-non-secretors. The 90% ethanol.precipitate contained all of the extracted glycoproteins but phosphorus (phospholipids) and cholesterol were not detected. A antigenic activity was also found in ethanol precipitates of fractions of the aqueous phase, obtained by gel filtration in the presence of I °/o sodium dodecyl sulfate. The highest A activity was found in glycoprotein III. The serologic activity may, thus, reside in a carbohydrate side chain of a specific glycoprotein or may be due to the A glycolipid which in that case, should be tightly bound to a specific glycoprotein. A definitive decision as to whether the erythrocyte membrane contains an ABH glycoprotein should await analysis for the presence of sphingosine or fatty acids in the ABH active glycoprotein preparation. The electrophoretic and immunologic analysis of the fractions obtained by gel filtration on I % sodium dodecyl sulfate-Sephadex G-Ioo columns indicates that it is possible to separate the erythrocyte membrane glycoproteins and to demonstrate that different glycoproteins are associated with different blood group antigens. The data suggest that the major glycoprotein, glycoprotein I, represents the so-called MN glycoprotein (refs 16-19, and H. Cleve, H. Hamaguchi and T. Htitteroth, unpublished). Glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 ooo, appears to be associated with the I and S activities. It is interesting to note that the M and S activities were separated by gel filtration. Glycoprotein III is also associated with the A antigenic activity. ACKNOWLEDGEMENTS
We thank Dr Alexander G. Bearn for his continuous advice and support. For expert technical assistance, we would like to thank Mrs Y. Thompson and Miss L. Bowes. We thank Mrs P. Geffner for the preparation of the manuscript. This work was supported by United States Public Health Service Grant: AM 11796 and aided by a grant from the National Foundation March of Dimes. Biochim. Biophys. Acta, 278 (1972) 271-28o
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REFERENCES I G. Fairbanks, T. L. Steck and D. F. H. Wallach, Biochemistry, IO (1971) 26o6. 2 T, L. Steck, G. F a i r b a n k s and D. F. H. Wallach, Biochemistry, io (1971) 2617. 3 M. S. Bretscher, Nature New Biol., 231 (1971) 229. 4 R. Kornfeld a n d S. Kornfeld, J. Biol. Chem., 245 (197 o) 2536. 5 0 . H. Lowry, N. J. Rosebrough, A. L. F a r r and R. J. Randall, J. Biol. Chem., 193 (1951) 2656 L. W a r r e n , J. Biol. Chem., 234 (1959) 1971. 7 G. R. Bartlett, J. Biol. Chem., 234 (1959) 466. 8 A. Zlatkis, B. Zak a n d A. J. Boyle, J. Lab. Clin. Med., 41 (1953) 486. 9 H. H a m a g u c h i a n d H. Cleve, Biochem. Biophys. Res. Commun., 47 (1972) 459. io H. H a m a g u c h i a n d H. Cleve, Biochim. Biophys. Acta, 233 (1971) 320. i i R. IV[. Zacharius, T. E. Zell, J. H. iV[orrison and J. J. Woodlock, Anal. Biochem., 3 ° (1969) 148. 12 A. Gardas a n d J. Kogcielak, Vox Sang., 20 (1971) 137. 13 M. S. Bretscher, J. Mol. Biol., 58 (1971) 775. 14 J. Lenard, Biochemistry, 9 (197 °) 1129. 15 K. W e b e r and M. Osborn, J. Biol. Chem., 244 (1969) 4406. 16 R. J. Winzler, in G. A. J a m i e s o n and T. J. Greenwalt, Red Cell Membrane, Structure and Function, Lippincott, Philadelphia and Toronto, 1969, p. 157. 17 R. H. K a t h a n and A. A d a m a n y , J. Biol. Chem., 242 (1967) 1716. 18 G. F. Springer, Y. Nagai a n d H. Tegtmeyer, Biochemistry, 5 (1966) 3254 • 19 E. Lisowska, Eur. J. Biochem., IO (1969) 574. 20 N. B. W h i t t e m o r e , N. C. Trabold, C. F. Reed and R. I. Weed, Vox Sang., 17 (1969) 289. 21 M. D. Poulik and C. Bron, in G. A. J a m i e s o n a n d T. J. Greenwalt, Red Cell Membrane, Structure and Function, Lippincott, Philadelphia a n d Toronto, 1969, p. 131.
