Vox Sang. 28: 122-132 (1975)
Reactions of Erythrocyte Glycoproteins and their Degradation Products with various Anti-I Sera ELWIRALISOWSKA, WANDADZIER~KOWA-BORODU, HALINASEYFRIED and ZOFIADRZENIEK Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, and Laboratory Department, District Blood Transfusion Centre, Wroclaw, and Department of Serology, Institute of Haematology, Warszawa
Abstract. Three fractions of erythrocyte glycoproteins obtained from Sepharose 4-B chromatography were tested for I activity with ten serologically differentiated anti-I sera. The most active was fraction I, eluted at the void volume and containing the lowest amount of alkali-labile oligosaccharide chains. The desialization of glycoproteins increased their activity toward anti-1' and anti-ID sera, and did not change or decreased the activity toward anti-IF sera. The most abundant fraction I1 (major sialoglycoprotein of erythrocyte membranes) showed no or only a very weak I activity, but I-active glycopeptides were isolated from products of digestion of fraction I1 with trypsin. The major product of digestion, sialoglycopeptide IIT-2 showed I activity only after alkaline elimination of alkali-labile oligosaccharide chains. The results indicate that I receptors are present in hindered form on apparently I-inactive components of erythrocyte membrane.
In our earlier studies we found that MN-active glycoproteins from human erythrocyte membrane inhibited weakly anti-I sera and that this inhibitory activity was significantIy increased after desialization of glycoproteins [121. The I activity of human erythrocyte membrane glycoproteins was also reported by other authors [16, 24, 251. Erythrocyte glycoproteins contain two kinds of oligosaccharide chains : (1) alkali-labile chains consisting of galactose, N-acetylgalactosamine and large proportion of sialic acid; (2) alkali-stable chains containing galactose, N-acetylglucosamine, mannose, and low amount of sialic acid and fucose [l, 20, 30, 311. Alkali-stable chains were required for I activity [12], whereas MN activity depended on alkali-labile chains [20, 311. Human erythrocytes contain one major glycoprotein and few minor ones, as has been shown by SDS-polyacrylamide gel electrophoresis of whole membranes and of glycoprotein preparations obtained from membranes by Rceived: June 16, 1974; accepted: July 15, 1974.
ELWRALISOWSKAet al.
123
different methods [7,8,16,26,27,33]. Membrane glycoproteins, due to their asymmetric structure, have a strong tendency to aggregate in water solutions. It makes difficult the separation of individual components and when some of them were obtained as electrophoreticallypure, the yield of preparation was very low [8, 161. HAMAGUCHI and CLEW[I61 reported that I activity was associated with minor glycoprotein. GARDMand K ~ C I E L A [I51 K found that I receptors were located on ABH-active blood group substances of human erythrocytes and not on MN-active sialoglycoprotein. Recently the same was indicated by experiments of ANSTEE and TANNER [2]. The heterogeneity and differences revealed among anti-I sera point to the great complexity of the I system. The serologic characterization of 12 anti-I sera described in our former paper [131revealed in some of them one dominant specificity (anti-18, anti-ID or anti-I*) or in others the combination of few specificities, anti-i included. Looking for I-specific receptors in components of erythrocyte membrane it may be unsufficient to test the activity with one, or even few serologically uncharacterized anti-I sera, since different anti-I subspecificities may be directed to different receptors of red cells. To shed some light on this problem, glycoprotein fractions prepared from 01 erythrocytes and some products of their enzymic and chemical degradation were tested for the inhibitory activity with various serologicallycharacterized anti-I sera.
