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Home , ART4, CD59, Colton antigen system, Duffy antigen system, Glycophorin A, Glycophorin C

Review: the function of group–specific RBC membrane components

J.R. S TORRY

Billions of RBCs course through the heart and Figure 1 shows the assignment of the blood group– arteries per second. Blood flow through and active molecules to a primary functional group. This is veins is somewhat slower but a RBC circulates somewhat artificial and it is important to keep in mind throughout the body many thousands of times during that many of these molecules may have more than one its 120-day lifespan. Thus, the contact that a RBC has function. There are two large groups: the transport with the vessel walls and with leukocytes and and the adhesion molecules. Aside from these is extremely intimate and unique. RBCs must be inert two groups, blood group–carrying proteins include enough to perform their function of gaseous exchange those involved in complement regulation and proteins without compromise but, as is becoming clearer, they that are known to be enzymes. There are two families must be active enough to respond to the of , the A (GPA) family, whose microenvironment in which they find themselves. function is not clearly defined, and the The extracellular surface of the RBC membrane (GPC) family, whose structural role, at least, has been consists of and glycolipids. Blood group identified. Finally, most of the integral membrane have been found on most of these proteins or membrane-associated proteins of the RBC components and have provided a useful “handle” by are glycosylated. These polysaccharides carry blood which to isolate and study them. There are few group determinants. A brief discussion of their proteins on the RBC membrane on which blood groups have not been identified, such as the glucose function is included. The role of blood group antigens transporter and MIRL (CD59). This review will as receptors for various pathogens is not discussed in describe the blood group–active RBC membrane this review. The interested reader is referred to the 4,5 components only and their relevance in a normal excellent reviews in the reference list. physiological state. Instead of being listed according to ISBT order, the blood groups have been grouped according to known or likely function (see Table 1). Blood group antigens will be discussed where they are relevant to the function of the carrier component. For more details regarding the blood group antigens themselves, two excellent reference texts (among many others) are recommended: The Blood Group Antigen FactsBook and Human Blood Groups. 1,2 The reference list is by no means exhaustive, since the functional groups are the subjects of detailed reviews themselves. In fact, these reviews have been cited wherever possible and original papers cited only where appropriate. The reader is also referred to another excellent and recent review that addresses both structure and function of RBC blood group antigens. 3 Fig. 1. Blood group systems grouped according to functional molecule.

206 IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 Function of blood groups a i m o e t n y e ? t e a i r n r e l i e l e i d u r l d b e u i u m t r n a c a e o a f o o e e e r i s h , s t l t i t d a n a m r o i d r r e n f o t l e y a p m o m b n i a s p e a r e m n r n n m r i d e c s o e t a e e o o d i n s u c t i r n e r t o e e a o a o r t r L d c a e l s t e w a c n i i d n i s v o c l d a i c t M m e e i t h ; f ; e s t t y m n i s s i m ; t i o i h ; i o s e s n t s l s i w t i i m p a s o o s f s s s t b t o n n n n n y o o y o a t y m t s o t c y w w w w i c n l e y d d t e c o o o o o o e l a c c a e e t r o i h n n n n c o p c c a r t n r e i c i t k k k k e e e e e e e e e e e e e e e e u u t e u e n v o l m p n i n n n n n n t t t n t n n n n n n n n n s a d c d i h s i r u l o o o o o o o o o o o o o o o o o o o o o l s c e e e l b t p h R a M N N A N N N N R N N N R E N N N N N t A N N S N S N N C N e l b a e b l e o u ? ? ? ? ? ? r h c e e e e e e t e p s s s s s s l f / n n n n n n o y o e e e e e e ? ? f f f f f f r l l l l l m e e e e e e a a a a a a n r r r r r d d d d d d d r o u u u u u i e t t t t t e e e e e e n t i t t t t t t c c c c c c o r a a a a a a u u u u u c r n n n n n n n r r r r r e a t t t t t u n n n n n n c S S S S I I I I I I S f S r e l i a r r r e r u t n a t r o c c i o t u f p a r ? r l