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BIOCHIMICA ET BIOPHYSICA ACTA BBA
36201
SOLUBILIZATION OF HUMAN E R Y T H R O C Y T E MEMBRANE GLYCOP R O T E I N S AND SEPARATION OF T H E MN GLYCOPROTEIN FROM A GLYCOPROTEIN W I T H I, S, AND A ACTIVITY*
H. H A M A G U C H I AND H. C L E V E
Division of Human Genetics, Department of Medicine, Cornell University Medical College, New York, N.Y. (U.S.A.) (Received March 23rd, 1972)
SUMMARY
Human erythrocyte membrane glycoproteins are recovered in high yields in the aqueous phase after extraction of the membranes with a mixture of chloroformmethanol. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the aqueous phase reveals the presence of three main glycoprotein components, glycoproteins I, II, and III, and one additional periodic acid-Schiff positive component, without contamination by other membrane proteins. Their apparent molecular weights correspond to 58 ooo, 37 ooo, 24 ooo, and 50 ooo, respectively. The blood group antigens I, Ss, and MN are recovered in the aqueous phase in high yields and with marked increase in their specific activities. The aqueous phase also contains moderate amounts of A and B and small amounts of H antigenic activities. Further separation of the glycoproteins of the aqueous phase was achieved by gel filtration on Sephadex G-ioo columns in the presence of I % dodecyl sulfate. The M and N antigenic activities were found in glycoprotein I. N activity as tested with a rabbit antiserum was also observed in glycoprotein III. The highest activities for A, I, and S antigens were found in glycoprotein III. The chloroform-methanol phase does not contain glycoproteins; only the blood group antigens A, B, and H are found in this phase. The interphase, which contains most of the membrane proteins except the main glycoproteins, shows only weak ABH, I, Ss, and MN activities. The data indicate that the major glycoprotein of the human erythrocyte membrane, glycoprotein I, is the so-called MN glycoprotein, and that the I and S antigenic activities are associated with glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 ooo. Glycoprotein III is also associated with the A antigenic activity.
* Presented in p a r t a t t h e 4th International Congress of H u m a n Genetics Paris, September 4 - I I , I971.
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H. HAMAGUCHI, H. CLEVE
INTRODUCTION
Recently, three electrophoretically distinguishable glycoprotein components have been demonstrated in the human erythrocyte membrane and all appear to extend from the external cell surface through the cell membrane to the inner surface 1-s. In the course of our studies of the human erythrocyte membrane, we noticed that these three glycoproteins and one additional glycoprotein component can be quantitatively recovered in the aqueous phase after extraction with a mixture, of chloroformmethanol. The preparations were almost completely free from contamination with other membrane proteins and the glycoprotein components contained the blood group specificities ABH, I, Ss, and MN. In this paper, the procedures for solubilization and separation of human erythrocyte membrane glycoproteins and their blood group activities are described. METHODS
Preparation of human erythrocyte membrane glycoproteins H u m a n blood of individual donors in acid-citrate dextrose, less than two weeks old, was used as starting material. Membrane glycoproteins were prepared b y a method adapted from Kornfeld and Kornfeld 4. The blood was centrifuged at 15oo rev./min for 15 min. Plasma and buffy coat were removed by aspiration. Erythrocytes were then suspended in 0.9% NaC1- 5 mM Tris-HC1 buffer at p H 7.4 and washed three times b y centrifugation at 15oo rev./min for IO min. Some erythrocytes were discarded in all wash steps to ensure complete removal of the buffy coat. The packed erythrocytes were hemolyzed in a 9-fold volume of IO mM Tris-o.i mM EDTA-HC1 buffer, p H 7.4. Ghosts were sedimented by centrifugation at 20 ooo × g for 20 min. Ghosts were washed four times by centrifugation at 2o ooo × g for 20 min in 2o-fold volume of the T r i s - E D T A buffer. The washed ghosts were mechanically homogenized with a Dounce homogenizer and diluted to a protein concentration of 2 mg/ml with IO mM Tris-o.I mM EDTA-HC1 buffer, p H 7.4. The entire procedure was performed at temperatures