3 Phosphatidylinositol-glycan linked proteins of the erythrocyte membrane

3 Phosphatidylinositol-glycan linked proteins of the erythrocyte membrane MARILYN WENDELL

J. T E L E N F. R O S S E

Erythrocytes provided one of the first model systems for the study of the varied roles of human membrane proteins. This was especially true because of the availability of large quantities of erythrocytes, as well as the ease with which erythrocyte membranes (or 'ghosts') could be isolated from their cytoplasmic contents (Dodge et al, 1963). Thus, erythrocyte glycophorin A was among the first human integral membrane proteins to be isolated and sequenced (Tomita and Marchesi, 1975). Erythrocyte band 3 protein (the anion channel protein) also provided an early functional model of a membrane transport protein (Cabantchik and Rothstein, 1974). Erythrocyte cytoskeletal proteins, such as spectrin and ankyrin, have also been the focus of pioneering research into the structure and function of that class of proteins (reviewed in Sheetz, 1983). Recently, a new class of membrane proteins has been described (reviewed in Low and Saltiel, 1988; Cross, 1990). These proteins have a unique structure, a phosphatidylinositol-glycan (GPI) anchor. Proteins attached to the membrane by such an anchor are widely distributed among animal species ranging from protozoa to mammals. Much early work on the nature of such proteins focused on the major phosphatidylinositol-anchored protein of trypanosomes, the so-called variant surface glycoprotein (VSG). Work on that protein, along with several such proteins found in mammalian cells, has now largely defined the structure of these anchors, along with how they and their proteins are synthesized and processed prior to insertion into the cell membrane. Erythrocytes have again become crucial to the investigation of the structure and function of several of these proteins.

THE PHOSPHATIDYLINOSITOL-GLYCAN ANCHOR

Although phosphatidylinositol-glycan anchors appear to vary somewhat from one species to another, as well as among different tissues within a single species, the general structural elements of the GPI anchor are constant (Ferguson et al, 1988; Homans et al, 1988; Roberts et al, 1988a, 1988b) (Figure 1). Baillibre's Clinical Haematology-Vol. 4, No. 4, Decembcr 1991 ISBN 0-7020-1545 8

849 Copyright9 1991,by BaillibreTindall All rightsof reproductionin anyformreserved

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I

Asp I

Ethanolamine

C=O I

NH I

OH 2

I

O i O=P=O

Glycan core

0

Phosphatidylinositol

O--P-el II

CH2_CH_CH20 I

I

O

O

Mannose ,=0

I

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Glucosamine Inositol

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Figure 1. Structure of a phosphatidylinositol-glycan anchor. The phosphatidylinositol-glycan anchor comprises a glycan core attached at one end to fatty acids via phosphoinositol and at the other end to ethanolamine via a phosphate group. The ethanolamine is attached to the terminal amino acid via an amide linkage. In erythrocytes, a third fatty acid (usually palmitate) is also present (indicated by dotted lines).

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Basic structure Two anchoring fatty acids are attached via a diacyl or alkylacyl glycerol bridge to a phosphate group attached in turn to inositol. The inositol is attached to a de-acetylated aminoglucose which is part of a tetrasaccharide, usually containing at least one or two mannoses. The last sugar is then attached to ethanolamine, again via a phosphate group. The ethanolamine is attached to the protein chain via an amide linkage to the carboxyl terminus of the last amino acid remaining after cleavage of the hydrophobic peptide domain (see below).

Synthesis Current evidence (reviewed in Doering et al, 1990), derived largely from studies of the synthesis of various trypanosomal structures, indicates that the GPI anchor is synthesized in the endoplasmic reticulum independently of the protein to which it will be attached. In trypanosomes, a glycolipid, termed glycolipid A, can be isolated and shown to contain many of the building blocks of glycolipid anchors, including glucosamine, mannose, ethanolamine, phosphate, and myristate. The similarity of glycolipid A to the glycolipid anchor of VSG protein suggests that the former is a precursor of the latter. Although not all the synthetic steps are completely known, it is clear that the N-acetylglucosamine is enzymatically added to phosphatidylinositol from UDP-N-acetylglucosamine. After deacetylation, the second and third sugars are added. When these are both mannoses, both GDPmannose and mannosyl phosphoryldolichol may be involved in these reactions. Phosphoethanolamine is then added to the fully glycosylated GPI intermediate, although again the donor of this structure is not clearly defined; it may be an ethanolamine-containing lipid, such as phosphatidylethanolamine. In trypanosornes, the resultant structure then undergoes 'remodelling', in which the fatty acid or fatty alkyl groups are replaced with myristate residues. Further modification of the GPI core, such as galactosylation in trypanosomes, may occur later, even after attachment of the anchor to protein. In humans, there is evidence that such anchor modifications may be governed by several factors, including the attached protein as well as the cell type.