s o t o e u s n t g ; a g n p r n r e r o e t a e r i e c l t g l t h ' e r o c e c n r C r o n n a n x / n e r e e o o p h n n n u i i c s o l n c a e f t t s n n n n n n y i t w w l a n n n n n n n n n l a a x h a r k l l p o o a u o o o o o o o o g w w w w w w e e e e n c g i i i i i i i i r u o u u e / n n c x o o o o o o t s s s s s s s s t r r / m m m g g c w n e k k e n n n n n n e e e e e e e e + c l m e e l y y y a e e e o 4 o t t t t k k k k k k e p i u h h h h h h h h r r r z z z e o a a r n o o H r n n n n n n ' ' ' h d d d d d n d d d n n n m t m W S N C A N U A A C C E E E U U U U U A N A U W A C A A K A o c ) l a M n A o i C ) t - 4 c B - n ) ( u M 4 9 f n ) 4 A i 9 e * 7 e C D D h ) t ) I 4 t e C ( o C 8 l G 1 f ( r , n 0 u o A i n D p n n i 1 n c ) i e i h n o C s e t e i e e 1 e t ( t R w c l t t e g o ( 5 i t n o ) ) y r o D o o , t l n r 1 o e 5 5 r i r p r E g l n C p n 5 3 D p p m o ( p a e D C B o o n o c o C l D D h P r s c 1 h P a ( A a y p c c t P l C C r e r y e 1 R G 7 y y G l o † i ( ( g / l g l 2 A e C / 1 3 m , 4 / E a r g r m e g g l T h 1 O O O O O O R P P o F R t C 1 h u T r r A D - l M t m c E n e P P e U A A a k g e 4 R H H H H H H R E h Q Q C i R u e W e A M G G C C R X A D X H N S D C K A A C C C C C H A C L E L n n a a s r i b t i m m t e e u r t b e s m s n y n e C s i e s B g r i p W R e t - u g n r g a d o e k n r c o n i p i n g y o u R r h a a e r r / ) o t r c e s n r n r d n i b s s o o B e T y p a i o H g a n f b o m S d d d g t i l h c B f m o P a i O r O l l H t d L o w e o i d n d N – S K i o u I e L i x n t g e o h r l B h A o u a e c I M n p o D B ( R K C K G L D X S L I O R J M G C C K K Y D A P L H I G l u b o r y r g r a d c o r t o o e e t o l t e r n n n r p t i B a o a e r r e s y r t n t e o d g n d r . m o o y l h y n r i o o e 1 e d i i t e l h h p l e p s l a i n o p o m e o l s G e a l r b y n b c u n A r m r h n r z b a y a g a h l a g o a d a r n h i e R C T C T * † c A s G C r C E

IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 207 J.R. S TORRY

Transporters and Channels RBC structure is demonstrated in people with The primary function of the RBC is to deliver hereditary spherocytosis (HS), where it has been shown oxygen to the tissues in exchange for metabolic wastes that 20 percent of HS cases are due to the heterozygous such as carbon dioxide and urea. The exchange of inheritance of one of numerous identified in oxygen for carbon dioxide occurs passively and is the SLC4A1 encoding band 3, and in Southeast based on oxygen dissociation from Hb, which in turn is Asian ovalocytosis, which is due to the inheritance of based on the oxygen affinity of the Hb molecules. one gene with a 27- deletion. 15 It was However, there are many transporters and channels on commonly thought that homozygosity for mutations in or through the membrane that move bioactive SLC4A1 would be incompatible with life; however, molecules to and from the extracellular domain and there are now living examples of natural “knockouts” in cytosol. Japanese black and in humans, as well as Transporter and channel proteins are multiple- experimental evidence from engineered “knockouts” in membrane–spanning proteins that either actively laboratory mice. 16–18 Studies of RBCs taken from the transport compounds across the membrane or provide cord blood of a severely anemic, hydropic baby girl a hydrophilic channel through the . 6 In showed the complete absence of band 3 and addition to their transmembrane functions, some of 4.2 as well as a significant reduction in , these proteins are connected to the and ankyrin, and GPA. 17 At the time of the report, she was 3 therefore play a role in the maintenance of RBC shape. years old and was sustained by regular RBC transfusions Transport proteins with and without a structural role as well as oral doses of bicarbonate to counteract the will be discussed in separate sections. renal acidosis that resulted from the absence of band 3 in her kidneys. Analysis of RBCs from Japanese black Transport proteins with structural functions cattle that had homozygous inheritance of a Structural function is not included in Figure 1; nonfunctional band 3 gene gave similar results: absence however, as Table 1 shows, there are RBC membrane of band 3 and protein 4.2, 44% , and 26% spectrin. 