Materials and Methods Anti4 sera. 10 out of 12 sera described in our former paper [13] were used for haemagglutination-inhibitiontests. According to the serological characterization the sera showed one dominating type of specificity (Woj., anti-In; Gaj., anti-ID; Ful. and Sob., anti-19, or the combination of few specificities (Zg., anti-ID+anti-I8; Cib. and Pyt., anti-ID+anti18+anti-IF; Hot, anti-ID+ anti-IF; Syp. and Buc., anti-ID+anti-i). Glycoproteins were obtained by phenolextraction of OM and ON erythrocyte membranes [4] and fractionated by gel filtration on Sepharose 4-B column. The well reproducible elution pattern gave four distinct peaks. Fractions represented by peaks I-III were glycoproteins and were used for haemagglutination-inhibitionstudies. Fraction IV. comprising about 5% of starting material, contained non-glycoprotein impurities and was serologically inactive. Trypsin fragments of fraction If. Products of digestion of fraction I1 from Sepharose 4-B were fractionated on Bio-Gel P-300, giving two fractions: IIT-1 - highly aggregated heterogeneous fraction eluted at the void volume of the column; IIT-2 - retarded glycopeptide fraction, containing over 60% of carbohydrate components of digested glycoprotein. Fraction IIT-l was fractionated further on Bio-Gel P-100 column equilibrated with 1% SDS. Three glycopeptide fractions were obtained, the major fraction IIT-1-B was used for haemagglutination-inhibition studies. Fraction IIT-2 was purilied by gel filtration on Bio-Gel P-100 without SDS.
ELWIRA LISOWSKA ct al.
124
Crude gly-oprotein
I I
N II
'
lalk
Ill
1 % SDS, Sephadex G-100
Z N NaOH, 2 h, 100 OC, reacetylation, get filtration
Ialk
Sepharose 4 - 0
Ill-1-A
Ill-1-8
Ill-1-C
IV
0.05 N NaOH-1M NaBH4 16 h, 4 5 OC, Ion -exchange chromatography
I IT - Z a l k -1
Fig. 1. Fractionation of erythrocyte glycoproteins and products of their degradation.
Alkaline-borohydride degradation. Fraction I was heated for 2 h in 2 N NaOH containing 1 M NaBH,, a t 100°C. The solution was neutralized with acetic acid and filtrated through Bio-Gel P-2 column. Carbohydrate-containing fractions (eluted at the void volume and free of salts and of the bulk of amino acids) were reacetylated with acetic anhydride at pH 7.5 at RT, concentrated in vacuo and rechromatographed on Bio-Gel P-6 column. Two carbohydratecontaining peaks were obtained, the eluates belonging to each peak were combined and lyophilized (Ialkl and I s l k 2). The major glycopeptide fraction of the trypsin digest of fraction I1 (IIT-2) was heated for 16 h at 45°C in 0.05 N NaOH containing 1 M NaBH, [18]. Glycopeptide containing exclusively alkali-stable oligosaccharides (11T-2s1k-1) was isolated from degradation products by ion-exchange chromatography on AG50 x 4(H+) and AG50 x l ( 0 H ) resins. All procedures concerning fractionation of glycoproteins and preparation of their et al., in preparation]. degradation products will be described in detail elsewhere [LISOWSKA Figure 1 presents the general scheme of fractionation and degradation of red cell glycoproteins. Desialization of samples was performed by mild acid hydrolysis, as described earlier [ 111. Analytical methods. Sialic acid was determined by periodate-resorcinol method [19], neutral sugars by phenol-sulphuric acid method [9], galactosamine and glucosamine by the slightly modified [20] method of LUDOWIEG and BENMAMAN [23]. Haemagglutination-inhibition tests. Twofold dilutions (25 pl) of samples tested for inhibitory activity were incubated with suitably diluted (to the penultimate dilution giving agglutination 3 +) anti-I sera (25 pl) for 1 h at l O T , after that 50 pl of 2% suspension of 01erythrocytes was added and agglutination was read microscopically after 2 h a t 10°C. The scale of agglutination was 3 2 + , + f,- . - and f were regarded as complete inhibition, + as partial inhibition, 2 and 3 as a lack of inhibition.
