Invited Review @ 1990 S . Karger AG, Basel 0042-9007/90/0581-01 $2.75/0
Vox Sang 1990;58:1-20
Blood Group-Active Surface Molecules of the Human Red Blood Cell David J. Anstee Blood Group Reference Laboratory, South Western Regional Blood Transfusion Centre, Southmead, Bristol, UK
Abstract. The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3 ) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately lo6 copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) has AB(H) activity and there are about 5 x lo5copiedred cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x lo5copieshed cell) are polypeptides of M, 30,000, polypeptides of MI 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of M, 30,000 and those of M, 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group ‘N’ antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately lo5 copiedred cell. The complete protein sequence of all the above-mentioned proteins is known, except for the MI 30,000 and M, 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C . Several of the minor blood group active proteins at the red cell surface (abundance 4 . 2 x 104/redcell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inbantigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.
Introduction Over 200 distinct blood group antigens have been described on the human red blood cell. Some of these antigens are found on the red cells of nearly all individuals (high frequency or public antigens), others occur very rarely (low frequency or private antigens), and still others are polymorphic in one or more ethnic populations. About 150 antigens have been accommodated within 17 blood group systems, whilst the remainder are either genetically distinct from those within the blood group systems, or have yet to be clearly shown to belong to one of these systems [l].Most of the studies which have associated antigens within blood group systems have involved classical genetics; however, in
some cases high frequency antigens have been assigned to a system because their respective antibodies fail to agglutinate rare red cells which lack all previously known antigens within that system (i.e., red cells expressing a null phenotype) Biochemicalanalysisof the constituents of the human red cell membrane has occurred in parallel with the genetic studies described above, with the result that the major surface membrane components, and several of the minor components have been extensively characterised. It is now possible to assign many blood group antigens to particular molecules of the red cell membrane. This review is an attempt to summarise the present situation regarding the nature and abundance of these blood group active molecules.
2
Anstee
Fig. 1. Models for the membrane orientation of the major erythrocyte transport proteins. Numbers denote putative membrane spanning regions. The model of the glucose transporter is drawn from Mueckler et al. [4]. The model of band 3 is courtesy of Dr. M. J . A. Tanner.
Antigens Which Are Exclusively Carbohydrate in Nature A B ( H ) Antigens It has been known for many years that the AB(H) antigens are exclusively carbohydrate in nature [for a recent review see 21, and since oligosaccharide structures in the red cell membrane are covalently linked to both protein and lipid, it is not surprising that AB(H) antigens are found on both glycolipid and glycoproteins. It is now evident that most of the AB(H) antigens on a red cell are located on glycoproteins, and, in particular, on the anion transport proteins (band 3) and the glucose transport protein (band 4.5; fig. 1). The complete amino acid sequences of both the anion transport protein and the glucose transport protein have recently been determined by nucleotide sequencing [3,4], each has a single highly branched poly-N-acetyl lactosaminyl N-glycosidically linked oligosaccharide (N-glycan) which expresses AB(H) antigen activity at its nonreducing termini [5]. There are about 1million monomers of the anion transporter and half a million monomers of the glucose transporter per red cell [6, 71 (table 1). AB(H) antigens are also found on a variety of glycolipids. There
are approximately half a million poly-N-acetyl lactosaminyl glycolipids in the red cell membrane [8]. Clearly, ABHactive oligosaccharides are likely to occur on a wide variety of additional red cell glycoproteins of lower abundance than the anion transporter and the glucose transphter if they carry similar poly-N-acetyl lactosaminyl N-glycans (for example, the Rh glycoproteins of M, 45,000-100,000, described by Moore and Green [9] see also table 1 and section on Rh complex below). The total number of potential AB(H) antigen sites on a red cell are therefore likely to be in excess of 2 million. PI, P, Pkand L K E Antigens The structure of the antigens of the P system [historically PI, P and Pk,although P is no longer considered to be part of the system, 11 have been reviewed by Marcus et al. [lo], and by Clausen and Hakomori [2]. The P antigen is found on the most abundant glycolipid in the red cell membrane, globoside I. The globoside series of glycolipids comprise about 14 x lo6 molecules per red cell [ll].The Pkantigen is on the glycolipid, ceramide trihexoside. The PI antigen has also been found on glycolipid. Recently, an explanation for the apparent association of the Luke agglutinating system