16 proteins that play a crucial role in maintenance of cell These examples show the importance of band 3 in the shape. It is becoming clear that our current perception stable assembly of the spectrin-actin web that makes up of the RBC skeleton and its interactions with the the RBC cytoskeleton. Surprisingly, they also showed integral membrane proteins is oversimplified, and there that while band 3 is not essential for life, it is critically is increasing evidence to demonstrate the association important for prolonged survival. The role of GPA in the of the Rh complex with the band 3 complex: band 3 complex is not clear, although there is evidence interaction of the Kell/Kx complex with GPC, Lu/B- to suggest that GPA acts as a chaperone in getting band CAM with spectrin, and so on. 7–9 Furthermore, 3 to the RBC membrane. 19 However, it is not essential phosphatidylserine in the lipid bilayer has been shown in this role, since persons who lack GPA have normal to interact directly with spectrin subunits. 10,11 This levels of band 3, although of the protein is information creates a picture in which the underlying altered. 19,20 More recently, the presence or absence of spectrin-actin web is in close and constant contact GPA has been shown to affect the efficacy of anion with the lipid bilayer as well as with an extracellular transport. 21 matrix of functional protein complexes which, in turn, The transport function of band 3 is the exchange of interact with themselves and other complexes. 7,12,13 anions: specifically, it exchanges equimolar concen- – – Band 3 (AE-1, anion transporter) carries the antigens trations of Cl ions for HCO 3 ions. This function is not 1 of the Diego blood group system. It is the most only important in the removal of CO 2 from tissues but abundant protein in the RBC membrane, with an also important in maintaining pH in the , where 6 14 estimated 1 × 10 copies per cell. This fascinating band 3 is located on the basolateral membrane of the protein has two distinct domains: an intracellular N- collecting tubule alpha-intercalated cells and abnor- terminal domain of 359 amino acids that forms struc- malities of band 3 cause distal renal acidosis. 15,22,23 turally important associations with the cytoskeleton The extracellular loops of band 3 carry the Diego and interacts with several glycolytic enzymes and a blood group antigens. Many low-frequency antigens of transmembrane, channel-forming domain that occurs as the Diego blood group system reside on the third homodimers and homotetramers to form the anion extracellular loop, which also carries sequences transporter. 14 Its contribution to the maintenance of thought to be important in RBC senescence and in the

208 IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 Function of blood groups adhesion of sickle RBCs. 3,24 Polymorphisms on the multi-system disorder known as the McLeod syndrome, intracellular domain or transmembrane domains are which is characterized by neurological abnormalities not as forgiving, and it is these mutations that lead to that develop, typically, in the fifth decade of life. 42,43 misfolding of the protein and have been shown to Xk is a multipass membrane–spanning protein with result in hereditary spherocytosis. 25 homology to neurotransmitter transporters. 44 The Rh complex consists of a core tetramer of two However, its substrate in RBCs has not been identified RhAG molecules and two molecules of either RhD or and the pattern of Xk expression in other tissues is RhCE. 26,27 In addition, the LW, CD47, glycophorin B different from that of Kell. (GPB), and Duffy glycoproteins interact with the complex, as evidenced in individuals with the Rh null Other transport proteins , whose RBCs not only lack the RhD, RhCE, The specific and controlled passage of water and RhAG proteins but also have reduced levels of molecules occurs through one of two water channels 28 expression of the accessory proteins. Rh null RBCs are in the RBC membrane. These proteins, named stomatocytic and are associated with a mild , were first identified by the discovery of compensated hemolytic . Although a role for 1 (AQP1, channel-forming integral protein, the complex in the maintenance of RBC structure was CHIP) in RBCs through the association of the Colton 45 evident from the abnormal cell shape in the Rh null blood group system. While AQP1 is the primary syndrome, interaction with the cytoskeleton has been water transporter in RBCs, it is also found in the kidney, shown only recently. Beckmann et al. 29,30 demonstrated lung, vascular , brain, and eye. The absence that in K562 cells the expression levels of Rh were of AQP1, first demonstrated in rare individuals with the increased if co-transfected with band 3, and Nicolas et Co(a–b–) phenotype, showed that AQP1 was not the al. 13 demonstrated that the cytoplasmic tails of Rh and only water transporter, because these individuals were RhAG polypeptides interacted with ankyrin-R. In seemingly healthy. However, the RBCs from Co(a–b–) addition to the direct attachment of the Rh proteins, persons demonstrate a markedly reduced osmotic CD47, another important member of the Rh complex, permeability. In addition, these individuals exhibit an has been shown to associate with protein 4.2. 