Intercellular adhesion molecule-4 (ICAM-4, syn. LW glycoprotein) interacts with the integrins alpha(L)beta(2), alpha(M)beta(2), A(4)beta(1), the alpha(V) family, and alpha(IIb)beta(3). Systematic mutagenesis of surface-exposed residues conserved between human and murine ICAM-4 defined 12 single amino-acid changes that affect the interaction of ICAM-4 with alpha(V) integrins. Mutation of 10 of these residues, 8 of which are spatially close on the surface of the molecule, led to a reduction in adhesion. Moreover, peptides corresponding to regions of ICAM-4 involved in its interaction with alpha(V) integrins inhibited these interactions. The other 2 mutations increased the extent of interaction of ICAM-4 with alpha(V) integrins. These mutations appear to prevent glycosylation of N160, suggesting that changes in glycosylation may modulate ICAM-4-alpha(V) integrin interactions. The region of ICAM-4 identified as the binding site for alpha(V) integrins is adjacent to the binding sites for alpha(L)beta(2) and alpha(M)beta(2). Selective binding of ICAM-4 to different integrins may be important for a variety of normal red cell functions and also relevant to the pathology of thrombotic disorders and vasoocclusive events in sickle cell disease. Our findings suggest the feasibility of developing selective inhibitors of ICAM-4-integrin adhesion of therapeutic value in these diseases.

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease.

The LW blood group glycoprotein, ICAM-4, is a member of the intercellular adhesion molecule (ICAM) family expressed in erythroid cells. To begin to address the function of this molecule, ligands for ICAM-4 on hemopoietic and nonhemopoietic cell lines were identified. Peptide inhibition studies suggest that adhesion of cell lines to an ICAM-4-Fc construct is mediated by an LDV-inhibitable integrin on hemopoietic cells and an RGD-inhibitable integrin on nonhemopoietic cells. Antibody inhibition studies identified the hemopoietic integrin as alpha(4)beta(1.) Antibody inhibition studies on alpha(4)beta(1)-negative, nonhemopoietic cell lines suggested that adhesion of these cells is mediated by alpha(V) integrins (notably alpha(V)beta(1) and alpha(V)beta(5)). The structure of ICAM-4 modeled on the crystal structure of ICAM-2 was used to identify surface-exposed amino acid residues for site-directed mutagenesis. Neither an unusual LETS nor an LDV motif in the first domain of ICAM-4 was critical for integrin binding. ICAM-4 is the first ICAM family member shown to be a ligand for integrins other than those of the beta(2) family, and the data suggest that ICAM-4 has a novel integrin-binding site(s). These findings suggest a role for ICAM-4 in normal erythropoiesis and may also be relevant to the adhesive interactions of sickle cells.

A CELL ADHESION molecule (CAM) has been defined as a cell surface receptor capable of attaching a cell either to another cell or to an extracellular matrix (ECM) substrate through interaction with its ligand. CAMs are often thought of as the glue holding cells to each other or to a matrix. Indeed, CAMs may simply integrate structural, intracellular proteins with substrate. Cell adhesive interactions also may result in the transmission of chemical and mechanical signals across membranes, thus providing important cues regulating many aspects of a cell's behavior such as proliferation, differentiation, and migration. The affinity of individual cell adhesion interactions is usually low (KD ranges from 10 -8 to 10 -5 mol/L), but because CAMs typically adhere by multiple attachments, high-avidity interactions result from individually weak ones. 1 This definition of CAMs excludes interactions between cell surface receptors and soluble molecules (cytokines and small peptides), although this distinction may be somewhat blurred. For example, the interaction between the membrane-bound form of the cytokine, steel factor, found on bone marrow stromal cells and its receptor, c-kit (CDI17), on hemopoietic progenitors, can lead to sequestration of stem cells in particular microenvironments, and in this context functions as an adhesion receptor of stromal cells. This is in addition to steel factor's well-defined function as a cytokine, which is essential for the progenitor cell's survival and proliferation. 2 In recent years, a large number of CAMs have been characterized, and their expression and function on cells of various lineages has been explored. The plethora of CAMs may be grouped into a number of superfamilies, based on their related primary structures. The different superfamilies mediate different types of functions, ranging from very stable interactions, through promigratory interactions that allow cells to move, to very transient interactions that serve to arrest cell movement (reviewed in references 1, 3, 4). Erythroid precursors express a number of CAMs, several of which have a functional role in erythroid differentiation. 5 From studies of blood group active membrane proteins, it is now clear that mature red blood cells (RBCs) also express a number of glycoproteins (gps) that either are known to function as CAMs [the LW, Lutheran (Lu) and Indian (CD44) gps] or by virtue of their close structural homology with known CAMs are thought to have similar function (the Ok a and Xg a gps). 6 Mature RBCs also express the non-blood group-active CAMs CD47 (Rh-associated gp, IAP), CD36 (thrombospondin receptor), and CD99 (Xga-associ ated sialomucin). This review describes the structure, tissue distribution, and ligand-binding properties of the different CAMs that are expressed on erythroid cells, and their function in erythropoiesis is discussed. We will discuss CAM deficiencies found in blood group null phenotypes and focus on the contribution of erythroid CAMs to the pathology of disease states.

