Intercellular adhesion molecule-4 binds alpha(4)beta(1) and alpha(V)-family integrins through novel integrin-binding mechanisms.

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.

Suppression of CDA II expression in a homozygote.

The CDAN2 gene, responsible for congenital dyserythropoietic anaemia, type II (CDA II), was recently mapped to 20q11.2. We report data on an additional member of a previously studied CDA II family. This member had always been regarded as haematologically normal. Unexpectedly, she had the same microsatellite assortments around the CDAN2 alleles as her three sisters with CDA II. In particular, she was a homozygote for microsatellites D20S863 and D20S841. This prompted an analysis of all facets of her phenotype. The Ham test was negative. The bone marrow smears contained a normal proportion of binucleate erythroblasts. Electron microscopy revealed the absence of extensive stretches of cisternae beneath and parallel to the inner surface of the erythroblast plasma membrane. Proteins of the endoplasmic reticulum, which contaminate the reticulocyte plasma membrane in CDA II patients, were missing. Only the shape of the band 3 peak appeared slightly altered. This case exemplifies that homozygosity (or compound heterozygosity) for a deleterious gene may be silenced, or almost completely silenced. In recessively inherited diseases, suppressed phenotypes tend to be overlooked in siblings where both patients and unaffected individuals are expected.

The Oka blood group antigen is a marker for the M6 leukocyte activation antigen, the human homolog of OX-47 antigen, basigin and neurothelin, an immunoglobulin superfamily molecule that is widely expressed in human cells and tissues.

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, G331AG-->AAG gives rise to a predicted E92-->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.

Expression of RHD and RHCE gene products using retroviral transduction of K562 cells establishes the molecular basis of Rh blood group antigens.

Retroviral-mediated gene transfer using cDNA transcripts of the RHD and RHCE genes resulted in the isolation of K562 clones expressing D and G or c and E antigens, respectively. These results represent the first direct demonstration that the RHD gene encodes the D and G antigens and the RHCE gene encodes the c and E antigens. Both c and E antigens were expressed after transduction of K562 cells with a single cDNA, indicating that the c antigen does not arise by alternative splicing (exon skipping) of the product of the RHCE gene, as has been suggested.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

A novel form of congenital dyserythropoietic anemia associated with deficiency of erythroid CD44 and a unique blood group phenotype [In(a-b-), Co(a-b-)].

We have used a panel of well-characterized monoclonal antibodies (MoAbs) to examine the blood cells of a patient with a novel form of congenital dyserythropoietic anemia (CDA) characterized by intra-erythroblastic and intra-erythrocytic membranous inclusions. Twelve antibodies defining three nonoverlapping epitope groups on the extracellular domain of CD44 all failed to react with the red blood cells (RBCs) of the patient. A rabbit antibody to the cytoplasmic domain of CD44 from normal RBCs failed to react with the patient's RBC ghosts. In contrast, the patient's lymphocytes, granulocytes, and monocytes showed apparently normal CD44 expression. Bone marrow preparations stained with CD44 antibodies and visualized with 125I antimouse Ig (F(ab')2) followed by autoradiography showed positive staining of lymphocytes and myeloid cells but not of most orthotolidine-positive erythroblasts. The patient's RBCs also gave weaker than normal reactions with MoAbs of anti-LWab specificity while MoAbs to glycophorins A, B, and C, Rh polypeptides, CD47, CD55, CD58, CD59, acetylcholinesterase, and Lutheran and Kell glycoproteins all gave normal reactions. Agglutination tests with human blood grouping sera demonstrated that the RBCs of the patient have the unique phenotype In(a-b-), Co(a-b-) and that they also lack the high incidence RBC antigen AnWj. The phenotype In(a-b-) would be expected because these antigens are known to be expressed on CD44. There is also some evidence associating the AnWj antigen with CD44. However, the CO blood group locus is on chromosome 7p whereas that for CD44 is on chromosome 11p. Quantitative binding assays using 125I-labeled Fab fragments of CD44 antibodies did not show any evidence for reduced levels of CD44 on RBCs from the parents of the patient or from her unaffected sister. The parents and sister had the common Colton blood group phenotype [Co(a+b-)]. Neither deficiency of CD44 nor absence of Colton antigens are general features of CDA because erythrocytes from patients with CDA I, CDA II, CDA III, and two other unclassified CDAs had normal expression of CD44 and normal Colton blood group phenotypes. Further analysis of the defect(s) present in the patient's erythroid cells may provide useful information regarding membrane assembly and the regulation of differentiation in normal erythroid cells.