12,31–33 inability to concentrate urine and decreased pulmon- The transport function of the Rh complex has still ary vascular permeability. 46 not been defined. However, RhAG shares The second aquaporin to be identified in the RBC approximately 40 percent evolutionary homology with membrane through its blood group “handle,” the Gil encoding ammonium transport proteins (Amt antigen, was AQP3. 47,48 AQP3 transports water, but its proteins) and there is growing experimental evidence primary function is the transport of glycerol. It also of its role as an ammonium transporter. 34–37 A role in transports urea with a low permeability. Apart from gas transport, specifically CO 2, has been suggested, RBCs, AQP3 is also found in the kidney, skin, lung, eye, possibly for a macromolecular complex involving band and colon. AQP1 and AQP3 are two members of a 3 and the Rh complex. 34,38 growing family of similar transmembrane proteins Another transmembrane protein that demonstrates involved in the transport of water, glycerol, urea, and evidential proof of structural and transport functions arsenite. Currently there are 11 members distributed (although neither has been proved experimentally) is across many different tissues. 46 the Xk protein. In RBCs, the Xk protein is covalently While AQP3 has a moderate affinity for urea, the linked to the Kell through a disulfide protein that is primarily responsible for urea transport bond. The K 0 phenotype, in which the Kell glyco- in the RBC is the urea transporter, or HUT11. This protein is absent from the RBC membrane, is not protein carries the antigens of the Kidd blood group associated with morphological change or with any system. 49 The relationship of the Kidd blood group pathology; however, the amount of Xk protein present system with a protein involved in urea regulation was is reduced. 39,40 In contrast, the absence of Xk protein is first demonstrated when Jk(a–b–) RBCs were shown to associated with a markedly reduced amount of Kell be resistant to lysis with 2M urea. 50 Further studies glycoprotein. The RBCs are acanthocytic, with variable showed that urea transport in Jk(a–b–) cells was greatly amounts of poikilocytes and other abnormal cells. 41 reduced when compared with that in RBCs of normal Abnormal RBC shape and reduced expression of Kell Jk phenotype and was consistent with simple diffusion blood group antigens are symptoms of a late-onset across the membrane. 51 Rapid transport of urea across

IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 209 J.R. S TORRY the RBC membrane is necessary in the kidney, where epithelial cells, homing of lymphocytes to lymphoid high urea concentrations make the RBCs susceptible to organs, and recruitment to sites of . 62 dehydration. 52 Additionally, CD44 plays a role in tumor , possibly via hyaluronan attachment to basement membranes. 61 There is also clear evidence that CD44 Adhesion and Molecules The view of the RBC as an inert bag of Hb has interacts with proteins that bind to actin in the changed considerably over the past decade, with the cytoskeleton. The role of CD44 on RBCs is not clear, identification of many blood group–bearing glyco- but it may play a role in anchoring erythroid proteins as adhesion molecules and receptors (Table 1). precursors. Furthermore, monoclonal to 63 The Duffy protein was identified as the exclusive RBC CD44 have been shown to stimulate erythropoiesis. receptor for vivax in 1976, when it was shown that the Fy(a–b–) RBCs of Africans were resistant Adhesion molecules of the immunoglobulin to invasion by the parasite. 53 Nearly 20 years later, the superfamily on RBCs physiological role of the Duffy glycoprotein as the The glycoproteins that carry the Lutheran, LW, a receptor was established when it was shown Scianna, and Ok blood groups are all adhesion that the IL-8, MGSA, RANTES, and MCP-1 molecules that belong to the immunoglobulin 63,64 bound with high affinity. 