of 0- 4 °C. To one volume of the ghost suspension, nine volumes of a chloroform-methanol mixture (2:1, v/v) were added. The mixture was stirred vigorously at room temperature for 30 rain and centrifuged afterwards at 15oo rev./ rain for IO min at room temperature. The aqueous layer was carefully aspirated and centrifuged again under the same conditions in order to remove contaminating interphase materials. The aqueous phase was concentrated in a rotary evaporator at 37 °C. to between one-tenth and one-forth the volume of the original ghost suspension. The concentrated aqueous phase was clear. Turbidity of the sample at this stage indicated the presence of contaminating interphase components. Ethanol precipitates of the aqueous phase components were prepared by addition of 9 vol. of ethanol, to I vol. of aqueous phase, followed by centrifugation at 3000 rev./min for 20 min at room temperature. The precipitates were air dried and distilled water was added; the obtained solution was clear. The interphase and chloroform-methanol phase were resuspended in distilled water and concentrated in a rotary evaporator. The procedure permits evaporation of organic solvents. Separation of the aqueous phase components, mostly glycoproteins, was carried out at room temperature by gel filtration on Sephadex G-Ioo columns in the presence
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of sodium dodecyl sulfate. The column, 4 cm × 115 cm, was packed and equilibrated with a 10/0 (w/v) sodium dodecyl sulfate solution in distilled water. Before application, the aqueous phase components were reduced for 4 h b y 1°/0 2-mercaptoethanol in the presence of 3°/0 sodium dodecyl sulfate and subsequently alkylated b y o.i M iodoacetic acid at p H 8.6 for I h at room temperature in the dark. p H was adjusted by addition of Tris-base. The flow rate of the column was 15-3o ml/h. The eluted materials were divided into three fractions as indicated (see below, Fig. 3), and concentrated b y ultrafiltration at room temperature to between one-tenth and one-sixth of the volume of the original eluates. Analytical methods Protein concentrations were measured b y the method of Lowry et al. ~. Three × crystallized bovine pancreatic ribonuclease was used as a standard. Sialic acid was assayed b y the method of Warren 6. Phosphorus was measured b y the method of Bartlett 7 and cholesterol was assayed by the method of Zlatkis et al. s. Sodium dodecyl sulfate-polyacrylamide gel electr0phoresis was performed in 1°/0 sodium dodecyl sulfate-o.I M sodium phosphate buffer at p H 7.1. The acrylamide concentration was either 7.5% or 6.5%. Tubes of 5 m m diameter and 12 cm length were used. Solubilization of samples and preparation of gels were performed according to the method described previously 9. Electrophoresis of the 7.5% gels was carried out at 3 V/cm for 8 h. The 7.5~o gels were fixed overnight b y 12.5% trichloroacetic acid dissolved in 50% ethanol and immersed in distilled water for i h. The gels were stained with 0.25% Coomassie brilliant blue for IO h 1°, or with the periodic acidSchiff reagent 11. In the 6.5% acrylamide gel system, electrophoresis was started with a current of 5 mA per tube. After 30 min the current was raised to IO mA per tube. Electrophoresis was stopped as soon as the B P B reached the 8.0 cm m a r k from the origin. Gels were stained with Coomassie brilliant blue or with the periodic acidSchiff reagent b y the method of Fairbanks et al3. The patterns of the erythrocyte membrane proteins were essentially identical in the two electrophoretic systems described. Blood group activities of glycoproteins were measured by hemagglutination inhibition tests. Two-fold serial dilutions were tested b y the conventional test tube method in the presence of four hemagglutination doses of antisera for A, B, I, S, and s antigens and two hemagglutination doses of antisera for M and N antigens and of anti-H lectin for H antigen. Samples in 0.9% NaC1 and antisera were incubated at 37 °C for I h for A, B. S, s, M, and N activities and at 4 °C for I h and 2 h for I and H activities, respectively. After these incubation periods, erythrocytes in 0.9°/0 NaC1 were added. The total volume of the mixture was 80-200/zl. The mixtures were incubated further at 37 °C for I h for A, B, S, s, M, and N activities and at 4 °C for I h, and 2 h for I