+,
+
+
I Receptors in Erythrocytes
125
Results Glycoprotein Fractionsfrom Sepharose 4-B The content of galactosamine and glucosamine in red cell glycoproteins is an indicator of the content of alkali-labile and alkali-stableoligosaccharide chains, respectively. As is shown in table I, fractions from Sepharose 4-B differed in proportions of both kinds of chains. fraction I showed the lowest content of sialic acid-rich alkali-labile oligosaccharides.This fraction showed also the highest I activity for all examined anti-I sera (fig. 2). Fractions I1 and I11 inhibited anti-I sera much weaker, however, fraction I11 was usually slightly more active than fraction 11. The distribution if M and N blood group activity among the fractions was entirely different: fractions I and I1 showed similar MN activity, equal to the activity of unfractionated glycoprotein, the activity of fraction I11 was lower (table I). The diagram (fig. 2) summarizing haemagglutination-inhibitionstudies of ten anti-I sera shows that the pattern of inhibition differed from one serum to another, which was compatible with individual differences between anti-I sera and also at certain degree with their subspecificities revealed by serological examination. The difference in the inhibition of anti-ID and anti-IF sera consisted in the different effect of removal of sialic acid from glycoproteins : desialization caused enhancement of ID (and IS) activity (sera Woj., Zg., Cib., Gaj.) and was without influence or even impaired IF activity (sera Ful., Sob.). The two sera containing anti-i besides anti-ID antibodies (Buc. and Syp.) were inhibited by fraction I similarly as anti-ID sera. Serum Buc. containing high proportion of anti-i antibodies was moreover much better inhibited by fractions I1 and I11 than anti-ID sera. The fraction I, showing the highest I activity, and fraction 11, representing the major sialoglycoprotein of erythrocyte membranes, were submitted to further examination. Fraction I To check whether I activity of fraction I was caused by contamination with the very highly I-active substance described by GARDAS and KOSCIELAK [151, the following experiments were performed. (1) Fraction I was chromatographed on DEAE-Sephadex A-50 under conditions used by GARDASand KOSCIELAK [I41 as an essential step for separation of ABH-I-active substance (eluted with 0.1 M NaCl) from MNactive sialoglycoprotein (eluted with 1 M NaCl). In our experiment only trace amount of sugars and no I activity were found in 0.1 M NaCl eluate.
ELWIRA LISOWSKA et al.
126 pg I rnl 40
Woj.
Cib.
Gaj.
BUC.
Ful.
HOE
Sob.
160
600
2,500 1234567 VX567
Pyt.
.. , . . ..
. . .. . . .
.
, . .. . .
...
.
Fig. 2. Inhibition of anti-I sera by glycoprotein fiactions. Inhibitory activity is given and partial (---) inhibition as a minimal concentration @g/ml) giving complete (-) of serum. 1:M-I, 2:M-11, 3:M-111, 4:N-I, 5:N-II, 6:N-111, 7:N-IIT-I-B, 1’-7‘:the same fractions desialized.
The I activity was found in 1M NaC1-eluted sialoglycoprotein, together with MN activity. (2) ABH- and I-active substance was reported to be stable under conditions of drastic alkaline degradation. The purpose of drastic alkaline degradation of M-I (as described in Materials and Methods) was to destroy most of sialoglycoprotein and to isolate the alkali-stable I-active substance. Two fractions (M-Ialk 1 and M-Ialk 2) isolated from degradation products contained carbohydrate components of alkalistable oligosaccharide chains and a very low amount of sialic acid. Only fraction M-Ialk 1 showed I activity, but weaker than the activity of undegraded fraction M-I (table 11). Fraction ZZ Digestion with trypsin increased I activity of fraction I1 and this I activity was found to be connected with aggregated product of digestion IIT-1, whereas the major (unaggregated) sialoglycopeptide IIT-2 did not inhibit anti-I sera, even after desialization, at conc. 10 mg/ml. The aggregated
I Receptors in Erythrocytes
127
Table I. carbohydrate composition (g/100 g) and MN activity of glycoprotein fractions from Sepharose 4-Bcolumn
M
N
crude I gp.
I1
10.0 20.7 Sialic acid 16.5 12.1 Neutral sugars 13.0 14.1 3.2 5.6 GalNH, 4.9 GlcNH, 2.3 3.6 2.4 GalNHJGlcNH, 2.1 0.9 2.3 Sialic acid/ neutral sugars 1.3 0.8 1.5 Activity M 80 160 80 Activity N 2,500 5,000 2,500
I11
crude
I
I11
I1
gP.