54–56 Duffy, which has homology superfamily (IgSF) of transmembrane proteins. IgSF with G-coupled signalling proteins, has been proposed proteins are defined as having one or more to act as a sink for excess circulating chemokines, thus immunoglobulin-like domains: discrete regions of the sequestering these highly bioactive molecules. protein that bear similarity to the V-set or C-set The high-incidence antigen JMH is carried on a domains of immunoglobulin molecules. The similarity glycosylphosphatidylinositol (GPI)-linked protein, is structural and they contain relatively few highly Sema7A (H-Sema-L, CD108). While Sema7A is conserved residues; however, the majority of the expressed on RBCs, it is preferentially expressed on proteins play a role in cell-cell recognition. 65 On RBCs, activated T lymphocytes and thymocytes. 57 Sema7A is the Lutheran antigens are found on two isoforms of the one of a large family of semaphorins, molecules whose Lu glycoprotein derived from alternate splicing of the primary function was considered to be cell-signalling LU gene. The shorter isoform (also known as B-CAM) in neural axonal development. More recently, however, lacks a cytoplasmic domain but the extracellular region it has been shown that several semaphorins are is identical to the Lu glycoprotein and has 5 IgSF important cell-signalling molecules in the immune domains. 62,63 Lu/B-CAM binds specifically to laminin system. 58 Sema7A binds specifically to plexin-C1, a 10/11 and is expressed relatively late in erythropoiesis. -encoded semaphorin protein receptor, and Thus, it has been suggested that the protein plays a role stimulates the production of proinflammatory cyto- in the migration of mature RBCs from the bone marrow kines such as IL-6 and IL-8, as well as being a potent to the circulation. Similarly, it may play a role in the monocyte activator. 59 trafficking of hemopoietic cells from fetal (where The antigens of the Indian blood group system it is upregulated) to the bone marrow during fetal (and, most likely, the AnWj antigen) are markers for development. 24,66 A pathophysiological role for Lu/B- CD44, another adhesion molecule on RBCs. CD44 is CAM has been explored in , where it found on lymphocytes and monocytes and is a marker has been shown that expression of the proteins is for some tumor cells. 1 Although CD44 is found in increased and might contribute to vaso-occlusive soluble forms, its role is not fully understood. 60 events in these patients. 67,68 Regulation of the CD44 gene is complex, and The LW glycoprotein has 2 IgSF domains and it alternative isoforms of the protein are produced in belongs to the intercellular adhesion molecule (ICAM) different cell lineages and at different stages of family designated ICAM-4. Unlike most of the adhesion development or activation. 61 CD44 is a receptor for molecules on the RBC, which have a wide tissue hyaluronan but interacts with many extracellular distribution, LW is restricted to RBCs and is weakly matrix proteins, such as fibronectin, collagens, growth expressed on the . 63 Like all ICAM family factors, chemokines, and metalloproteinases. It is an members, LW is a ligand for LFA-1 but it is also a important signalling molecule for leukocytes and is specific ligand for VLA-4 on hemopoietic cells and αV involved in leukocyte rolling and attachment to integrins on nonhemopoietic cells. 24,66 LW is expressed

210 IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 Function of blood groups early in erythropoiesis and it has been proposed that its reduction in the expression of Kell. 75 function is to tether erythroid progenitors to the Acetylcholinesterase, a GPI-linked protein on RBCs, erythroblastic islands for their subsequent matu- carries the antigens of the Yt blood group system. 76,77 ration. 66 There is recent evidence that ICAM-4 exists as While its function is well explored with regard a secreted isoform that may play a regulatory role in to regulation of acetylcholine at neuromuscular cell attachments, particularly in erythroblast junctions, 78 its role on RBCs is not known. One attachment. 69 Furthermore, ICAM-4 has been shown to example of theYt(a–b–) phenotype has been described be a ligand for the αIIb/ β3 integrin on platelets, which and no obvious pathology was observed; however, this suggests an active role for RBCs in thrombosis and was a transient reduction of the protein. 