and H activities, respectively. Readings were taken after centrifugation at 1500 rev./min for I min. Presence of s antigen was determined by indirect Coombs test. In order to calculate the approximate recoveries of blood group activities, total hemagglutination inhibition units were determined according to Gardas and Kogcielak 12. Removal of sodium dodecyl sulfate and measurement of serological activities of glycoproteins fractionated by gel filtration were performed as follows : To I vol. of concentrated fraction (40-200/zl) in test tubes with I.O cm diameter and 7.5 cm length, 9 vol. of ethanol were added and centrifuged at 2500 rev./min for 30 min at room Biochim. Biophys. Acta, 278 (1972) 271-28o
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H. HAMAGUCHI, H. CLEVE
temperature. The supernatant was carefully decanted, I ml of 9 o~o ethanol was added, and centrifugation at 2500 rev./min for 30 min was repeated. After decantation of the supernatant, the tubes were put upside-down on filter paper for 3o min. The ethanolwashed precipitates did not cause hemolysis and could be tested for inhibition activities. 5 o - I o o / A of antiserum with eight hemagglutination doses were added to the precipitates in the tubes. The precipitates dissolved in the antiserum. The tubes were shaken overnight at 4 °C to ensure the reaction of antibody and inhibiting components. Equal volumes of a 2% suspension of erythrocytes were added. Hemagglutination inhibition tests were read after appropriate incubation times. Human anti-A, anti-B and anti-S sera, rabbit anti-M, anti-N, and anti-immunoglobulin sera, and anti-H lectin (Ulex europeaus) were purchased from Behring Diagnostics, Inc. Human anti-s serum for indirect Coombs test was purchased from Pfizer Inc. A human cold agglutinin was used as anti-I reagent. RESULTS
Electrophoretic and chemical analysis of erythrocyte membrane glycoproteins Fig. I presents the 1% sodium dodecyl sulfate-7.5~/o polyacrylamide gel electro-
Fig. I. E l e c t r o p h o r e t i c p a t t e r n s of h u m a n e r y t h r o c y t e m e m b r a n e s on I ~o s o d i u m dodecyl s u l f a t e 7.5~o p o l y a c r y l a m i d e gels. (i) C o o m a s s i e brilliant blue stain, (2) Periodic a c i d - S c h i f f stain. G P = glycoprotein.
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phoresis patterns of human erythrocyte membranes. Staining with Coomassie brilliant blue reveals the presence of at least 15 polypeptide components in red cell ghosts. Components I-VI are designated in accordance with the notation used by Fairbanks et al. 1. In order to show the periodic acid-Schiff stain positive components clearly, 15o pg of the membrane proteins were applied to the gel. The periodic acid-Schiff positive proteins are named glycoproteins I, II, and III. The less intensely stained zones and glycolipid components are named Components A, B, and C. Glycoproteins I, II, and III correspond to the components periodic acid-Schiff-I, periodic acid-Schiff-2, and periodic acid-Schiff-3, respectively, described as by Fairbanks et al. 1. Component III corresponds to the component a reported by BretscherS, 13. Component A reacts only weakly with the periodic acid-Schiff reagent. Electrophoretically, Components III and A show complete correspondance. It may be assumed that they are identical and that their carbohydrate content is relatively low. On sodium dodecyl sulfate gel electrophoresis, Component B is not clearly separated from glycoprotein I. By gel filtration, however, the two components were more distinctly separated (see below, Fig. 3). The periodic acid-Schiff positive Component C is supposed to be a glycolipid 14. Coomassie blue stain of this component is positive. Apparent molecular weights of the glycoproteins were determined from the migration rates of the proteins in i%
A
Fig. 2. ElectrophoreUc p a t t e r n s of the three phases obtained by chloroform-methanol extraction of h u m a n erythrocyte membranes, on I ~o sodium dodecyl sulfate-6.5 ~o polyacrylamide gels. (A) Coomassie brilliant blue stain, (B) Periodic acid-Schiff stain. Samples and amounts of protein applied are: (I) h u m a n erythrocyte membrane 6o pg, (2) aqueous phase 25/~g, (3) ethanol precipitates of the aqueous phase which contained 25/~g proteins, (4) interphase 60 t'g, and (5) chlorof o r m - m e t h a n o l phase.