Observations on two members of the Swedish family with congenital dyserythropoietic anaemia, type III.

Two affected individuals of the Swedish family with CDA, type III, in which the disease is transmitted as an autosomal dominant character, were studied. Both cases displayed features hitherto undescribed in this family but described in patients with CDA, type III, in whom the inheritance may have been as an autosomal recessive character. Such features were: (a) haemosiderinuria, (b) grossly disorganised erythroblast nuclei, (c) differences in the ultrastructural appearances of individual nuclei within the same multinucleate erythroblast and (d) intraerythroblastic inclusions resembling precipitated globin chains. In both cases the giant mononucleate erythroblasts and the multinucleate erythroblasts had total DNA contents up to 28c (1c = haploid DNA content) and 48c respectively, and some DNA synthesising bi- and multinucleate erythroblasts contained one or more nuclei which were unlabelled with 3H-thymidine. These findings are similar to those in patients with the autosomal recessive type of disease. Thus no major phenotypic differences are yet apparent between cases of CDA, type III, with different patterns of inheritance. Analysis of the surface erythrocyte proteins of the 2 Swedish CDA, type III, patients with monoclonal antibodies recognising Band 3, glycophorins A, B, C and D, Rh, CD44, CD47, CD55, CD58, CD59, Lutheran, Kell, LW and acetylcholinesterase did not reveal any gross abnormality of expression of these proteins. A slightly altered expression of blood group antigens A and H was revealed by the lectins Dolichos biflorus and Ulex europaeus and the Mr of Band 3 as judged by SDS polyacrylamide gel electrophoresis was also slightly reduced, suggesting that there may be minor alterations in the degree of N-glycosylation of some red cell membrane constituents.

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.

New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes.

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.

Immunochemical studies on the differential binding properties of two monoclonal antibodies reacting with Tn red cells.

Two monoclonal antibodies (MoAbs), BRIC 66 (IgM) and BRIC 111 (IgG1), were produced by immunizing mice with ovarian cyst blood group A1 glycoprotein and Tn red cells (RBCs), respectively. Their specificities were determined by inhibitions using Tn sialoglycoproteins (SGPs), mucins (armadillo [ASG] and ovine [OSG] submaxillary glycoproteins), and monosaccharides. BRIC 66 agglutinated both Tn and group A RBCs and reacted immunohistochemically with both the vascular endothelium and tumor cells from a group A adenocarcinoma, BRIC 66 was inhibited by N-acetylgalactosamine (GalNAc), Tn SGPs, and mucins on both hemagglutination inhibition tests and radioimmunoassay. BRIC 111 agglutinated Tn RBCs only, and it specifically stained tumor cells from a group O patient's breast carcinoma and a group A patient's adenocarcinoma. In hemagglutination inhibition tests, BRIC 111 was readily inhibited by Tn SGPs, only partially inhibited by GalNAc, and not inhibited by mucins. In a sensitive radioimmunoassay, BRIC 111 was inhibitable by GalNAc. Tn SGP was 2000-fold more effective as an inhibitor than the mucins (ASG and desialized OSG), which contain a high content of terminal alpha-GalNAc-O-serine (threonine) residues. It is postulated that BRIC 66 is specific for terminal alpha-GalNAc units in carbohydrate chains. The exclusive reaction of BRIC 111 with Tn SGP indicates a combining site larger than GalNAc alpha-1, which probably includes amino acid residues in juxtaposition to GalNAc in Tn SGP. In view of its specific agglutination of Tn RBCs, BRIC 111 is a useful reagent for the examination of polyagglutinable RBCs.

Effect of endoglycosidase F-peptidyl N-glycosidase F preparations on the surface components of the human erythrocyte.

Endo-N-acetyl-beta-D-glucosaminidase F-Peptidyl N-glycosidase F preparations (abbreviated Endo F) and endo-beta-D-galactosidase were used to study the major human erythrocyte membrane glycoproteins and the components carrying the blood group A, B, Rhesus (D), and Duffy (Fya) antigens. The results are consistent with the known presence of an N-glycosyl-linked oligosaccharide on sialoglycoprotein alpha and the absence of such an oligosaccharide from sialoglycoprotein delta. Under the conditions used, only a portion of the N-glycosyl-linked oligosaccharides on band 3 molecules were cleaved by Endo F alone or by Endo F in combination with endo-beta-D-galactosidase. Immunoblotting experiments showed that treatment of red cells with Endo F alone had little effect on the components carrying blood group A and B antigen activity. However, Endo F used in combination with endo-beta-D-galactosidase caused a substantial reduction in the binding of monoclonal anti-A and anti-B antibodies. The results clearly show that sialoglycoproteins alpha and delta carry little or no blood group A or B activity. Endo F alone, or in combination with endo-beta-D-galactosidase, had no effect on the electrophoretic mobility of the Rh(D) polypeptide, supporting previous suggestions that this membrane polypeptide is unusual in not being glycosylated. Endo F had a dramatic effect on the electrophoretic mobility of the component(s) carrying blood group Fya activity. The diffuse Fya component of Mr 38,500-90,000 was sharpened to a band of Mr 26,000. Either endo-beta-D-galactosidase or neuraminidase treatment reduced the Mr of the Fya component(s) but did not significantly sharpen the bands, suggesting that the Fya component contains between 40-50% by mass of N-glycosyl-linked oligosaccharides.