Glycolipid anchor-protein attachment Much research on human proteins has attempted to delineate the signals which direct the cell to attach a particular protein to a GPI anchor. However, we have as yet only a general understanding of the signaling process (Caras et al, 1987a; Caras and Weddell, 1989; Caras et al, 1989). In order to be linked to a GPI anchor, a protein must have at its carboxyl (C) terminus 20-30 predominantly hydrophobic amino acids. However, no specific signal sequence has yet been determined, despite study of several different GPIlinked proteins. Neither is it clear whether other sequences farther removed from the C-terminus may also play a role in determining which proteins are

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GPI-anchored. Attachment of the anchor occurs in a process which cleaves the C-terminal hydrophobic domain and then attaches the glycolipid anchor to the new terminal amino acid. ERYTHROCYTE PROTEINS WITH GPI ANCHORS

Numerous proteins with GPI anchors have been found to be expressed by human erythrocytes, and it is reasonable to expect that more such proteins will be identified in the near future. Table 1 shows their structural and functional diversity. One of the first erythrocyte membrane proteins shown to have a GPI anchor was the enzyme acetylcholinesterase. Erythrocyte acetylcholinesterase has been shown to be similar to muscle acetylcholinesterase, but why it should be present on erythrocytes remains a mystery. Other erythrocyte enzymes since shown to have a GPI anchor include NAD + glycohydrolase, which catalyses the hydrolysis of NAD to form nicotinamide and adenosine diphosphoribose (Kim et al, 1988). Table 1. Erythrocyte membrane proteins with phosphatidylinositol-glycan

anchors.

Complement regulatory proteins Decay-accelerating factor (DAF; CD55 antigen) Protectin (membrane inhibitor of reactive lysis, CD59 antigen) C8 binding protein (homologous restriction factor, HRF) Membrane-bound enzymes Acetylcholinesterase NAD glycohydrylase Other Lymphocyte function-associated antigen-3 (LFA-3, CD58 antigen) JMH protein Hy/Gy" protein(s)

The next category of erythrocyte membrane proteins found to be bound to the cell by a GPI anchor were complement regulatory proteins. Three such proteins are now known: decay-accelerating factor (DAF, CD55 antigen); membrane inhibitor of reactive lysis (MIRL, CD59 antigen); and C8 binding protein (also known as homologous restriction factor). The three proteins inhibit the activation of complement at different points in the system, and thus prevent the lysis of cells by autologous complement. Finally, several other GPI-anchored proteins are also known to be expressed by human erythrocytes. These include lymphocyte-functionassociated antigen-3 (LFA-3, CD58 antigen) and folate-binding protein. Although neither of these appear to play an important role in the mature erythrocyte, they may be important during erythrocyte maturation, the former as a contact protein in the bone marrow, and the latter as a receptor for a nutrient vital to erythrocyte development.

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Blood group antigens on GPI-anchored proteins

In addition to the above well-characterized proteins, transfusion-induced human alloantibodies, as well as autoantibodies detected during pretransfusion testing, identify a number of structures that appear likely to be GPIanchored. The first example of a blood group system resident on a phosphatidylinositol-anchored protein was the Cromer blood group system. Initial work with monoclonal antibodies that appeared Cromer-related identified an erythrocyte glycoprotein of Mr about 70000 (Spring et al, 1987). This protein has since been shown to be decay-accelerating factor (Telen et al, 1988). Further work has explored the relationship between individual Cromer phenotypes and antigens and polymorphisms of DAF (Telen and Green, 1989; Lublin et al, 1991). Using PNH I and PNH III erythrocytes isolated separately from the blood of patients with paroxysmal nocturnal haemoglobinuria (PNH) (Kabakci et al, 1972; Chow et al, 1986), investigators have also found that antigens of the JMH, Yt (Cartwright), Hy/Gy (Holley/Gregory) and Do (Dombrock) systems appear to reside on phosphatidylinositoMinked membrane proteins (Telen et al, 1990). These antigens are all absent from PNH III cells; PNH I cells, however, express these antigens, demonstrating that persons who acquire PNH both inherit and express functional genes for these antigens. Because PNH erythrocytes are known to have a broad defect in expression of phosphatidylinositol-anchored proteins (Rosse, 1990b), these data provide strong evidence that all these antigens reside on such proteins.