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H. HAMAGUCHI, H. CLEVE
sodium dodecyl sulfate-7.5% polyacrylamide gels according to Weber and Osborn 15. The molecular weights for glycoprotein I, Component B, glycoprotein I I and glycoprotein III were found to be 58 ooo, 50 ooo, 37 ooo, and 24 ooo, respectively. Fig. 2 presents the electrophoretic analysis of the chloroform-methanol extracts. I°/0 sodium dodecyl sulfate~6.5°/0 polyacrylamide gels were used and the gels were stained with Coomassie brilliant blue for protein (Fig. 2A) and with the periodic acidSchiff reagent for carbohydrate (Fig. 2B). Glycoproteins I, II, and III are recovered in the aqueous phase of chloroform-methanol extracts almost quantitatively. The patterns also reveal that glycoprotein I, glycoprotein II, and glycoprotein III are precipitable by 90% ethanol at room temperature. In order to obtain clearly visible protein bands, 25/zg aqueous phase proteins were applied to the gels. Application of relatively large protein quantities leads to a retardation of the electrophoretic migration of glycoprotein III and two bands appear in the glycoprotein III region. Component A was present only in the interphase and Component B was present in two phases, aqueous phase and interphase. Component C is present in the chloroformmethanol phase only. Apart from the glycoproteins, the aqueous phase does not contain any additional protein components. The electrophoretic patterns of the aqueous phase components show complete correspondance in mobilities on periodic acid-Schiff stain and Coomassie brilliant blue stain. The recovery of ghost proteins in the aqueous phase of chloroform-methanol extracts was on the average 3.7~o. The range observed in seven preparations was from 2.6-4.90/0 . The recovery of sialic acid was 83% with a range from 79-90% in three preparations. Cholesterol was not recovered in detectable quantities; the sensitivity of the assays excluded the presence of more than o.15% of ghost cholesterol. Phosphorus recovery was on the average 3.3%- In the ethanol precipitates of the aqueous phase, however, phosphorus could not be detected; the sensitivity of the phosphorus assay indicated a molar N/P ratio of more than 300.