23.1 18.6 5.7 3.0 1.9
17.3 12.5 5.9 3.2 1.8
8.6 9.9 3.4 3.2 1.o
19.3 12.8 5.9 2.6 2.3
25.3 16.9 7.1 4.1 1.7
0.9 1.5 1.5 1.2 1.4 1,250
Reactions of Erythrocyte Glycoproteins and their Degradation Products with various Anti-I Sera ELWIRALISOWSKA, WANDADZIER~KOWA-BORODU, HALINASEYFRIED and ZOFIADRZENIEK Department of Immunochemistry, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, and Laboratory Department, District Blood Transfusion Centre, Wroclaw, and Department of Serology, Institute of Haematology, Warszawa
Abstract. Three fractions of erythrocyte glycoproteins obtained from Sepharose 4-B chromatography were tested for I activity with ten serologically differentiated anti-I sera. The most active was fraction I, eluted at the void volume and containing the lowest amount of alkali-labile oligosaccharide chains. The desialization of glycoproteins increased their activity toward anti-1' and anti-ID sera, and did not change or decreased the activity toward anti-IF sera. The most abundant fraction I1 (major sialoglycoprotein of erythrocyte membranes) showed no or only a very weak I activity, but I-active glycopeptides were isolated from products of digestion of fraction I1 with trypsin. The major product of digestion, sialoglycopeptide IIT-2 showed I activity only after alkaline elimination of alkali-labile oligosaccharide chains. The results indicate that I receptors are present in hindered form on apparently I-inactive components of erythrocyte membrane.
In our earlier studies we found that MN-active glycoproteins from human erythrocyte membrane inhibited weakly anti-I sera and that this inhibitory activity was significantIy increased after desialization of glycoproteins [121. The I activity of human erythrocyte membrane glycoproteins was also reported by other authors [16, 24, 251. Erythrocyte glycoproteins contain two kinds of oligosaccharide chains : (1) alkali-labile chains consisting of galactose, N-acetylgalactosamine and large proportion of sialic acid; (2) alkali-stable chains containing galactose, N-acetylglucosamine, mannose, and low amount of sialic acid and fucose [l, 20, 30, 311. Alkali-stable chains were required for I activity [12], whereas MN activity depended on alkali-labile chains [20, 311. Human erythrocytes contain one major glycoprotein and few minor ones, as has been shown by SDS-polyacrylamide gel electrophoresis of whole membranes and of glycoprotein preparations obtained from membranes by Rceived: June 16, 1974; accepted: July 15, 1974.
ELWRALISOWSKAet al.
123
different methods [7,8,16,26,27,33]. Membrane glycoproteins, due to their asymmetric structure, have a strong tendency to aggregate in water solutions. It makes difficult the separation of individual components and when some of them were obtained as electrophoreticallypure, the yield of preparation was very low [8, 161. HAMAGUCHI and CLEW[I61 reported that I activity was associated with minor glycoprotein. GARDMand K ~ C I E L A [I51 K found that I receptors were located on ABH-active blood group substances of human erythrocytes and not on MN-active sialoglycoprotein. Recently the same was indicated by experiments of ANSTEE and TANNER [2]. The heterogeneity and differences revealed among anti-I sera point to the great complexity of the I system. The serologic characterization of 12 anti-I sera described in our former paper [131revealed in some of them one dominant specificity (anti-18, anti-ID or anti-I*) or in others the combination of few specificities, anti-i included. Looking for I-specific receptors in components of erythrocyte membrane it may be unsufficient to test the activity with one, or even few serologically uncharacterized anti-I sera, since different anti-I subspecificities may be directed to different receptors of red cells. To shed some light on this problem, glycoprotein fractions prepared from 01 erythrocytes and some products of their enzymic and chemical degradation were tested for the inhibitory activity with various serologicallycharacterized anti-I sera.