DECAY ACCELERATING FACTOR Decay-accelerating factor was first described by Nicholson-Weller and colleagues (1981, 1982), who demonstrated that an extract of human erythrocyte stroma contained a protein that accelerated the decay of the classical complement pathway C3 convertase. Shortly after, workers demonstrated that PNH erythrocytes lacked this complement regulatory protein present on normal erythrocytes (Nicholson-Weller et al, 1985a). Protein and gene structure

The general structure of DAF is illustrated in Figure 2. N-terminal peptide sequencing and cloning and sequencing of the gene for this glycoprotein (Davitz et al, 1987; Caras et al, 1987a; Medof et al, 1987a) have shown DAF to contain, starting at the N-terminus, four homologous regions of about 66 amino acids each; similarly repeated 'short consensus repeats' (SCRs) are common to several proteins which interact with C3b and C4b (including complement component C2, factor B, factor H, complement receptors types 1 and 2, and C4 binding protein), as well as to several non-complementrelated proteins (Nicholson-Weller et al, 1987). A single site for Nglycosylation is present as the C-terminal amino acid of the first SCR. The SCRs are followed by a 70-amino acid region rich in serine and threonine

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sho

consensus repeats

I t 0 N-linkedpolysaccharide II Dr aantigen i t

9 O-linkedpolysaccharide (~ Ethanolamine

Figure 2. Structure of decay-accelerating factor (DAF). The DAF protein contains four short consensus repeats with multiple disulfide bonds. C-terminal to this region is a highly-serine/ threonine-rich domain that is highly O-glycosylated. A single N-linked oligosaccharide is present between the first and second short consensus repeats. The Dr" antigen polymorphic site resides in the terminal portion of the third consensus repeat.

residues and thus probably highly O-glycosylated. There is then a stretch of highly hydrophobic amino acids. No hydrophilic intracytoplasmic peptide appears to be encoded by the DAF gene. However, one group has proposed, based on observations made on cDNA, that a hydrophilic C-terminal peptide may be present in some DAF molecules because of an unspliced intron and a resultant shift in the reading frame (Caras et al, 1987a). This hypothesis, however, is unproven. DAF is encoded by a gene within the RCA (regulation of complement activation) gene locus on the long arm of chromosome 1, band q3.2 (Lublin et al, 1987; Rey-Campos et al, 1987). This locus contains genes for numerous complement regulatory proteins, including factor H, C4 binding protein, CR1, CR2, membrane cofactor protein (MCP), and DAF. Thus far, the order of these genes has been determined to be MCP-CR1-CR2-DAF-C4 binding protein (Carroll et al, 1988). The only other erythrocyte protein encoded by this gene cluster is CR1, which has recently been shown to carry Knops/McCoy blood group antigens (Rao et al, 1991). Three RFLPs located in the non-coding region of the DAF gene have been identified. Two are for the enzyme HindlII, and one is for BamHI (Rey-Campos et al, 1987). At least one other RFLP is dependent on a polymorphism associated with an unusual blood group antigen phenotype, D r ( a - ) (see below).

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Biosynthesis and expression

Experiments using endo- and exoglycosidases have shown that DAF is a highly glycosylated protein. Endoglycosidase F, which cleaves only Nlinked oligosaccharides, lowers the apparant Mr of DAF by about 3000 (Lublin et al, 1986). This is consistent with there being one N-linked complex-type oligosaccharide attached to the amino acid sequence inferred from the eDNA. In contrast, treatment of the protein with endo-Nacetylgalactosaminidase lowers its Mr by about 26 000, indicating the presence of numerous O-linked oligosaccharides (Lublin et al, 1986). Biosynthetic studies have shown that the earliest form of DAF is a 43 000 Mr form, which undergoes very early modification to a 46 000 form by a step not involving glycosylation (Lublin et al, 1986; Medof et al, 1986). The protein then enters the Golgi apparatus and undergoes glycosylation. Conformation of the native DAF molecule is also highly dependent on intrachain disulfide bonds. This is clearly evident in SDS-PAGE, where DAF migrates more slowly under reducing conditions than non-reducing conditions. Furthermore, all human alloantibodies to DAF recognize their target antigens only when disulfide bonds are preserved. DAF is expressed by nearly all circulating haematopoietic cells (Kinoshita et al, 1985; Nicholson-Weller et al, 1985b), as well as by numerous other tissues. DAF-positive tissues include oral and gastrointestinal mucosa, corneal and conjunctival epithelia, and serosa of renal tubules, bladder, ureter, uterus and cervix (Medof et al, 1987b). Extracellular fluids, such as plasma, urine, tears, saliva, and synovial fluid contain a soluble form of DAF (Medof et al, 1987b). However, natural killer cells do not express DAF (Nicholson-Weller et al, 1986). Thus, DAF is a widely distributed molecule with functional importance as a regulator of activation of the complement cascade. However, its physiological importance to nonhaematopoietic tissues remains largely unexplored. Identification of blood group antigens on DAF