The Ina and Inb blood group antigens are located on a glycoprotein of 80,000 MW (the CDw44 glycoprotein) whose expression is influenced by the In(Lu) gene.

The Ina and Inb blood group antigens were found to be located on an erythrocyte membrane glycoprotein of 80,000 MW by immunoblotting with human anti-Ina and anti-Inb antibodies under non-reducing conditions. This glycoprotein is shown here to be identical to that defined by monoclonal antibodies to CDw44, and a new murine monoclonal antibody (BRIC 35) is added to this cluster. Experiments with endo-beta-galactosidase and Endo F preparations suggest that the glycoprotein contains one or more N-glycans but that these oligosaccharides do not contain extensive poly-N-acetyllactosaminyl sequences. Experiments using membranes prepared from sialidase-treated normal erythrocytes, from Tn erythrocytes and from Cad erythrocytes suggest that the glycoprotein does not contain a substantial content of O-glycans. The Inb antigen and the epitope defined by a murine monoclonal antibody (BRIC 35) show reduced expression on Lu(a-b-) erythrocytes which result from the effect of the dominant inhibitor gene In(Lu). Evidence is presented here that the Inb antigen is expressed on normal granulocytes and lymphocytes and on the haemopoietic cell lines HEL, K562 and HL-60, a lymphoblastoid cell line and lymphocytes from two patients with B-CLL.

Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.

1. Rhnull human erythrocytes lack all of the antigens of the Rh and LW blood group systems and have abnormal shape and an increased osmotic fragility. In this paper two murine monoclonal antibodies raised against intact human erythrocytes were used to investigate further the abnormalities in these cells. BRIC 125 reacts weakly with Rhnull erythrocytes and BRIC 69 does not react at all. The results showed that BRIC 125 reacts with a component of Mr 47,000-52,000 which has a substantial content of N-glycans. In contrast, BRIC 69 reacted with a band of Mr 31,000 together with a very diffuse band of Mr 35,000-52,000. Treatment of BRIC 69 immunoprecipitates with endoglycosidase F/peptidyl-N-glycosidase F resulted in the loss of both BRIC 69 reactive components and the appearance of a new band of Mr similar to that of the Rh(D) polypeptide. 2. BRIC 125 had a broad reactivity with cells in peripheral blood, whereas the reactivity of BRIC 69 was confined to erythrocytes. BRIC 125, but not BRIC 69, reacted with human kidney tissue and bound to endothelium in peritubular capillaries, arteries and veins as well as the epithelial tissue of distal tubules. BRIC 125 stained haemopoietic cells, foetal hepatocytes and megakaryocytes in foetal liver and sinusoidal cells, hepatocytes and portal tracts in adult liver. In contrast, BRIC 69 reactivity was confined to haemopoietic cells in foetal liver. The BRIC 125 epitope has a wide tissue distribution, suggesting the occurrence of a related group of polypeptides which have a general functional role on cell surfaces. 3. Rhnull erythrocytes are deficient in at least four different membrane polypeptides.

A human cell-surface glycoprotein that carries Cromer-related blood group antigens on erythrocytes and is also expressed on leucocytes and platelets.

A new human erythrocyte glycoprotein has been identified by immunoblotting with murine monoclonal antibodies under non-reducing conditions. The glycoprotein has a MW of 70,000 and carries Cromer-related blood group antigens. The monoclonal antibodies also react with normal peripheral blood leucocytes and platelets and several haemopoietic cell lines. The glycoprotein has a reduced MW after sialidase treatment. The MW is markedly reduced in Tn erythrocyte membranes and slightly increased in Cad erythrocyte membranes. These results suggest that the glycoprotein has a substantial content of O-glycans. The glycoprotein appears to be absent from, or grossly altered in, the erythrocytes of two individuals with the rare Inab phenotype.