Download Structure and function of red cell surface antigens

ISBT Science Series (2006) 1, 3–8
ORIGINAL PAPER
1ED-07-02
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing
Structure and function of red cell surface antigens
Blackwell Publishing Ltd
G. Daniels
Bristol Institute for Transfusion Sciences, National Blood Service, Bristol, UK
Introduction
Currently there are almost 300 red cell surface antigenic
determinants or blood group specificities recognized by the
International Society of Blood Transfusion; most of these
belong to 1 of 29 blood group systems. Each system represents
a single gene or cluster of 2 or 3 closely linked homologous
genes, giving a total of 34 gene loci. Each of these genes has
been cloned and sequenced, with the exception of one, P, and
there is a candidate gene for this. Consequently, we have a
tremendous amount of structural information about the red
cell surface. Despite this, there is still much we do not know
about the functions of these antigens; much of what we do know
has been deduced from their structures. We know almost
nothing about the functions of the blood group polymorphisms. The blood group systems are listed in Table 1,
with information on the structures of their antigens and their
possible functions.
Almost all blood group systems have a null phenotype,
in which no antigen of that system is expressed on the red
cells. Null phenotypes are generally rare. They are usually
only found when individuals with these phenotypes may
make antibodies to the missing proteins following immunization by blood transfusion or pregnancy. Such antibodies
are then identified in immunohaematology reference laboratories. Null phenotypes usually result from homozygosity
for inactivating mutations or even a gene deletion, so the whole
protein is absent from the red cells and from everywhere else
in the body, at any stage of development. Yet in many cases
individuals with these rare phenotypes are apparently healthy.
This reflects the functional redundancy of many cell surface
proteins. Often, when one protein is missing, another can
perform the same function in its absence. However, this is not
always the case and null phenotypes have been very informative
about the functions of blood group antigens both on red cells
and in other tissues.
Red cell surface antigens are macromolecules anchored in
the lipid bilayer of the red cell envelope. There are three main
types of macromolecules expressing blood group activity:
proteins, glycoproteins, and glycolipids. Most blood group
antigens are glycoproteins, with the specificity determined
primarily either by the oligosaccharide sequence (e.g. ABO)
or by the amino acid sequence (e.g. MN, Kell, Duffy, Kidd, Diego).
The Rh antigens are non-glycosylated proteins, though the
presence of an associated glycoprotein is required for antigenic
expression. ABO antigens are also expressed on the carbohydrate
moiety of glycosphingolipids.
In this article red cell surface antigens will be discussed in
terms of their functions or potential functions, under the
following five headings: transporters and channels; receptors
and adhesion molecules; enzymes; structural proteins that
link the lipid bilayer to the membrane skeleton; and those
structures that contribute to the glycocalyx, or cell coat.
Membrane transporters and channels
Membrane transporters and channels facilitate the transport
of biologically important molecules in or out of cells. They
are typically polytopic: that is, they go in and out of the
membrane several times, have both termini inside the cytoplasm,
and they generally have an N-glycan on one of the extracellular
loops (Fig. 1).
The Diego antigen, band 3, the red cell anion
exchanger
The Diego antigen, known as band 3, is one of the most abundant
red cell surface glycoproteins. It crosses the membrane 12 or
14 times, is glycosylated on the fourth extracellular loop, and
has a long cytoplasmic N-terminal domain that interacts with
the membrane skeleton (Fig. 1).
One of the major functions of the blood is to transport
respiratory gases. In addition to carrying oxygen to the
tissues, carbon dioxide must be carried away from the tissues,
to the lungs. Carbon dioxide in the blood is hydrated to bicarbonate by carbonic anhydrase II (CAII), which is only present
in the red cell cytoplasm. Band 3 acts as an anion exchanger,
an antiporter that permits bicarbonate ions to cross the
membrane in exchange for chloride ions. The bicarbonate
that accumulates in the red cells after conversion from carbon
dioxide is rapidly transported out of the cell. This increases
the amount of bicarbonate that can be transported in the
plasma, which greatly increases the quantity of carbon
dioxide that the blood can convey to the lungs.