Decay-accelerating factor bears the Cromer blood group antigens (Telen et al, i988). Prior to the demonstration of Cromer blood group antigens on DAF, it was known that these antigens (then called Cromer-related) most likely comprised a family of antigens expressed by most or all circulating haematopoietic cells (reviewed by Daniels, 1989; Reid, 1990). Most of the Cromer antigens defined to date are high incidence antigens present on the cells of > 90% of blood donors. However, a few antigens produced by rare alleles have also been identified. The Cromer antigens and their interrelationships are listed in Table 2. Cromer antigens were initially related to one another because of serological work on two rare blood group phenotypes. One, termed Inab, failed to express any Cromer antigens (Daniels et al, 1982). This phenotype was sometimes associated with production of an antibody, anti-IFC, which agglutinated red cells from all other donors, even those lacking individual Cromer antigens. Thus, Inab was serologically a 'null' phenotype for this group of antigens. In a second phenotype, D r ( a - ) ,

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M . J . TELEN AND W. F. ROSSE Table 2. T h e C r o m e r blood group system. High-frequency antigens

Antithetical low-frequency antigens

Cr a Tc a Dr a Es a IFC WES b UMC

Tc b, Tc c (Drb) *

WES a

9 T h e allele encoding this variant has been identified by sequence analysis of D r ( a - ) individuals D A F ; it has never been identified by an antibody.

one Cromer antigen, Dr a, was absent (Levene et al, 1984, 1987). However, D r ( a - ) erythrocytes expressed all other high incidence Cromer antigens very weakly. Thus, all antigens recognized by sera which failed to react with Inab cells and reacted weakly (or in the case of anti-Dr a, not at all) with D r ( a - ) , cells were termed Cromer-related. These antigens have now been officially gathered into the Cromer blood group, based on evidence of their relationship to DAF and of their genetic independence from other established bJood group systems (Lewis et al, 1989). Initial biochemical characterization of the Cromer antigens was accomplished by Spring and colleagues (1987), who used murine monoclonal antibodies with anti-IFC-like reactivity, as well as human Cromer-related antisera, to demonstrate that all these antibodies identified a protein of Mr 70 000 in immunoblots of erythrocyte membrane proteins. This M r 70 000 protein was then proven to be DAF by a number of methods. Using biochemically-purified DAF, Telen and colleagues (1988) demonstrated that binding to erythrocytes by human anti-Cr a and anti-Tc a was inhibitable in a dose-dependent fashion by purified DAF but not by other purified erythrocyte membrane proteins. These antibodies also recognized purified D A F but not other proteins in immunoblots. They further demonstrated that PNH III cells, which lack DAF, are negative for all high incidence Cromer antigens. Lack of a genetic basis for this was proven by demonstrating that PNH I cells from the same donors expressed Cromer antigens normally. Further evidence that Inab was indeed a null phenotype and did not express DAF was provided when it was shown that rabbit polyclonal anti-DAF did not bind to Inab erythrocytes in either radioimmunoassays or in immunoblots.

Inherited DAF deficiency: the Inab phenotype The Inab phenotype is extremely rare: only six individuals have been found worldwide. Although none of these persons have ever been found to have haemolytic disease, many of them have had severe bowel disease, usually of an inflammatory nature. Existence of this phenotype has allowed close examination of the physiological importance of DAF, as well as investigation of a defect with apparent linkage to gastrointestinal disease.