79 hemostasis. 70 Another GPI-linked protein with enzyme activity is the Dombrock glycoprotein (ART4), an ADP- ribosyltransferase that carries the antigens of the Enzymes 80 There are two GPI-linked proteins and one integral Dombrock blood group system. As yet, no RBC on the RBC surface that carry blood enzyme activity has been identified for this protein. group antigens and are known to have enzymatic The absence of the glycoprotein is characterized by the activity, although their role on erythrocytes is not clear. Gy(a–) phenotype and individuals of this phenotype do The Kell blood group protein, discussed briefly in not appear clinically affected in any way. context with the Xk glycoprotein, is a type II integral membrane protein that belongs to a family of neutral Complement-Related Proteins , specifically the (M13) The activation and regulation of the complement family of zinc metalloproteinases. 40 It acts as a highly cascade is tightly monitored by both cell-bound and specific -3–converting enzyme although it plasma proteins. The primary erythrocyte receptor is converts both endothelin-1 and endothelin-2, albeit less 1 (CR1, CD35), which carries the efficiently. 71 Although the role of the Kell glycoprotein antigens of the Knops blood group system. 81–83 CR1 is on the RBC has not been elucidated, are a member of a large family of complement control potent vasoconstrictors and it is possible that Kell may proteins (CCP). It is a large protein that consists of 30 be involved in local vascular tone. Maternal antibodies homologous domains known as CCP domains or short to Kell blood group antigens on fetal RBCs have been consensus repeat domains. These are arranged into 4 shown not only to cause immune-mediated destruction long homologous repeat domains. 84 CR1 not only has of these RBCs but also to inhibit proliferation and a high binding affinity for and C4b, and is therefore maturation of erythrocyte precursors. 72 The early important in the removal of immune complexes, but appearance of the Kell glycoprotein in erythropoiesis also acts a cofactor in their breakdown, thus protecting suggests that Kell might participate in lineage the RBC from autohemolysis. The Sl(a–) phenotype, determination. 40,73,74 found only on the RBCs of persons of African origin, In contrast to the Xk protein, to which it is has been shown in vitro to confer some protection covalently linked in the RBC membrane, the absence of against invasion of erythrocytes by Plasmodium the Kell glycoprotein is not associated with any falciparum. Rosetting of uninfected RBCs around an morphological changes or with any clinical pathology. infected cell has been proposed as an important Interestingly, however, the Kell glycoprotein has been mechanism for transmission. Sl(a–) RBCs or those with shown to associate with protein 4.1 and thus, low CR1 copy number show reduced rosetting. 4,85 indirectly, with both the cytoskeleton and GPC. 8 The The antigens of the increasingly polymorphic significance of this is not understood yet, although Cromer blood group system are carried on decay both GPC and Kell appear early in erythropoeisis. 73,74 accelerating factor (DAF, CD55). 86,87 This GPI-linked Twenty-four antigens have been assigned to the Kell protein is present not only on RBCs but also on the glycoprotein, each of which is associated with a single vascular and gastrointestinal and urinary change. None of these polymorphisms has epithelia. It is also a soluble protein in plasma. DAF is been shown to affect function; however, the amino acid an important regulatory protein in both the classic and substitution responsible for the Kp a antigen has been the alternative pathways of complement activation, shown to affect intracellular trafficking of Kell to the serving to accelerate the decay of C3 and C5 RBC membrane and consequently results in or causes a convertases. 3 Interestingly, DAF is also found in high