Blood Group-Active Red Cell Molecules
3
Tablel. Major surface proteins of the red cell ~~
Protein
~
Copieskell x 105
~
~~~
Blood group antigens
Interaction with red cell skeleton
Red cell shape abnormality resulting from defect in skeletal interaction atypical hereditary elliptocytosis results from defective ankyrin - band3 interaction [181]
Anion transport protein (band 3)
10
ABH Ii
through Ankyrin (abundance 105 copiedcell)
Glycophorin A
10
MN
none
Glucose transporter (band 4.5)
5
ABH Ii
none
Glycophorin B
2.5
" ss
none
Rh polypeptides M, 30,000 M, 45,000-100,OOO
1-2 1-2
\1
Rh antigens
yes, but linking skeletal protein unknown
stomatocytes in Rh,,,, syndrome
Glycophorin C Glycophorin D
0.6-1.2 0.154.2
Ge antigens
through band 4.1
elliptocytosis associated with Glycophorin C and D deficiency and with band 4.1 deficiency
with P has been provided by Tippett [12]. A monoclonal antibody MC813-70 defining the glycolipid known as SSEA-4 was shown to have the same specificity as the antibody in Luke serum and the antigen recognised by these antibodies was designated LKE. LKE may be derived from globoside I (P antigen) by addition of galactose (p, 1-3) and sialic acid ( a 2-3), respectively [12].
I and i Antigens The I and i antigens are associated with both glycolipids and poly-N-acetyl lactosaminyl N-glycans of the type found on the anion transporter and the glucose transporter. Anti-i sera recognise an unbranched poly-N-acetyl lactosaminyl structure, whilst anti-I sera recognise a branched structure. The branched structure is created by addition of an Nacetyl glucosamine residue to carbon 6 of a galactosyl residue in the unbranched chain [reviewed in 21.
Antigens Associated with Sialoglycoproteins Structure, Function and Antigens of Glycophorins A and B The MN and Ss antigens are located on two highly homologous sialoglycoproteins known as Glycophorin A and Glycophorin B (syn: a , MN glycoprotein and 6, Ss glycoprotein), respectively [fig. 2; for a recent review and discussion of nomenclature see 13,141. Glycophorin A ex-
presses M or N antigen and this expression is governed by the nature of the amino acid residues at positions 1 and 5 (serine and glycine, respectively, in the case of M, and leucine and glutamic acid, respectively, in the case of N). Glycophorin B has leucine and glutamic acid at positions 1 and 5 , respectively, and so has N antigen activity (usually denoted 'N' to distinguish it from N activity on Glycophorin A), but not M antigen activity. Glycophorin B also expresses the S and s antigens and this expression is governed by amino acid substitutions at residue 29 (methionine for S; threonine for s; fig. 2). The complete amino acid sequences of both these proteins have been published [15,16] and nucleotide sequencing of isolated cDNA clones for both proteins have confirmed these sequences except for one or two minor corrections [17-201. Rahuel et al. [19] used in situ hybridisation to confirm the location of the gene for Glycophorin A to chromosome 4 (q28331). Tate and Tanner [20] isolated almost full length cDNA clones for both Glycophorin A and Glycophorin B, and the sequences of these clones suggest that the two proteins have almost identical N-terminal leader sequences. The cDNAs isolated for Glycophorin A and Glycophorin B show very strong homology, and it is clear that the two proteins are coded by separate, but closely linked genes [21] Both Glycophorin A and Glycophorin B are heavily glycosylated with numerous 0-glycans, the predominant form of which is a disialotetrasaccharide [22]. Fukuda
4
Anstee
Fig. 2. Diagrammatic representation of the structures of erythrocyte glycophorins. * =These antigens probably result from the association of these polypeptides with other molecules. Glycophorin A with band 3 and Glycophorin B with the 2D10-reactive polypeptides of the Rh complex; 4 = trypsin cleavage site on intact red cells;
Vox Sang 1990;58:1-20
Blood Group-Active Surface Molecules of the Human Red Blood Cell David J. Anstee Blood Group Reference Laboratory, South Western Regional Blood Transfusion Centre, Southmead, Bristol, UK
Abstract. The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3 ) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately lo6 copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) has AB(H) activity and there are about 5 x lo5copiedred cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x lo5copieshed cell) are polypeptides of M, 30,000, polypeptides of MI 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of M, 30,000 and those of M, 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group ‘N’ antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately lo5 copiedred cell. The complete protein sequence of all the above-mentioned proteins is known, except for the MI 30,000 and M, 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C . Several of the minor blood group active proteins at the red cell surface (abundance 4 . 2 x 104/redcell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inbantigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.