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The Inab phenotype has now been shown to arise from an inherited defect involving DAF but not other GPI-linked proteins. Thus, Inab erythrocytes express acetylcholinesterase, LFA-3, and CD 59 at normal levels, unlike PNH erythrocytes, which have a broad defect in expression of GPIanchored proteins (Telen and Green, 1989: Tate et al, 1989). Examination of the complement sensitivity of Inab erythrocytes indicates that DAF probably plays only a minor role in complement regulation when the other complement regulatory proteins are normally present. In the complement lysis sensitivity test of Rosse and Dacie (1966), Inab erythrocytes were only modestly more sensitive to lysis by complement than were normal erythrocytes and were most comparable to PNH II cells (Telen and Green, 1989). In the sucrose haemolysis test, Inab cells gave an abnormal result but were nevertheless only slightly more sensitive than normal cells (Merry et al, 1989; Telen and Green, 1989). Inab cells have given uniformly normal results with the acidified serum lysis (Ham's) test. Telen and Green (1989) further defined the physiological defect of Inab cells by comparing the rate of decay of the alternate pathway C3 convertase on normal, Inab and PNH III cells. By measuring the fixation of C3, they were able to show that roughly equivalent numbers of C3 convertases went on to fix more C3 on Inab cells than on normal cells. Thus, lnab cells have an identifiable defect in complement regulation at the level of regulation of C3 convertase; this is probably not associated with haemotysis due to the normal activity of regulators of the terminal phases of complement activation, such as CD59 antigen and C8 binding protein. The mechanism whereby DAF is lacking from lnab cells is not yet completely understood. Tate and colleagues demonstrated that at least one individual with the lnab phenotype appeared to have a grossly intact DAF gene, associated with normal RFLPs and even normal (although quantitatively decreased) DAF mRNA. Other evidence that there is not a gross deletion in the RCA gene cluster is the demonstration that other RCA genes, including CR1, factor H, and C4 binding protein, arc normally expressed in the Inab phenotype (Telen, Rosse and Moulds, unpublished data). Some evidence exists suggesting that at least some persons with the Inab phenotype can produce soluble (i.e. plasma) but not membrane DAF (Telen, unpublished data). Thus, further investigation is underway to determine what defect (or defects) leads to the Inab phenotype. The specificity of anti-IFC also remains to be elucidated; if Inab individuals do indeed produce soluble but not membrane DAF, then this antibody may react with a segment of membrane DAF not present in soluble (plasma) DAF.

The Dr(a-) phenotype The Dr a antigen is one of the high frequency antigens of the Cromer blood group. Several individuals lacking this antigen have been identified among Israeli Jews who emigrated from Bukhara (Levene et al, 1984, 1987). As mentioned above, erythrocytes from these individuals also expressed all other high incidence Cromer antigens very weakly. Measurement of the ability of these cells to bind monoclonal and polyclonal antisera to DAF and

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other GPI-linked proteins has demonstrated that D r ( a - ) cells express only about 40% as much DAF as normal cells. Other GPI-linked proteins, however, are expressed normally. Assays of the complement sensitivity of these cells, such as in the complement lysis sensitivity assay, demonstrated that D r ( a - ) cells do not have an in vitro complement regulatory defect (Lublin et al, 1991). Biochemical techniques such as immunoblotting have confirmed that D r ( a - ) cells express grossly normal but quantitatively reduced DAF. SDSPAGE analysis is suggestive, however, of a minimally lower molecular weight, consistent perhaps with minor glycosylation differences. This was not due to lack of N-glycosylation, as the expected decrease in Mr was obtained after treatment with endoglycosidase F. Analysis of genomic DNA from persons with the D r ( a - ) phenotype has elucidated its genetic basis. When each of the nine exons encoding mature protein were sequenced (Post et al, 1990), a single base pair mutation was found in the exon encoding the latter part of the third SCR. This point mutation, a C to T change in codon 165, changes a serine to a leucine. This mutation constitutes the first reported polymorphism in the coding region of DAF and corresponds to a Taq I RFLP. Thus, future examples of the D r ( a - ) phenotype can now be confirmed by RFLP analysis. By transfecting the normal and Dr(a-)-type DNAs into Chinese Hamster ovary (CHO) cells, investigators have also established that it is this single point mutation which is responsible for the antigenic difference between D r ( a - ) and common-type cells (Lublin et al, 1991). Human anti-Dr a could be adsorbed only by the cells transfected with the Dr(a+)-type DAF gene. Likewise, in immunofluorescent assays, only the cells transfected with the Dr(a+) gene bound anti-Dr a, while cells transfected with either gene bound other anti-DAF antibodies. Thus far, however, work with transfectants has been unable to elucidate the cause of decreased expression of the D r ( a - ) variant of DAF. The processing of the DAF molecule appears similar in both types of transfectants. THE PROTEIN-BEARING JMH ANTIGENS

The JMH antigen, named after one of the original producers of the antibody (John Milton Hagen), was first identified by autoantibodies found most often in elderly patients whose own erythrocyte JMH antigen expression was weakened or absent (Sabo et al, 1978). To date, only one family has been identified as illustrative of an inheritable JMH-negative phenotype (Kollmar et al, 1981). However, since the original descriptions of JMH antibodies, Moulds and colleagues have identified several sera which appear to recognize JMH polymorphisms (Moulds et al, 1982). None of these sera react with JMH-negative cells. Among JMH-positive cells, however, there are rare examples of cells (including those of the antibody-makers) that fail to react with such antibodies, suggesting that these antibodies recognize polymorphic determinants on the JMH structure.