IMMUNOHEMATOLOGY, VOLUME 20, NUMBER 4, 2004 211 J.R. S TORRY levels on the apical surface of trophoblast cells in the elliptocytosis: the RBCs are more rigid and placenta, where is it thought to protect the fetus from mechanically unstable. Absence of protein 4.1 results maternal complement-mediated attack. 88 While there is in a more pronounced elliptocytosis and these RBCs no definitive pathology associated with the null have ~70 percent of normal levels of GPC. 3 phenotype (Inab phenotype, IFC–), a gastrointestinal disorder has been reported for the majority of the IFC– 2 Carbohydrate Antigens probands. Interestingly, DAF has been shown to be a The carbohydrate antigens include those antigens receptor for Dr-fimbriated strains of Escherichia coli, in the ABO, P, Lewis, H, I, and GLOB blood group a 89 specifically the Dr . systems. With the exception of I antigen, which is Although not strictly a RBC membrane component, defined by a tri-mannose structure at the branch points the Chido and Rodgers antigens are polymorphisms of of complex carbohydrates, these antigens are defined C4, one of the complement proteins found in plasma. by terminal or subterminal sugar residues on both C4 is adsorbed onto the RBC surface and thus the simple and complex oligosaccharides N-linked to 90 antigens are detected on its surface. Two isoforms of membrane proteins or attached to extracellular lipids. C4 are found, and C4B, of which C4A is more Carbohydrate antigens are not restricted to RBCs but efficient in solubilizing immune complexes and are widely distributed on many tissues and in body preventing immune precipitation. The absence of C4 fluids. For instance, ABH antigens are found on RBCs; has been associated with a number of autoimmune platelets; lymphocytes; respiratory, digestive, and 91 diseases. Additionally, an association of C4 and GPA urinary epithelia; vascular endothelium; sensory was demonstrated by GPA-deficient RBCs that were receptors; and epidermis; and in saliva, plasma, tears, shown to have reduced amounts of Ch antigens, and semen. 1 although Rg antigen expression was variable. 92 While the function of the carbohydrate antigens has not been definitively assigned, it is thought that Glycophorins they may have a role as adhesins in embryogenesis and As mentioned in the introduction, there are two malignancy. 24,94 It is also likely that they provide a first families of glycophorins on the RBC membrane. The line of defense against invading pathogens. The GPA family consists of GPA and GPB, which are single- naturally occurring antibodies produced by individuals pass membrane proteins and carry the antigens of the who lack these carbohydrate antigens are not present MNS blood group system. Although GPA and GPB have at birth, and are produced in the first 6 months of life been shown to be components of larger protein as the infant’s immune system becomes active. 95 It is complexes, respectively, band 3 and the Rh complex, thought that they are made in response to the constant their functions have not been defined clearly. Both bacterial, viral, and other pathogenic challenges of 96 proteins are heavily glycosylated by sialic-acid– everyday life. No pathology has been associated with containing oligosaccharides, and it has been suggested the null of each of these blood group K K that their function on the mature RBC is to provide a systems, with the exception of women with the P 1 , P 2 , highly negatively charged barrier that limits interaction and p phenotypes, who are prone to spontaneous with other cells and prevents cell aggregation. 24 abortion. However, the absence of an expected antigen 97 Alternative splicing of the unrelated GYPC gene can serve as an indicator of disease. generates two glycophorin molecules: glycophorin C and D (GPC, GPD), both of which carry the antigens of Conclusion the Gerbich system. Both proteins are also Initially, the identification and study of erythrocyte glycosylated by sialic acid–containing oligosaccharides blood group antigens taught us much about cell and contribute to the negatively charged glycocalyx. 93 membrane structure and immunology. I hope that this However, GPC has also been shown to be another review makes clear that the continuing study of these important site of attachment of the RBC membrane to molecules is teaching us about the complex the cytoskeleton, mediated through the cytoplasmic interactions between the RBC and its environment in tail, which forms a complex with protein 4.1 and p55. 93 health. Although they have not been discussed in this This complex is important in maintaining cell shape review, the roles of blood group antigens as receptors and mechanical stability. Absence of GPC, i.e., the for pathogens and as susceptibility/resistance markers Leach phenotype (Gerbich null) results in a mild for disease has also taught us much about the

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