Band 3 is part of the Rh protein macrocomplex described
below.
3
4 G. Daniels
Table 1 The blood group systems
System
Gene(s)
Structure
Possible functions
ABO
MNS
P
Rh
Lutheran
Kell
Lewis
Duffy
Kidd
Diego
Yt
Xg
Scianna
Dombrock
Colton
LW
Ch/Rg
H
Kx
Gerbich
Cromer
Knops
Indian
Ok
Raph
JMH
ABO
GYPA, GYPB
P
RHD, RHCE
LU
KEL
FUT3
DARC
SLC14A1
SLC4A1
ACHE
XG, CD99
ERMAP
ART4
AQP1
ICAM4
C4A, C4B
FUT1
XK
GYPC
CD55
CR1
CD44
BSG
CD151
SEMA7A
GCNT2
B3GALNT1
AQP3
Carbohydrate
Sialoglycoproteins
Carbohydrate
Polytopic proteins
IgSF glycoprotein
Enzyme
Carbohydrate
G protein-coupled superfamily
Solute carrier
Solute carrier
Enzyme
Sialoglycoproteins
IgSF glycoprotein
Enzyme
Aquaporin
IgSF glycoprotein
Complement protein
Carbohydrate
Polytopic protein
Sialoglycoproteins
Complement control protein
Complement control protein
Glycoprotein
IgSF glycoprotein
Tetraspanin
Semaphorin
Carbohydrate
Carbohydrate
Aquaporin
Glycocalyx
Glycocalyx
Unknown
NH3/NH4+ or CO2 gas channel/transporter
Adhesion/receptor – binds laminin
Endopeptidase – processes endothelin 3
Unknown
Chemokine receptor
Urea transporter
Anion exchanger, anchor to membrane skeleton
Acetylcholinesterase
Receptors
Adhesion/receptor
ADP-ribosyltransferase
Water channel
Adhesion/receptor – binds integrins
C4 component of complement
Glycocalyx
Neurotransmitter transporter
Anchor to membrane skeleton
Complement inactivation
Complement inactivation
Adhesion/receptor – binds hyaluronan
Adhesion/receptor
Complexes with integrins in the membrane
Adhesion/receptor
Glycocalyx
Unknown
Water/glycerol channel
Globoside
Gill
Band 3 in red cells has other functions: anchoring the
plasma membrane to the membrane skeleton; acting as a
binding site for haemoglobin, CAII, and glycolytic enzymes;
and playing a role in red cell senescence.
Kidd antigen: urea transporter
The Kidd antigen spans the membrane 10 times and is
glycosylated on the third extracellular loop (Fig. 1). It is a
urea transporter: it transports urea rapidly into or out of the
cell. When red cells enter the renal medulla, where there is a
high concentration of urea in the blood, urea is transported
rapidly into those cells. This prevents the rapid loss of water
in this hypertonic environment and shrinkage of the cells.
As the cells leave the renal medulla, urea is transported
rapidly out of the cells, which prevents the entry of water and
excessive swelling of the cells and defends against the cells
from taking urea away from the kidney, which would reduce
the urea concentrating capability of the kidney.
Colton antigen: aquaporin 1 (AQP1) a water
channel
The Colton antigen is a member of the Aquaporin family of
membrane channels. Aquaporins have six membrane-spanning
domains (Fig. 1), but also have two cytoplasmic loops that
contain Ala-Pro-Asn motifs. Tetramers of AQP1 in the red
cell membrane form channels that enhance osmotically
driven water transport.
GIL antigen: aquaporin 3 (AQP3) a glycerol
channel
The GIL antigen is aquaporin 3, a glycerol and water channel.