BACKGROUND: Three women have been identified with an antibody to a "new" high-incidence antigen found on multiple cell lines.CASE REPORTS: The proposita, M.A.M., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own. She delivered a thrombocytopenic infant with a 3+ DAT, but without symptoms of HDN. The second example, A.N., presented during her third pregnancy with an antibody reacting with all RBCs tested except her own and those of M.A.M. She delivered a slightly thrombocytopenic but severely anemic infant. The third example, F.K., a sister of A.N., has an antibody reacting with all RBCs tested except her own and those of M.A.M. and A.N.CONCLUSION: This "new" high-incidence antigen has been named MAM and assigned high-incidence antigen number 901016 by the International Society of Blood Transfusion. The corresponding antibody, anti-MAM, has been shown to cause HDN and has the potential to shorten RBC survival after the transfusion of incompatible RBC units, as determined by monocyte monolayer assay. Immunoblotting and flow cytometry show that this new antibody reacts with various WBC lines in addition to RBCs. This antibody also appears to react with platelets in some assays.

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.

The high-frequency blood group antigen Ok(a) is carried on a red cell membrane glycoprotein (gp) of 35-69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Ok(a) gp that appears to be nonlineage-restricted, although certain tissues show differentiation-related expression. Ok(a) gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N-terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX-47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Ok(a) mutation was established by sequencing M6 cDNA derived from normal and Ok(a-) EBV-transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G(331)AG --> AAG gives rise to a predicted E-92 --> K amino acid change in the first Ig-like domain of the Ok(a-) form of the protein. Transfection of mouse NS-0 cells with normal or Ok(a-) cDNA confirmed the identity of the protein and only the Ok(a-) transfectants failed to react with monoclonal anti-Ok(a) Ab.

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.

Blood group antigens are structural variants in surface carbohydrate or amino acid polymorphisms on extracellular domains of membrane proteins. The red cell water channel-forming integral protein (Aquaporin CHIP) is a homotetramer with only one N-glycosylated subunit, however no CHIP-associated blood group antigens have yet been identified. Immunoblotting, monosaccharide composition analysis, and selective glycosidase digestions revealed that the CHIP-associated oligosaccharide contains ABH determinants and resembles a band 3-type glycan that cannot be cleaved from intact membranes by Peptide:N-glycosidase F. The molecular structure of the Colton antigens was previously unknown, but CHIP was selectively immunoprecipitated with anti-Coa or anti-Co(b). The DNA sequence from Colton-typed individuals predicted that residue 45 is alanine in the Co(a+b-) phenotype and valine in the Co(a-b+) phenotype. The nucleotide polymorphism corresponds to a PflMI endonuclease digestion site in the DNA from Co(a-b+) individuals. These studies have defined antigens within two blood group systems on CHIP: (a) an ABH-bearing polylactosaminoglycan attached to a poorly accessible site in the native membrane; and (b) the Colton antigen polymorphism which may permit the identification of rare individuals with defective water channel expression.

The CD47 glycoprotein was isolated from human erythrocytes by immunoprecipitation using monoclonal antibody (mAb) BRIC-125. Enzymic deglycosylation of the protein showed it contained N-linked oligosaccharides, and trypsin proteolysis of the protein in situ in the erythrocyte membrane cleaved it into two portions, one of which was glycosylated. Both the intact protein and the glycosylated fragment had blocked N-termini. Amino acid sequence was obtained from several proteolytic fragments of CD47. Comparison with the sequence database showed the protein to be very similar to or identical with OA3, a multispanning membrane protein. The protein also appears to be the same as the integrin-associated protein, which has a role in cell adhesion in non-erythroid cells. CD47 has six potential N-glycosylation sites, five of which are in an Ig superfamily domain. We show that three of these sites carry N-glycans in erythrocytes. Immunocytochemical staining of human tissues showed that CD47 was broadly distributed on mesenchyme and epithelia at multiple sites. Reactivity was particularly prominent in surface and ductular epithelia, and in the brain. The possible roles of the CD47 glycoprotein are discussed.