Materials and Methods Anti4 sera. 10 out of 12 sera described in our former paper [13] were used for haemagglutination-inhibitiontests. According to the serological characterization the sera showed one dominating type of specificity (Woj., anti-In; Gaj., anti-ID; Ful. and Sob., anti-19, or the combination of few specificities (Zg., anti-ID+anti-I8; Cib. and Pyt., anti-ID+anti18+anti-IF; Hot, anti-ID+ anti-IF; Syp. and Buc., anti-ID+anti-i). Glycoproteins were obtained by phenolextraction of OM and ON erythrocyte membranes [4] and fractionated by gel filtration on Sepharose 4-B column. The well reproducible elution pattern gave four distinct peaks. Fractions represented by peaks I-III were glycoproteins and were used for haemagglutination-inhibitionstudies. Fraction IV. comprising about 5% of starting material, contained non-glycoprotein impurities and was serologically inactive. Trypsin fragments of fraction If. Products of digestion of fraction I1 from Sepharose 4-B were fractionated on Bio-Gel P-300, giving two fractions: IIT-1 - highly aggregated heterogeneous fraction eluted at the void volume of the column; IIT-2 - retarded glycopeptide fraction, containing over 60% of carbohydrate components of digested glycoprotein. Fraction IIT-l was fractionated further on Bio-Gel P-100 column equilibrated with 1% SDS. Three glycopeptide fractions were obtained, the major fraction IIT-1-B was used for haemagglutination-inhibition studies. Fraction IIT-2 was purilied by gel filtration on Bio-Gel P-100 without SDS.
ELWIRA LISOWSKA ct al.
124
Crude gly-oprotein
I I
N II
'
lalk
Ill
1 % SDS, Sephadex G-100
Z N NaOH, 2 h, 100 OC, reacetylation, get filtration
Ialk
Sepharose 4 - 0
Ill-1-A
Ill-1-8
Ill-1-C
IV
0.05 N NaOH-1M NaBH4 16 h, 4 5 OC, Ion -exchange chromatography
I IT - Z a l k -1
Fig. 1. Fractionation of erythrocyte glycoproteins and products of their degradation.
Alkaline-borohydride degradation. Fraction I was heated for 2 h in 2 N NaOH containing 1 M NaBH,, a t 100°C. The solution was neutralized with acetic acid and filtrated through Bio-Gel P-2 column. Carbohydrate-containing fractions (eluted at the void volume and free of salts and of the bulk of amino acids) were reacetylated with acetic anhydride at pH 7.5 at RT, concentrated in vacuo and rechromatographed on Bio-Gel P-6 column. Two carbohydratecontaining peaks were obtained, the eluates belonging to each peak were combined and lyophilized (Ialkl and I s l k 2). The major glycopeptide fraction of the trypsin digest of fraction I1 (IIT-2) was heated for 16 h at 45°C in 0.05 N NaOH containing 1 M NaBH, [18]. Glycopeptide containing exclusively alkali-stable oligosaccharides (11T-2s1k-1) was isolated from degradation products by ion-exchange chromatography on AG50 x 4(H+) and AG50 x l ( 0 H ) resins. All procedures concerning fractionation of glycoproteins and preparation of their et al., in preparation]. degradation products will be described in detail elsewhere [LISOWSKA Figure 1 presents the general scheme of fractionation and degradation of red cell glycoproteins. Desialization of samples was performed by mild acid hydrolysis, as described earlier [ 111. Analytical methods. Sialic acid was determined by periodate-resorcinol method [19], neutral sugars by phenol-sulphuric acid method [9], galactosamine and glucosamine by the slightly modified [20] method of LUDOWIEG and BENMAMAN [23]. Haemagglutination-inhibition tests. Twofold dilutions (25 pl) of samples tested for inhibitory activity were incubated with suitably diluted (to the penultimate dilution giving agglutination 3 +) anti-I sera (25 pl) for 1 h at l O T , after that 50 pl of 2% suspension of 01erythrocytes was added and agglutination was read microscopically after 2 h a t 10°C. The scale of agglutination was 3 2 + , + f,- . - and f were regarded as complete inhibition, + as partial inhibition, 2 and 3 as a lack of inhibition.