The Rh protein macrocomplex or Rh metabolon
The proteins expressing the D and CE antigens of the Rh
system cross the membrane 12 times but, unlike transporters,
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing Ltd. ISBT Science Series (2006) 1, 3–8
Red cell surface antigens 5
Fig. 1 Diagrammatic representation of the proposed configuration of some
polytopic proteins and glycoproteins with blood group activity. N = N-glycan.
they are not glycosylated (Fig. 1). They are closely associated
in the membrane with a glycoprotein, called the Rh-associated
glycoprotein (RhAG), that has a similar structure to the Rh
proteins, but is glycosylated and does not carry Rh antigen
activity (Fig. 1). The Rh proteins are encoded by genes on
chromosome 1, whereas the RhAG is encoded by a gene on
chromosome 6. The Rh protein complex has generally been
considered to be a tetrameric structure, with two molecules
of RhD and/or RhCE and two molecules of RhAG, but recent
structural analyses has suggested that a trimeric structure is
most likely. The Rh genes, RHD and RHCE, have about 30%
sequence identity to RHAG. Two more genes of the human
Rh gene family, RHCG and RHBG, both with about 50%
sequence identity to RHAG, are nonerythroid, but are expressed
in kidney, liver, skin, and testis. RhD, RhCE, RhAG, RhBG, and
RhCG are part of a large family of proteins found in archaea,
bacteria, plants, and animals, known as the ammonium transporter/
methylamine permease/Rh (Amt/Mep/Rh) family.
RhAG has about 25% homology with ammonium transporters
in lower animal and plants, called the Mep proteins. Yeast cells
require ammonium for growth and yeast cells lacking Mep
proteins fail to grow in low levels of ammonium. The growth
defect in mep-deletion yeast cells could be repaired by transfection of the cells with the human RHAG gene. Furthermore,
Xenopus oocytes transfected with the RHAG gene took up
methylamine, an analogue of ammonium, which untransfected
cells did not. Rhnull cells, which lack Rh proteins and RhAG,
have reduced rates of ammonium transport through the mem-
brane. These results suggest that RhAG has the capability to
mediate ammonium transport, but whether this is in the pro+
tonated ( NH4 ) or unprotonated (NH3) form is controversial.
It has been suggested that RhAG promotes retention of
ammonium in red cells in order to maintain a low level of
+
total blood ammonia ( NH4 + NH3), and possibly for transport
of ammonium to the liver or kidney and subsequent removal
from the body, thus protecting against ammonia toxicity in
the brain. However, no direct evidence exists that the Rh
complex functions to facilitate transfer of ammonium in or
out of the red cells in vivo.
In contrast, there is also some evidence that the Rh protein
complex might function as a gas channel for CO2. The photosynthetic green alga Chlamydomonas reinhardtii depends
on CO2 for photosynthesis. It has a protein homologous to the
Rh family of proteins, encoded by a gene called RH1. When
this alga is cultured in air, with low levels of CO2, there is high
expression of RH1, but when it is cultured in high levels of
CO2, there is low expression of RH1. When it is moved from
low to high levels of CO2, there is substantial reduction in
RH1 expression, but when moved from high to low levels of
CO2, there is a substantial increase in RH1 expression. This
suggests that the Rh1 protein might function as a CO2 channel
in this microbe.
The complex of Rh proteins and RhAG are part of a
macrocomplex or metabolon of proteins in the red cell
membrane, which also contains band 3, LW (ICAM-4), CD47,
and glycophorins A and B, with band 3 at its core. It has been
proposed that the macrocomplex involving Rh and band 3
functions as an oxygen/carbon dioxide gas exchange channel.
The presence of such a gas exchange channel might be
expected, as the primary purposes of the red cell are transport
of oxygen and conversion of carbon dioxide to bicarbonate.
The macrocomplex is ideally located to channel CO2 to and
from carbonic anhydrase and oxygen to and from haemoglobin, as band 3 is anchored to both CAII and haemoglobin.
There is no doubt that ancestral Rh proteins function as
ammonium transporters. It is reasonable to speculate that, as
a result of its evolutionary origins, RhAG is able to mediate
transport of ammonium, and that its nonerythroid homologues
RhBG and RhCG may still serve this function. However, the
likely primary function of RhAG in red cells is to provide a
gas channel for CO2, which like NH3 is a readily hydrated gas,
and possibly for O2. It is unlikely that the more rapidly evolving Rh proteins have any direct transport or channel-forming
functions, and may have a role in maintaining the correct
shape of the red cells.