Band 3 Memphis is a commonly occurring polymorphic form of the human red cell anion transporter (band 3, AE1). Band 3 Memphis migrates more slowly on an SDS-polyacrylamide gel than normal band 3 and results from a point mutation Lys(56) --> Glu. TWO types of band 3 Memphis, variants I and II, can be distinguished by their susceptibility to covalent labeling with H2DIDS (4,4'-diisothiocyanato-2,2'-dihydrostilbene disulfonate). Memphis variant II is more readily labeled than Memphis variant I or normal band 3. The Memphis variant II is also associated with the presence of the Diego (Di(a)) blood group antigen on the red cells. We have shown that Memphis variant II carries the polymorphism Pro(854) --> Leu as well as Lys(56) --> Glu. The blood group antigen (Di(a)) present at the surface of Memphis variant II type red cells suggests the mutation Pro(854) --> Leu causes a change in the structure of an extracellular loop of Memphis variant II band 3. We discuss possible ways in which the mutation Pro(854) --> Leu affects the reactivity of Lys(539) to covalent reaction with H2DIDS.

Summary: Antisera to high‐ and low‐incidence blood‐group antigens were used in immunoblotting and immune precipitation studies to identify novel erythrocyte membrane components and to assign antigens to known proteins. Several antibodies identified well‐characterized membrane proteins which are widely expressed on other cells and tissues. The Cromer system antigens were found to reside on the complement regulatory protein decay accelerating factor (DAF). Antigens of the Indian collection were located to the cell adhesion molecule, CD44. The Cartwright system antigens were assigned to acetylcholinesterase, the function of which, on erythrocytes, remains unclear. Two novel blood‐group‐active glycoproteins were identified. One carries the Scianna system antigens whilst the second carries the high‐incidence antigens, Gy a , Hy and Jo a . The low‐incidence antigens, Dh a and Rd, were assigned to Glycophorin C and to the Scianna‐active glycoprotein, respectively. The existence of Cromer‐null, DAF‐deficient erythrocytes greatly facilitated the study of the function of DAF on erythrocytes. Location of the YT locus and hence of the AChE gene to chromosome 7q22 may be of significance in leukaemias and myelodysplasias since this region is a mutational ‘hot spot’ in these disorders. The novel proteins identified, for which no monoclonal antibodies are available, may also prove to be of functional significance on erythrocytes or on other cells and tissues. Several of the rare phenotype cells, such as the In(a ––– b –––) cells, Gy(a–––) cells and the Sc‐null cells, may prove to be of great value in defining the function of these molecules on erythrocytes, in the way that Inab cells have been for studying the function of DAF.

1. We have shown that the Di(a) antigen of the Diego blood group system is associated with the presence of red cell band 3 Memphis, but not all band 3 Memphis samples carry the Di(a) antigen. 2. The band 3 Memphis associated with the Di(a) antigen was covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) more readily than was normal band 3 or band 3 Memphis not associated with the Di(a) antigen. This altered reactivity with H2DIDS has previously been noted for a band 3 Memphis sub-type designated variant 2. 3. This is the first example of a band 3 polymorphism associated with an antigenic change in the extracellular region of the band 3 polypeptide and with altered H2DIDS binding.

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.

The Yt blood group system comprises two antigens, Yta and Ytb. Human anti-Yta and human anti-Ytb immune precipitate a component of the same apparent molecular weight as acetylcholinesterase from radioiodinated erythrocytes of appropriate Yt phenotype. Immune precipitates obtained with anti-Yta and anti-Ytb contained acetylcholinesterase activity. In contrast, immune precipitates obtained with human anti-Gya and murine monoclonal anti-CD55, which identify other glycosylphosphatidylinositol- linked erythrocyte surface proteins, did not have acetylcholinesterase activity. Quantitative binding assays using murine monoclonal antiacetylcholinesterase antibodies (AE-1 and AE-2) gave 3,000 to 5,000 binding sites/cell for IgG and 7,000 to 10,000 sites/cell for Fab fragments. Endo F digestion of immune precipitates obtained with AE-1 and anti-Yta indicated that approximately 10% of the enzyme comprises N- glycans. These results indicate that the Yt antigens define an inherited polymorphism on erythrocyte acetylcholinesterase and that the recent assignment of the Yt blood group locus to the long arm of chromosome 7 (Zelinski et al, Genomics 11:165, 1991) provisionally identifies the position of the acetylcholinesterase gene.