Blood group activities of erythrocyte membrane glycoproteins Table I presents the results of hemagglutination inhibition assays performed with ghosts and the aqueous phase of chloroform-methanol extracts. In the aqueous phase the blood group specificities A, B, H, I, S, s, M, and N were found. High recoveries in the aqueous phase were observed for the I, S, s, M, and N activities. Specific activities for A, B, I, S, s, M, and N antigens were increased in the aqueous phase. Recovery and specific activity of A blood group antigens in the aqueous phase were not significantly different when A-secretor and A-non-secretor were compared. Ethanol precipitates of the aqueous phase also contained A, B, H, I, S, s, M, and N activities. The chloroform-methanol phase is rich in membrane lipids. This fraction did not show any activities for I, S, s, M, and N, but contained inhibitory activities for ABH. Weak A, B, H, I, S, s, M, and N activities were observed in the interphase fraction which contained most of the membrane proteins except the main glycoproteins. The ethanol soluble fraction of the aqueous phase, the supernatant after precipitation with 90% ethanol, contained inhibitory activities for A and B, suggesting that at least parts of the A and B activities of the aqueous phase are attributable to contaminating A and B glycolipids. The aqueous phase of chloroform-methanol extracts also showed weak activity for the P antigen. Rh (D), k, and Fy a antigenic activities were not detected in the aqueous phase. Biochim. Biophys. Acta, 278 (1972) 271-28o
277
SOLUBILIZATION OF ERYTHROCYTE GLYCOPROTEINS TABLE I
ACTIVITIES AND RECOVERIES OF BLOOD GROUP ANTIGENS IN THE AQUEOUS PHASE OF CHLOROFORM-METHANOL EXTRACTS Figures for activities a n d recoveries are m e a n values f r o m three p r e p a r a t i o n s (A-secretor, B, H, I, S, a n d M) or f r o m t w o p r e p a r a t i o n s (A-non-secretor, s a n d 1~). The activity is expressed as the m i n i m u m a m o u n t of p r o t e i n ( i n / , g ) which inhibits h e m a g g l u t i n a t i o n b y four h e m a g g l u t i n a t i n g doses of a n t i s e r a for A, B, I, S, and s antigens and b y t w o h e m a g g l u t i n a t i n g doses of antisera for M, N, a n d H antigens. Total i n h i b i t o r y activities are s h o w n as total h e m a g g l u t i n a t i o n u n i t s of g h o s t p r e p a r a t i o n s or a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l extracts, respectively, which were o b t a i n e d f r o m IO ml a c i d - c i t r a t e - d e x t r o s e blood. The recoveries of blood g r o u p s u b s t a n c e s in the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s are expressed as percentage of h e m a g g l u t i n a t i o n inhibition units of the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s against h e m a g g l u t i n a t i o n inhibition u n i t s of ghosts.
Antigen
A, secretor A, non-secretor B H I S s M N
Aqueous phase ofchloroform-methanol extracts
Ghosts
Activity (l~g/ml)
Total activity (hemagglutination units)
Recovery (%)
Activity (t~g/ml)
13 16 34 53 53 62 56 35 53
380 240 250 90 lO5 ioo 83 177 131
4° 32 29 9 75 77 75 93 73
i2o 14o 19o 12o iooo 12oo 99 ° 670 64o
Total activity (hemagglutination units) 960 74 ° 870 97 ° 14o 13o IiO I9o 18o
Separation of the glycoproteins with blood group activities The aqueous phases of chloroform-methanol extracts of erythrocyte ghosts from several persons with the phenotype A, MN, S were fractionated by gel filtration on Sephadex G-Ioo columns in the presence of 1% sodium dodecyl sulfate. A representative diagram is shown in Fig. 3. 1% sodium dodecyl sulfate- 7.5 % polyacrylamide gel electrophoresis revealed the presence ofglycoprotein I in Fraction I, glycoprotein II and Component B in Fraction II, and glycoprotein n I in Fraction I n (Fig. 3). Table n shows the results of hemagglutination inhibition assays performed with the fractionated materials from a representative experiment. In several experiments the highest activity for A antigen was always found in Fraction n I . Fraction n and, to a lesser degree, Fraction I also showed some A activity. The I antigenic activity was also associated with Fraction III. The M blood group antigen was associated with Fraction I. Peculiarly, in several experiments it was found that all three fractions strongly inhibited rabbit N-antisera. It is further noteworthy that S antigen was not associated with Fraction I. The strongest S antigenic activity was associated with Fraction III.