XK protein
The XK protein crosses the membrane 10 times (Fig. 1). It is
not glycosylated, but is linked through a single disulphide
bond to the Kell glycoprotein. The function of XK is not
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing Ltd. ISBT Science Series (2006) 1, 3– 8
6 G. Daniels
The IgSF is a very large family of adhesion molecules and
receptors with different numbers of repeating immunoglobulinlike domains.
ICAM-4 can bind LFA-1 (α1β2), α4β1, and αV integrins.
Integrins are adhesion molecules present on haemopoietic
and non-haemopoietic cells, but not mature red cells. They
have a heterodimeric structure, consisting of different types
of α and β chains, both of which cross the cell membrane once.
αV subunit forms heterodimers with a variety of β subunits.
Erythroblasts express both ICAM-4 (LW) and α4β1 integrin,
whereas macrophages express αV integrins. Erythroblastic
islands in the bone marrow comprise a cluster of erythroblasts
around a macrophage, which is important for enucleation of
the erythroblasts. Erythroblastic islands involve cell adhesion
events and cytokine interactions that are critical in the
regulation of erythropoiesis and apoptosis. It is possible that
ICAM-4 on erythroblasts can bind integrins on macrophages
and on other erythroblasts in the bone marrow and help to
stabilize the erythroblastic islands. Furthermore, downregulation of α4β1 integrin on enucleated erythroblasts may
assist in their release from the erythroblastic islands.
ICAM-4 is part of the Rh glycoprotein macrocomplex and
it has been speculated that, together with another adhesion
IgSF molecule, CD47, it might assist in maintaining close
contact between the red cell membrane and the endothelium
of the capillary wall.
It is probable that both Lutheran and ICAM-4 (LW) have a
pathophysiological role in sickle cell disease by contributing
to red cell adhesion to endothelial cells and development of
vaso-occlusion.
Lutheran
Scianna
The Lutheran glycoproteins, which have five immunoglobulin
superfamily domains (V-V-C2-C2-C2), bind laminin, a glycoprotein of the extracellular matrix. Laminin consists of three
chains, α, β, and γ, and exists in at least 12 different isoforms,
derived from combinations of different α, β, and γ chains.
The Lutheran glycoproteins bind specifically and with high
affinity to isoforms of laminin that contain α-5 chains, called
laminin 10 and 11.
During in vitro erythropoiesis, Lutheran is the last erythroid
surface marker to appear, probably at the orthochromatic
erythroblast stage. For mature erythroblasts to leave the bone
marrow they must cross the sinusoidal endothelium. Bone
marrow sinusoidal endothelium has laminin isotypes 10 and
11 on its surface and this has led to speculation that the
Lutheran glycoproteins are involved in the trafficking of
mature erythroid cells from the erythroblastic islands of the
bone marrow, across the sinusoidal endothelium, and to the
peripheral blood.
The Scianna antigen has one IgSF domain (V) and is
erythrocyte membrane-associated protein (ERMAP).
LW, intercellular adhesion molecule-4 (ICAM-4)
Complement control proteins are cell surface glycoproteins
that regulate the complement cascade. Glycoproteins of
the complement control protein superfamily have between two
and 30 repeating domains, which are characteristic of this
superfamily.
known, but XK shares some sequence homology with a family
of neurotransmitter transporters. Absence of XK results in
late onset muscular, neurological, and psychiatric symptoms,
together with acanthocytic red cells.
Receptors and adhesion molecules
Duffy glycoprotein, the Duffy antigen receptor for
chemokines (DARC)
Like the transporters, the Duffy glycoprotein is polytopic; unlike
the transporters, the amino-terminus is outside the membrane.
This structure is characteristic of a very large superfamily of
receptors, called the G protein-coupled superfamily, that bind
many different ligands. The Duffy glycoprotein, also known as
DARC, acts as a receptor for a variety of pro-inflammatory
cytokines of both C-X-C and C-C types, including IL-8, MGSA,
MCP-1, and RANTES. Duffy is present in many organs, and
is abundant on postcapillary venules. On red cells it could act
as a clearance receptor for inflammatory mediators.