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Using both human antisera and a murine monoclonal antibody, H8, with JMH-like reactivity, Bobolis et al (in press) have identified a GPI-linked protein present on JMH-positive cells but absent on JMH-negative cells. This protein exhibits an Mr of 76 000 under both reducing and non-reducing conditions, indicating it is comprised of a single protein molecule. Although JMH antigens have been reported to be denatured by sulfhydryl-reactive reagents, such reducing reagents did not change the apparent Mr of the protein as analyzed by SDS-PAGE. Radio-immunoprecipitation experiments also confirmed that this protein is present on both erythrocytes and leukocytes, as a similar protein was isolated from peripheral blood mononuclear cells. The suspected presence of a GPI anchor was confirmed by studies using phosphatidylinositol-specific phospholipase C (PI-PLC) (Davitz et al, 1986). This enzyme was able to cleave a small portion of the JMH protein from erythrocytes, although the majority of the protein was resistant to cleavage, similar to erythrocyte acetylcholinesterase (Walter et al, 1990). The PI-PLC cleavage product had a similar Mr to protein solubilized from the membrane by detergents. Presumably, a change in protein conformation is responsible for the lack of reduction in Mr. Antigenic activity was also found to be unaffected by PI-PLC cleavage, indicating that epitopes recognized by both human and monoclonal anti-JMH are not dependent on the presence of an intact GPI anchor. Bobolis and colleagues (1991) also demonstrated that the Mr 76 000 JMH protein appeared to be different from previously-described GPI-anchored proteins, including both DAF and C8 binding protein, which have somewhat similar Mrs. Furthermore, JMH-negative cells were demonstrated not to have a defect in expression of GPI-anchored proteins, as they expressed DAF, protectin, LFA-3, and acetylcholinesterase at normal levels.

THE PROTEIN(S) BEARING Hy AND Gy ANTIGENS The Hy and Gy antigens, like the Cromer and JMH antigens, have been shown to be absent from the complement-sensitive erythrocytes of patients with paroxysmal nocturnal haemoglobinuria, thus suggesting that they reside on one or more GPI-linked proteins. The Hy and Gy antigens have been associated with one another, as erythrocytes which are G y ( a - ) are most often or always Hy-negative as well. Spring and Reid have recently described that both anti-Gy a and anti-Hy antisera identify a broad protein component of Mr 46 850-57 750 in Western blots of membrane proteins from antigen-positive erythrocytes (Spring and Reid, 1990). Although it is not yet clear that anti-Hy and anti-Gy a recognize the same rather than similar protein components, in both cases this component was destroyed when erythrocytes were treated with chymotrypsin, pronase, or trypsin prior to the preparation of membrane protein extracts. N-glycosylation was also demonstrated in both cases by sensitivity of the blotted proteins to the action of endoglycosidase F, which reduced their Mr by approximately 11 000. As in serological studies, antibodies did not recognize the protein in Western

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blots of gels run under reducing conditions. It appears that the protein(s) recognized by anti-Hy and anti-Gya are not identical to any already recognized GPI-linked proteins of similar molecular weight, such as LFA-3. THE CD59 COMPLEMENT REGULATORY PROTEIN