+,
+
+
I Receptors in Erythrocytes
125
Results Glycoprotein Fractionsfrom Sepharose 4-B The content of galactosamine and glucosamine in red cell glycoproteins is an indicator of the content of alkali-labile and alkali-stableoligosaccharide chains, respectively. As is shown in table I, fractions from Sepharose 4-B differed in proportions of both kinds of chains. fraction I showed the lowest content of sialic acid-rich alkali-labile oligosaccharides.This fraction showed also the highest I activity for all examined anti-I sera (fig. 2). Fractions I1 and I11 inhibited anti-I sera much weaker, however, fraction I11 was usually slightly more active than fraction 11. The distribution if M and N blood group activity among the fractions was entirely different: fractions I and I1 showed similar MN activity, equal to the activity of unfractionated glycoprotein, the activity of fraction I11 was lower (table I). The diagram (fig. 2) summarizing haemagglutination-inhibitionstudies of ten anti-I sera shows that the pattern of inhibition differed from one serum to another, which was compatible with individual differences between anti-I sera and also at certain degree with their subspecificities revealed by serological examination. The difference in the inhibition of anti-ID and anti-IF sera consisted in the different effect of removal of sialic acid from glycoproteins : desialization caused enhancement of ID (and IS) activity (sera Woj., Zg., Cib., Gaj.) and was without influence or even impaired IF activity (sera Ful., Sob.). The two sera containing anti-i besides anti-ID antibodies (Buc. and Syp.) were inhibited by fraction I similarly as anti-ID sera. Serum Buc. containing high proportion of anti-i antibodies was moreover much better inhibited by fractions I1 and I11 than anti-ID sera. The fraction I, showing the highest I activity, and fraction 11, representing the major sialoglycoprotein of erythrocyte membranes, were submitted to further examination. Fraction I To check whether I activity of fraction I was caused by contamination with the very highly I-active substance described by GARDAS and KOSCIELAK [151, the following experiments were performed. (1) Fraction I was chromatographed on DEAE-Sephadex A-50 under conditions used by GARDASand KOSCIELAK [I41 as an essential step for separation of ABH-I-active substance (eluted with 0.1 M NaCl) from MNactive sialoglycoprotein (eluted with 1 M NaCl). In our experiment only trace amount of sugars and no I activity were found in 0.1 M NaCl eluate.
ELWIRA LISOWSKA et al.
126 pg I rnl 40
Woj.
Cib.
Gaj.
BUC.
Ful.
HOE
Sob.
160
600
2,500 1234567 VX567
Pyt.
.. , . . ..
. . .. . . .
.
, . .. . .
...
.
Fig. 2. Inhibition of anti-I sera by glycoprotein fiactions. Inhibitory activity is given and partial (---) inhibition as a minimal concentration @g/ml) giving complete (-) of serum. 1:M-I, 2:M-11, 3:M-111, 4:N-I, 5:N-II, 6:N-111, 7:N-IIT-I-B, 1’-7‘:the same fractions desialized.
The I activity was found in 1M NaC1-eluted sialoglycoprotein, together with MN activity. (2) ABH- and I-active substance was reported to be stable under conditions of drastic alkaline degradation. The purpose of drastic alkaline degradation of M-I (as described in Materials and Methods) was to destroy most of sialoglycoprotein and to isolate the alkali-stable I-active substance. Two fractions (M-Ialk 1 and M-Ialk 2) isolated from degradation products contained carbohydrate components of alkalistable oligosaccharide chains and a very low amount of sialic acid. Only fraction M-Ialk 1 showed I activity, but weaker than the activity of undegraded fraction M-I (table 11). Fraction ZZ Digestion with trypsin increased I activity of fraction I1 and this I activity was found to be connected with aggregated product of digestion IIT-1, whereas the major (unaggregated) sialoglycopeptide IIT-2 did not inhibit anti-I sera, even after desialization, at conc. 10 mg/ml. The aggregated
I Receptors in Erythrocytes
127
Table I. carbohydrate composition (g/100 g) and MN activity of glycoprotein fractions from Sepharose 4-Bcolumn
M
N
crude I gp.
I1
10.0 20.7 Sialic acid 16.5 12.1 Neutral sugars 13.0 14.1 3.2 5.6 GalNH, 4.9 GlcNH, 2.3 3.6 2.4 GalNHJGlcNH, 2.1 0.9 2.3 Sialic acid/ neutral sugars 1.3 0.8 1.5 Activity M 80 160 80 Activity N 2,500 5,000 2,500
I11
crude
I
I11
I1
gP.
23.1 18.6 5.7 3.0 1.9
17.3 12.5 5.9 3.2 1.8
8.6 9.9 3.4 3.2 1.o
19.3 12.8 5.9 2.6 2.3
25.3 16.9 7.1 4.1 1.7
0.9 1.5 1.5 1.2 1.4 1,250