Blood group antigens belonging to the
immunoglobulin superfamily (IgSF)
ICAM-4 has two IgSF domains (C2-C2) and is part of the Rh
glycoprotein macrocomplex. During erythropoiesis ICAM-4
first appears on erythroid cells on late proerythroblasts or
early erythroblasts, about the same time as the Rh antigens.
Ok
The Ok antigen has two IgSF domains (C2-V) and is basigin
(CD147).
The Indian antigen, CD44
The hyaloadherin CD44 is a member of the Link Module
superfamily of proteoglycans that contain a Link module, a
structural domain of about 100 amino acids that is involved
in ligand binding. CD44 is the predominant cell surface
receptor for the extracellular matrix (ECM) glycosaminoglycan
hyaluronan. The functions of CD44 are diverse, although the
function of CD44 on RBCs remains unknown.
Complement control proteins
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing Ltd. ISBT Science Series (2006) 1, 3–8
Red cell surface antigens 7
Cromer
The Cromer antigen is CD55 or decay-accelerating factor
(DAF), which has four complement control protein domains
and is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. CD55 helps to protect the red cells from
lysis by autologous complement by inhibiting the action
of C3-convertases. However, the glycoprotein that is most
important in the function of protecting the cell from autologous
complement is another GPI-linked glycoprotein, CD59,
which is not polymorphic and does not have blood group
activity. CD59 prevents assembly of the membrane-attack
complex.
Dombrock
The sequence of the Dombrock protein suggests that it
belongs to a family of ADP-ribosyltransferases that transfer
ADP-ribose to various protein acceptors. It is not known
whether this structure is enzymatically active on red cells.
Kell
The Kell glycoprotein is an active endopeptidase and can
process the vasoconstrictor endothelin-3 in vitro, but it is
not known whether the Kell glycoprotein serves this function
in vivo.
Knops
CR1 or CD35, the Knops blood group antigen, is a very large
glycoprotein with about 30 repeating complement control
protein domains. The major function of red cell CR1 is to bind
and process immune complexes and transport them to the
liver and spleen for removal from the circulation.
Xg, CD99, and JMH
The structures of the Xg and CD99, and JMH (CD108 or
Sema7a) molecules suggest they might also have receptor or
adhesion functions.
MER2
The MER2 blood group antigen, CD151, belongs to the
tetraspanin superfamily. Tetraspanins span the cell membrane
four times, have short, internal N- and C-termini and one small
and one large extracellular loop (Fig. 1). Although CD151 is
probably not an adhesion or receptor molecule, it associates
closely with certain integrins in cell membranes to generate
complexes that bind laminin in basement membranes and
are important in maintaining the integrity of the basement
membrane. The very rare MER2-null phenotype results in
disruption of basement membranes in the kidney, causing
hereditary nephritis (all the patients required dialysis and
kidney transplants), in the skin, causing epidermolysis bullosa,
a severe blistering, and in the inner ear, leading to neurosensory
deafness. The function of CD151 in the red cell membrane
remains unknown.
Red cell surface antigens with putative
enzymatic activity
Some red cell surface glycoproteins appear to be enzymes.
Yt
Yt is acetylcholinesterase, an enzyme that plays an essential
role in neurotransmission. This structure is enzymatically
active on red cells, but its function on red cells is not
known.
Structural proteins
There are some membrane proteins that have a structural
function to link the membrane to its skeleton. The membrane
skeleton is a network of glycoproteins, mostly spectrin and
actin, beneath the plasma membrane, which is responsible for
maintenance of the shape of the red cell. Effective attachment
of the plasma membrane to its skeleton is very important in
maintaining the shape and flexibility of the red cell as it
squeezes through the smallest capillaries.
Band 3, the Diego glycoprotein, has an extended N-terminal
domain attached to the membrane skeleton through ankyrin
and proteins 4·2 and 4·1R. Individuals heterozygous for
mutations with the gene for band 3 often have hereditary
elliptocytosis or Southeast Asian ovalocytosis.