After the characterization of DAF, two other complement regulatory proteins of erythrocytes were described. By far the most physiologically important of the two is the protein variously designated as MIRL (membrane inhibitor of reactive lysis), protectin, MEM 21, H19, HRF20, and CD59 antigen (the latter being its leukocyte differentiation cluster designation) (Sugita et al, 1986; Groux et al, 1989; Holguin et al, 1989; Stefanova et al, 1989). Like DAF, CD59 antigen is expressed by most or all circulating haematopoietic cells as well as by a variety of other tissues, such as endothelium (Nose et al, 1990). This protein comprises a single, highly-glycosylated peptide chain whose Mr when mature is about 18 000. The protein appears to be attached to the membrane by a GPI anchor in both erythrocytes and leukocytes. CD59 antigen cDNA has been cloned and the CD59 antigen amino acid sequence deduced (Davies et al, 1989; Sawada et al, 1989, 1990). These studies demonstrate that this protein has no known homologies to other previously described proteins except for the murine Ly6 lymphocyte membrane protein. The gene for CD59 antigen maps to human chromosome 11 (Forsberg et al, 1989; Philbrick et al, 1990). The CD59 protein regulates the effects of complement activation by inhibiting the insertion of C9, the final component of the complement cascade, into the cell membrane (Meri et al, 1990; Rollins and Sims, 1990; Whitlow et al, 1990). On lymphocytes, however, CD59 antigen also appears to be important in immunological activation. Antibodies to CD59 antigen can block rosetting of human lymphocytes to both human erythrocytes as well as sheep erythrocytes. Such antibodies also block anti-CD3-mediated stimulation of lymphocytes, as measured by incorporation of tritiated thymidine (Groux et al, 1989). However, this inhibitory activity appears due to the antibodies' effect on accessory cells rather than on the target lymphocytes (Groux et al, 1989). Recently, an individual has been reported to have a paroxysmal nocturnal haemoglobinuria-like syndrome due to inherited deficiency of the CD59 protein (Taguchi et al, 1990; Yamashina et al, 1990). Unlike individuals with the Inab phenotype, who are DAF-deficient but have no apparent haemolyric syndrome, the person deficient in CD59 antigen exhibited haemolysis in vivo similar to that seen in PNH patients. LFA-3

The LFA-3 (lymphocyte function-associated antigen-3) molecule is another molecule with extremely broad tissue distribution, being expressed by haematopoietic cells, endothelial and epithelial cells, and connective tissue

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cells in most organs (Krensky et al, 1983). LFA-3 is the ligand for the T lymphocyte surface molecule CD2 and plays an important role in T cell activation (reviewed in Springer et al, 1987). In monocytes, LFA-3 is involved in the process which allows monocytes to produce interleukin 1 (IL-1) in response to T-lymphocyte signalling. Ligand binding to LFA-3 also induces IL-1 production by thymic epithelial cells (Le et al, 1990). The LFA-3 protein has an Mr on SDS-PAGE of 55 000-70 000, and its gene has been cloned and mapped to chromosome 1 (Wallner et al, 1987). While all LFA-3 molecules are GPI-anchored on erythrocytes, a subpopulation of LFA-3 molecules on tymphocytes are integral membrane proteins with a transmembrane domain (Dustin et al, 1987). Its function on erythrocytes and what erythrocyte blood group antigens it might carry remain unidentified. C8 BINDING PROTEIN

Human erythrocyte C8 binding protein (C8bp), also called homologous restriction factor (HRF), is another GPI-anchored complement regulatory protein (Schonermark et al, 1986; Zalman et al, 1987a; Hansch et al, 1988). Expressed by erythrocytes as well as other circulating blood cells, such as platelets and leukocytes, C8bp acts by interfering with the assembly or activity of the terminal complement components C5-9. However, this protein remains somewhat poorly characterized, and its relationship to blood group antigen expression is still unknown. ROLE OF G P I - A N C H O R E D PROTEINS IN DISEASE

It is now clear that the acquired haemolytic disease, paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the absence or marked deficiency in expression of GPI-anchored proteins from affected haematopoietic cells of multiple lineages (Rosse, 1991). The characteristic manifestation of this disease is intravascular haemolysis, often resulting in frank haemoglobinuria. This haemolysis is due to an unusual sensitivity of the erythrocytes to the haemolytic action of complement. The diagnosis of PNH is traditionally made by one or more of several tests that assess this susceptibility of the patient's cells to lysis by human complement: the acidified serum lysis (Ham) test (Ham, 1937), the sucrose lysis test (Hartman et al, 1970), the complement lysis sensitivity (CLS) test (Rosse and Dacie, 1966). Paroxysmal nocturnal haemoglobinuria is a clonal stem cell disorder (Hartmann and Arnold, 1977), and a variable proportion of the red cells (ranging from 1-2% to over 90%) are affected. Further, the abnormal cells vary in their sensitivity to complement. About 85% of patients have some cells that are markedly sensitive, so-called 'PNH III' cells (Rosse, 1990a); these cells lack all expression of the GPI-linked proteins. At least 60% of patients have cells intermediate in sensitivity, some of which are classified as PNH II cells; these cells are markedly deficient in the expression of the GPI-linked proteins (Rosse et al, 1991).