Introduction Over 200 distinct blood group antigens have been described on the human red blood cell. Some of these antigens are found on the red cells of nearly all individuals (high frequency or public antigens), others occur very rarely (low frequency or private antigens), and still others are polymorphic in one or more ethnic populations. About 150 antigens have been accommodated within 17 blood group systems, whilst the remainder are either genetically distinct from those within the blood group systems, or have yet to be clearly shown to belong to one of these systems [l].Most of the studies which have associated antigens within blood group systems have involved classical genetics; however, in
some cases high frequency antigens have been assigned to a system because their respective antibodies fail to agglutinate rare red cells which lack all previously known antigens within that system (i.e., red cells expressing a null phenotype) Biochemicalanalysisof the constituents of the human red cell membrane has occurred in parallel with the genetic studies described above, with the result that the major surface membrane components, and several of the minor components have been extensively characterised. It is now possible to assign many blood group antigens to particular molecules of the red cell membrane. This review is an attempt to summarise the present situation regarding the nature and abundance of these blood group active molecules.
2
Anstee
Fig. 1. Models for the membrane orientation of the major erythrocyte transport proteins. Numbers denote putative membrane spanning regions. The model of the glucose transporter is drawn from Mueckler et al. [4]. The model of band 3 is courtesy of Dr. M. J . A. Tanner.
Antigens Which Are Exclusively Carbohydrate in Nature A B ( H ) Antigens It has been known for many years that the AB(H) antigens are exclusively carbohydrate in nature [for a recent review see 21, and since oligosaccharide structures in the red cell membrane are covalently linked to both protein and lipid, it is not surprising that AB(H) antigens are found on both glycolipid and glycoproteins. It is now evident that most of the AB(H) antigens on a red cell are located on glycoproteins, and, in particular, on the anion transport proteins (band 3) and the glucose transport protein (band 4.5; fig. 1). The complete amino acid sequences of both the anion transport protein and the glucose transport protein have recently been determined by nucleotide sequencing [3,4], each has a single highly branched poly-N-acetyl lactosaminyl N-glycosidically linked oligosaccharide (N-glycan) which expresses AB(H) antigen activity at its nonreducing termini [5]. There are about 1million monomers of the anion transporter and half a million monomers of the glucose transporter per red cell [6, 71 (table 1). AB(H) antigens are also found on a variety of glycolipids. There
are approximately half a million poly-N-acetyl lactosaminyl glycolipids in the red cell membrane [8]. Clearly, ABHactive oligosaccharides are likely to occur on a wide variety of additional red cell glycoproteins of lower abundance than the anion transporter and the glucose transphter if they carry similar poly-N-acetyl lactosaminyl N-glycans (for example, the Rh glycoproteins of M, 45,000-100,000, described by Moore and Green [9] see also table 1 and section on Rh complex below). The total number of potential AB(H) antigen sites on a red cell are therefore likely to be in excess of 2 million. PI, P, Pkand L K E Antigens The structure of the antigens of the P system [historically PI, P and Pk,although P is no longer considered to be part of the system, 11 have been reviewed by Marcus et al. [lo], and by Clausen and Hakomori [2]. The P antigen is found on the most abundant glycolipid in the red cell membrane, globoside I. The globoside series of glycolipids comprise about 14 x lo6 molecules per red cell [ll].The Pkantigen is on the glycolipid, ceramide trihexoside. The PI antigen has also been found on glycolipid. Recently, an explanation for the apparent association of the Luke agglutinating system