Glycophorins C and D, the Gerbich glycoproteins, have a
long C-terminal domains attached to the membrane skeleton
through protein 4·1R and p55. A proportion of Gerbich-null
red cells are spherocytic.
In addition there is evidence that RhAG interacts with
ankyrin, the Lutheran and XK proteins with spectrin, and
CD44 with protein 4·1. Red cells lacking RhAG, XK, or, to a
lesser degree, Lutheran glycoproteins and CD44 [In(Lu)
phenotype] have some degree of abnormal morphology.
Carbohydrate antigens
The ABO and H antigens are carbohydrate histo-blood group
antigens present on many tissues. On red cells most ABO and
H antigens are on the N-glycans of band 3 and the glucose
transporter (GLUT1), as well as being on minor glycoproteins
and on glycolipids. The extracellular domains of glycophorins, that express the MNS antigens and are abundant on
the red cell surface, are also heavily glycosylated. The
functions of these carbohydrates on red cells are not known,
apart from contributing to the glycocalyx or cell coat, an
extracellular matrix of carbohydrate that surrounds the cell
and protects it from mechanical damage and microbial
attack.
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing Ltd. ISBT Science Series (2006) 1, 3– 8
8 G. Daniels
Conclusion
The red cell surface is highly complex and the red cell membrane proteins serve a variety of functions: some designed to
make the red cell an efficient transporter of respiratory gases,
others to provide the red cell with additional functions, such
as the removal of excess cytokines and immune complexes
from the peripheral blood, and probably many others that we
do not know about. Some structures on the red cell may be
vestigial, having served their function during erythropoiesis
or even at an earlier evolutionary stage of the organism. In
addition, we know almost nothing about the biological
importance of the polymorphisms that make the red cell
surface proteins alloantigenic.
Bibliography
1 Daniels G: Human Blood Groups, 2nd edn. Oxford, Blackwell
Science, 2002
2 Daniels G: Functional aspects of red cell antigens. Blood Rev
1999; 13:14–35
3 Denomme GA: The structure and function of the molecules that
carry human red blood cell and platelet antigens. Transfus Med
Rev 2004; 18:203–231
4 Reid ME, Mohandas N: Red blood cell blood group antigens:
structure and function. Semin Hematol 2004; 41:93–117
5 Storry JR: Review: the function of blood group-specific RBC
membrane components. Immunohematology 2004; 20:206–216
6 Tanner MJA: Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders. Curr Opin Hematol 2002; 9:133–139
7 Wintour EM: Water channels and urea transporters. Clin Exp
Pharmacol Physiol 1997; 24:1–9
8 Proceeding of the International Conference on the Rh protein
superfamily. Transfus Clin Biol 2006; 13:1–178
9 Westhoff CM: The Rh blood group system in review: a new face
for the next decade. Transfusion 2004; 44:1663–1673
10 Bruce LJ, Beckmann R, Ribeiro ML, Peters LL, Chasis JA,
Delaunay J, Mohandas N, Anstee DJ, Tanner MJA: A band 3based macrocomplex of integral and peripheral proteins in the
RBC membrane. Blood 2003; 101:4180 –4188
11 Hadley TJ, Peiper SC: From malaria to chemokine receptor: the
emerging physiologic role of the Duffy blood group antigen.
Blood 1997; 89:3077–3091
12 Parsons SF, Spring FA, Chasis JA, Anstee DJ: Erythroid cell
adhesion molecules Lutheran and LW in health and disease.
Baillière’s Clin Haemat 1999; 12:729–745
13 Eyler CE, Telen MJ: The Lutheran glycoprotein: a multifunctional
adhesion receptor. Transfusion 2006; 46:668– 677
14 Jentoft N: Why are proteins O-glycosylated? Trends Biochem Sci
1990; 15:291–294
© 2006 The Author.
Journal compilation © 2006 Blackwell Publishing Ltd. ISBT Science Series (2006) 1, 3–8