DISCUSSION
Human erythrocyte membranes have at least three electrophoretically distinguishable glycoprotein components in which most of the erythrocyte membrane sialic Biochim. Biophys. Acta, 278 (1972) 271-28o
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H. HAMAGUCHI, H. CLEVE
GPI GP 1[ GPlU
030
0.08
I
0.06
I
I CME
I
II
Ill
0.04
0.02
0.00
//.__j
250
A___~r
300
I
I
I
I
I
350
400
4 50
500
550
"~-
L
_____
600
Effluent Volume (ml) Fig. 3. E l u t i o n d i a g r a m of the a q u e o u s p h a s e of c h l o r o f o r m - m e t h a n o l e x t r a c t s (CME) fractionated b y gel filtration on a S e p h a d e x G - i o o c o l u m n in the presence of 1% s o d i u m dodecyl sulfate. 2.6 m g reduced a n d alkylated p r o t e i n s in 4.5 ml of 2% s o d i u m dodecyl sulfate were applied. Sodium dodecyl s u l f a t e - p o l y a c r y l a m i d e gel electrophoresis p a t t e r n s stained b y periodic acid-Schiff are s h o w n in the u p p e r right. G P = glycoprotein. TABLE II DISTRIBUTION OF BLOOD GROUP SPECIFICITIES IN ERYTHROCYTE GLYCOPROTEINS FRACTIONATED BY GEL FILTRATION ON SODIUM DODECYL SULFATE SEPHADEX G-Ioo COLUMNS. RESULTS OF FIEMAGGLUTINATION INHIBITION TESTS*.
Tests were carried o u t at four h e m a g g l u t i n a t i n g doses of antisera.
Fraction I
(glycoprotein I) F r a c t i o n I I (glycoprotein II, B) F r a c t i o n I I I (glycoprotein I I I ) Control (saline) Protein c o n c e n t r a t i o n (/2g/ml) used for inhibition tests
A
I
M
N
S
+ +
+ + + +
--
--
+ + + +
+ -++++
+ + + -++++
+ + + + + + ++++
--++++
+ + -++++
5°
5°
ioo
IOO
60
acid is present 1,16. In this paper, the three main glycoproteins and one additional periodic acid-Schiff stain positive component are shown to be extractable without detectable contamination with other membrane proteins by a rather simple procedure. Electrophoretic patterns indicate a high degree of purity and sialic acid analysis as well as the comparison of the electrophoretic patterns show recovery of the glycoproteins in high yields. The procedure is also applicable for the solubilization and separation of other mammalian erythrocyte membrane glycoproteins 9. The M and N antigens of the human erythrocyte reside in an oligosaccharide side chain of a membrane glycoprotein 16-19. The data presented in this paper demonBiochim. Biophys. Acta, 278 (1972) 271-28o
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279
strate that the Ss and I antigens of the human erythrocyte membrane are glycoproteins also. The antigenic activities of Ss and I are recovered in high yields in the aqueous phase of chloroform-methanol extracts, similar to the MN activities. High recoveries of these antigens are associated with an increase in their specific activities. The interphase of chloroform-methanol extracts which contains most of the other membrane proteins with the exception of the main glycoproteins, possesses only weak activities for these antigens; the activities might be explained by contamination of the interphase with low concentrations of these glycoproteins. The lack of Ss and I activities in the chloroform-methanol phase indicates that these antigens are not lipid or glycolipid in nature. Recently, several laboratories have shown that the ABH blood group antigen of the erythrocyte membrane may be present, in part, as a glycoprotein12,2°,~1. After chloroform-methanol extraction, moderate activities of A and B and low activities of H are recovered in the aqueous phase. There are no significant differences in recovered A activities between A-secretor and A-non-secretor. Part of the A and B activities of the aqueous phase can be extracted by 9O~o ethanol. This observation suggests that the aqueous phase of chloroform-methanol extracts contains a certain amount of AB glycolipids. The precipitate after 90% ethanol extraction of the aqueous phase, however, reveals ABH activities