Blood Group-Active Red Cell Molecules
3
Tablel. Major surface proteins of the red cell ~~
Protein
~
Copieskell x 105
~
~~~
Blood group antigens
Interaction with red cell skeleton
Red cell shape abnormality resulting from defect in skeletal interaction atypical hereditary elliptocytosis results from defective ankyrin - band3 interaction [181]
Anion transport protein (band 3)
10
ABH Ii
through Ankyrin (abundance 105 copiedcell)
Glycophorin A
10
MN
none
Glucose transporter (band 4.5)
5
ABH Ii
none
Glycophorin B
2.5
" ss
none
Rh polypeptides M, 30,000 M, 45,000-100,OOO
1-2 1-2
\1
Rh antigens
yes, but linking skeletal protein unknown
stomatocytes in Rh,,,, syndrome
Glycophorin C Glycophorin D
0.6-1.2 0.154.2
Ge antigens
through band 4.1
elliptocytosis associated with Glycophorin C and D deficiency and with band 4.1 deficiency
with P has been provided by Tippett [12]. A monoclonal antibody MC813-70 defining the glycolipid known as SSEA-4 was shown to have the same specificity as the antibody in Luke serum and the antigen recognised by these antibodies was designated LKE. LKE may be derived from globoside I (P antigen) by addition of galactose (p, 1-3) and sialic acid ( a 2-3), respectively [12].
I and i Antigens The I and i antigens are associated with both glycolipids and poly-N-acetyl lactosaminyl N-glycans of the type found on the anion transporter and the glucose transporter. Anti-i sera recognise an unbranched poly-N-acetyl lactosaminyl structure, whilst anti-I sera recognise a branched structure. The branched structure is created by addition of an Nacetyl glucosamine residue to carbon 6 of a galactosyl residue in the unbranched chain [reviewed in 21.
Antigens Associated with Sialoglycoproteins Structure, Function and Antigens of Glycophorins A and B The MN and Ss antigens are located on two highly homologous sialoglycoproteins known as Glycophorin A and Glycophorin B (syn: a , MN glycoprotein and 6, Ss glycoprotein), respectively [fig. 2; for a recent review and discussion of nomenclature see 13,141. Glycophorin A ex-
presses M or N antigen and this expression is governed by the nature of the amino acid residues at positions 1 and 5 (serine and glycine, respectively, in the case of M, and leucine and glutamic acid, respectively, in the case of N). Glycophorin B has leucine and glutamic acid at positions 1 and 5 , respectively, and so has N antigen activity (usually denoted 'N' to distinguish it from N activity on Glycophorin A), but not M antigen activity. Glycophorin B also expresses the S and s antigens and this expression is governed by amino acid substitutions at residue 29 (methionine for S; threonine for s; fig. 2). The complete amino acid sequences of both these proteins have been published [15,16] and nucleotide sequencing of isolated cDNA clones for both proteins have confirmed these sequences except for one or two minor corrections [17-201. Rahuel et al. [19] used in situ hybridisation to confirm the location of the gene for Glycophorin A to chromosome 4 (q28331). Tate and Tanner [20] isolated almost full length cDNA clones for both Glycophorin A and Glycophorin B, and the sequences of these clones suggest that the two proteins have almost identical N-terminal leader sequences. The cDNAs isolated for Glycophorin A and Glycophorin B show very strong homology, and it is clear that the two proteins are coded by separate, but closely linked genes [21] Both Glycophorin A and Glycophorin B are heavily glycosylated with numerous 0-glycans, the predominant form of which is a disialotetrasaccharide [22]. Fukuda
4
Anstee
Fig. 2. Diagrammatic representation of the structures of erythrocyte glycophorins. * =These antigens probably result from the association of these polypeptides with other molecules. Glycophorin A with band 3 and Glycophorin B with the 2D10-reactive polypeptides of the Rh complex; 4 = trypsin cleavage site on intact red cells;