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At least four erythrocyte proteins are known to be deficient in PNH. The first to be found was acetylcholinesterase (Auditore et al, 1960); the deficiency was later proved to be restricted to the complement-sensitive ceils (Kunstling and Rosse, 1969). The deficiency of this protein did not explain the sensitivity to complement but was an important clue to the role of the GPI-linkage in the pathogenesis of the disease. Nicholson-Weller and her colleagues (1983) were the first to demonstrate that PNH cells were deficient in a complement regulatory protein, namely decay-accelerating factor (DAF, CD55). This protein was later shown to be GPI-linked, like acetylcholinesterase (Medof et al, 1986), thus contributing to the eventual realization that the PNH defect was a failure to express GPI-anchored proteins. DAF inhibits the action of the C3 convertases on cell surfaces, thus interfering with the major amplification step of both the classical and alternative complement pathways (Medof et al, 1984). Thus, larger amounts of C3b are activated and deposited on the membranes of affected PNH erythrocytes than on normal erythrocytes (Parker et al, 1982). However, the lack of DAF does not completely account for the greatly increased sensitivity to complement of the affected PNH cells, especially the PNH III cells. Two other GPI-linked complement regulatory proteins that are also normally expressed by erythrocytes are decreased or absent on PNH II and PNH III ceils. The more important is a glycoprotein of Mr 19 000 variously called membrane inhibitor of reactive lysis (MIRL) (Holguin et al, 1990), protectin (Meri et al, 1990), or CD59 antigen. This molecule inhibits the formation of the polymeric complex of C9 molecules that, in penetrating the lipid bilayer of the membrane, effects lysis of the cell (Meri et al, 1990; Rollins and Sims, 1990). Inhibition of this molecule on normal cells by specific antibody renders the ceils nearly as sensitive to complement as PNH III cells; further, the addition of this molecule to the membranes of PNH III cells renders them nearly normal in their sensitivity to complement. Another protein, C8 binding protein or homologous restriction factor, has been described and found to be lacking from abnormal PNH cells (Hansch et al, 1987; Zalman et al, 1987b). The identity and role of this protein in the regulation of the membrane attack complex of complement is not clear. All the clinical manifestations of PNH cannot be explained by the abnormalities on the red cells, and it is clear that many (thrombosis, proclivity to infection, diminished haematopoiesis) are due to defects in other haematopoietic cells. Since all cells derived from the abnormal stem cells are deficient in GPI-linked proteins, platelets, granulocytes, monocytes, and lymphocytes of PNH patients lack GPI-anchored proteins (NicholsonWeller et al, 1985a). Presumably, the resultant abnormalities in these cells lead to the other manifestations of PNH. It is clear that the lack of GPI-linked proteins in PNH is due to abnormalities in post-translational processing of the protein; the apparently normal mRNA for DAF is present in the abnormal cells (Stafford et al, 1988). Further, protein without the glycolipid anchor may be found in the cytoplasm of abnormal cells (Carothers et al, 1990). Recently, evidence has been adduced that PNH cells cannot make the complete anchor and that any of several biosynthetic defects in the pathway leading to the construction of

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the p r e f o r m e d a n c h o r m a y be the reason for the defect in expression of G P I - a n c h o r e d proteins ( M a h o n e y et al, 1991). T h e exact biochemical abnormalities that can lead to complete and partial lack of expression remain to be elucidated.

SUMMARY The h u m a n e r y t h r o c y t e bears a n u m b e r of proteins a n c h o r e d to the o u t e r m e m b r a n e surface via a phosphatidylinositol-glycan linkage. This class o f proteins includes several c o m p l e m e n t regulatory proteins (including decayaccelerating factor, C D 5 9 antigen (protectin), and C8 binding protein) as well as several e n z y m e s and at least one protein important in cell-cell interaction. In addition, a n u m b e r of blood g r o u p antigens have been identified to reside on proteins with phosphatidylinositol anchors. O n e blood g r o u p ( C r o m e r ) resides on D A F . Study of variants in this blood g r o u p system has led to interesting information about the function and expression of this protein. Several o t h e r blood groups, such as J M H and Holley/ G r e g o r y , a p p e a r to reside on as yet unidentified phosphatidylinositol linked proteins. In paroxysmal nocturnal haemoglobinuria, a variable p r o p o r t i o n of red cells fail to express or express weakly all phosphatidylinositol-linked proteins. T h e origin of this deficiency is now being worked out. In addition, individuals with inherited deficiency of D A F or C D 5 9 (protectin) have been identified, Only the latter deficiency leads to a PNH-like s y n d r o m e

Acknowledgements This work has been funded in part by grants ROI H L35572 and RO1 HL44042 (MJT) from the National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). MJT is the recipient of Research Career Development Award KO4 HL02233 (NHLBI, N1H). WFR is the recipient of MERIT Award R37 DK31379 from the National Institute of Diabetes and Digestive and Kidney Disease, NIH.

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