also, including the precipitate of the aqueous phase obtained from A-non-secretors. The 90% ethanol.precipitate contained all of the extracted glycoproteins but phosphorus (phospholipids) and cholesterol were not detected. A antigenic activity was also found in ethanol precipitates of fractions of the aqueous phase, obtained by gel filtration in the presence of I °/o sodium dodecyl sulfate. The highest A activity was found in glycoprotein III. The serologic activity may, thus, reside in a carbohydrate side chain of a specific glycoprotein or may be due to the A glycolipid which in that case, should be tightly bound to a specific glycoprotein. A definitive decision as to whether the erythrocyte membrane contains an ABH glycoprotein should await analysis for the presence of sphingosine or fatty acids in the ABH active glycoprotein preparation. The electrophoretic and immunologic analysis of the fractions obtained by gel filtration on I % sodium dodecyl sulfate-Sephadex G-Ioo columns indicates that it is possible to separate the erythrocyte membrane glycoproteins and to demonstrate that different glycoproteins are associated with different blood group antigens. The data suggest that the major glycoprotein, glycoprotein I, represents the so-called MN glycoprotein (refs 16-19, and H. Cleve, H. Hamaguchi and T. Htitteroth, unpublished). Glycoprotein III, a minor glycoprotein with an apparent molecular weight of 24 ooo, appears to be associated with the I and S activities. It is interesting to note that the M and S activities were separated by gel filtration. Glycoprotein III is also associated with the A antigenic activity. ACKNOWLEDGEMENTS
We thank Dr Alexander G. Bearn for his continuous advice and support. For expert technical assistance, we would like to thank Mrs Y. Thompson and Miss L. Bowes. We thank Mrs P. Geffner for the preparation of the manuscript. This work was supported by United States Public Health Service Grant: AM 11796 and aided by a grant from the National Foundation March of Dimes. Biochim. Biophys. Acta, 278 (1972) 271-28o
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H. HAMAGUCHI, H. CLEVE
REFERENCES I G. Fairbanks, T. L. Steck and D. F. H. Wallach, Biochemistry, IO (1971) 26o6. 2 T, L. Steck, G. F a i r b a n k s and D. F. H. Wallach, Biochemistry, io (1971) 2617. 3 M. S. Bretscher, Nature New Biol., 231 (1971) 229. 4 R. Kornfeld a n d S. Kornfeld, J. Biol. Chem., 245 (197 o) 2536. 5 0 . H. Lowry, N. J. Rosebrough, A. L. F a r r and R. J. Randall, J. Biol. Chem., 193 (1951) 2656 L. W a r r e n , J. Biol. Chem., 234 (1959) 1971. 7 G. R. Bartlett, J. Biol. Chem., 234 (1959) 466. 8 A. Zlatkis, B. Zak a n d A. J. Boyle, J. Lab. Clin. Med., 41 (1953) 486. 9 H. H a m a g u c h i a n d H. Cleve, Biochem. Biophys. Res. Commun., 47 (1972) 459. io H. H a m a g u c h i a n d H. Cleve, Biochim. Biophys. Acta, 233 (1971) 320. i i R. IV[. Zacharius, T. E. Zell, J. H. iV[orrison and J. J. Woodlock, Anal. Biochem., 3 ° (1969) 148. 12 A. Gardas a n d J. Kogcielak, Vox Sang., 20 (1971) 137. 13 M. S. Bretscher, J. Mol. Biol., 58 (1971) 775. 14 J. Lenard, Biochemistry, 9 (197 °) 1129. 15 K. W e b e r and M. Osborn, J. Biol. Chem., 244 (1969) 4406. 16 R. J. Winzler, in G. A. J a m i e s o n and T. J. Greenwalt, Red Cell Membrane, Structure and Function, Lippincott, Philadelphia and Toronto, 1969, p. 157. 17 R. H. K a t h a n and A. A d a m a n y , J. Biol. Chem., 242 (1967) 1716. 18 G. F. Springer, Y. Nagai a n d H. Tegtmeyer, Biochemistry, 5 (1966) 3254 • 19 E. Lisowska, Eur. J. Biochem., IO (1969) 574. 20 N. B. W h i t t e m o r e , N. C. Trabold, C. F. Reed and R. I. Weed, Vox Sang., 17 (1969) 289. 21 M. D. Poulik and C. Bron, in G. A. J a m i e s o n a n d T. J. Greenwalt, Red Cell Membrane, Structure and Function, Lippincott, Philadelphia a n d Toronto, 1969, p. 131.
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