Role of Lu/BCAM in abnormal adhesion of sickle red blood cells to vascular endothelium.

Lutheran (Lu) blood group and Basal Cell Adhesion Molecule (BCAM) antigens are both carried by two glycoprotein (gp) isoforms of the immunoglobulin superfamily representing receptors for laminin alpha5 chain. They are expressed in red blood cells, in endothelial cells of vascular capillaries and in epithelial cells of several tissues. Lu/BCAM gps are overexpressed in sickle red blood cells (SS RBCs). Stimulation of SS RBCs by epinephrine activates the PKA depending signaling pathway and induces reinforced Lu/BCAM-mediated adhesion to laminin10/11. We have analyzed the phosphorylation state of Lu/BCAM long isoform cytoplasmic tail and showed that it is phosphorylated by CKII, GSK3b and PKA. Phosphorylation of this isoform in transfected K562 cells is stimulated by effectors of the PKA pathway and induces cell adhesion to laminin10/11. Lu/BCAM gps are highly expressed in endothelial cells and exhibit potential integrin binding motifs. We showed that they interact with integrin alpha4beta1, the unique integrin expressed on the surface of young reticulocytes. Adhesion assays under flow conditions showed that SS RBCs adhere to primary human endothelial cells (HUVEC) after selective activation of intergin alpha4beta1 and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid or on endothelial cells are involved in SS RBC-endothelium interactions and could play a role in the abnormal adhesion of SS RBCs to vascular endothelium contributing to the vaso-occlusive crises reported for sickle cell disease patients.

Red cell and endothelial Lu/BCAM beyond sickle cell disease.

Recent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) blood group antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were first identified as laminin-10/11 erythroid receptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated by epinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated that constitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), a frequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation of erythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PV disease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of the Lu/BCAM gene provided evidence that Lu/BCAM gps, as laminin-alpha5 receptors, are involved in vivo in the maintenance of normal basement membrane organization in different non erythroid tissues since up to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerular basement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minor blood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues as well.

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression.

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.

Promoter sequence and chromosomal organization of the genes encoding glycophorins A, B and E.

The promoter and exon 1 sequences of the genes encoding erythrocyte glycophorins GPA, GPB and GPE were investigated in detail, both from a genomic clone sorted out of a human leukocyte library and from genomic clones obtained by polymerase chain reaction amplification of total genomic DNA from control individuals and from GAP and/or GPB deletion variants. The three exons 1 and upstream sequences were shown to be highly homologous with only a few point mutations that did not affect the potential cis-acting elements (CACCC, NF-E1 and NF-E2) that are present in the same position within the three genes. Moreover, these genes share the same transcription start point. Analysis of the exon 1 and promoter sequences together with the gene defects occurring in the GP variants indicate that unequal cross-overs between the three genes are responsible for deletions and the generation of hybrid gene structures in which the promoter of one gene is brought close to another gene of the family. On the basis of these studies, a model of the gene organization is proposed to explain the rearrangements occurring in the variants.

Structure of the 5' flanking region of the gene encoding human glycophorin A and analysis of its multiple transcripts.

Glycophorin A (GPA), the major sialoglycoprotein of human erythrocytes, is the carrier for blood group MN antigens and a receptor for viruses, bacteria and parasites. (1) Three distinct GPA mRNAs (1.0, 1.7 and 2.2 kb) have been previously identified in erythroid tissues by Northern-blot analysis. It is shown here by sequence analysis of several human fetal liver cDNAs, and by transcription start point (tsp) determination using primer extension analysis, that the production of the multiple GPA mRNAs is governed by poly(A) site choice generating 3'-untranslated regions of different length, and not by the tsp heterogeneity, since all messages exhibit the same cap site (tsp). (2) The structural gene encoding GPA has been recently cloned [Vignal et al., Eur. J. Biochem. 184 (1989) 337-344; Kudo and Fukuda, Proc. Natl. Acad. Sci. USA 86 (1989) 4619-4623] and we have now determined the sequence of a DNA genomic fragment upstream from the tsp. This fragment does not contain the typical TATA and CAAT boxes found in a number of tissue-specific genes, but contains typical motifs like the CACC, nuclear factor erythroid 1 and 2 elements, which have been identified recently in several erythroid-specific promoters, therefore suggesting that transcription of these genes might be regulated by the same or analogous factors.

Human erythrocyte glycophorins: protein and gene structure analyses.

Human RBCs glycophorins are integral membrane proteins rich in sialic acids that carry blood group antigenic determinants and serve as ligands for viruses, bacteria, and parasites. These molecules have long been used as a general model of membrane proteins and as markers to study normal and pathological differentiation of the erythroid tissue. The RBC glycophorins known as GPA, GPB, GPC, GPD, and GPE have recently been fully characterized at both the protein and the DNA levels, and these studies have demonstrated conclusively that these molecules can be subdivided into two groups that are distinguished by distinct properties. The first group includes the major proteins GPA and GPB, which carry the MN and Ss blood group antigens, respectively, and a recently characterized protein, GPE, presumably expressed at a low level on RBCs. All three proteins are structurally homologous and are essentially erythroid specific. The respective genes are also strikingly homologous up to a transition site defined by an Alu repeat sequence located about 1 Kb downstream from the exon encoding the transmembrane regions. Downstream of the transition site, the GPB and GPE sequences are still homologous, but diverge completely from those of GPA. The three glycophorin genes are organized in tandem on chromosome 4q28-q31, and define a small gene cluster that presumably evolved by duplication from a common ancestral gene. Most likely two sequential duplications occurred, the first, about 9 to 35 million years ago, generated a direct precursor of the GPA gene, and the second, about 5 to 21 million years ago, generated the GPB and GPE genes and that involved a gene that acquired its specific 3' end by homologous recombination through Alu repeats. Numerous variants of GPA and GPB usually detected by abnormal expression of the blood group MNSs antigens are known. An increasing number of these variants have been structurally defined by protein and molecular genetic analyses, and have been shown to result from point mutations, gene deletions, hybrid gene fusion products generated by unequal crossing-over (not at Alu repeats), and microconversion events. The second group of RBC membrane glycophorins includes the minor proteins GPC and GPD both of which carry blood group Gerbich antigens. Protein and nucleic acid analysis indicated that GPD is a truncated form of GPC in its N-terminal region, and that both proteins are produced by a unique gene called GE (Gerbich), which is present as a single copy per haploid genome and is located on chromosome 2q14-q21.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical characterisation of monoclonal antibodies directed to glycophorins A and/or B.

Among sixty-nine monoclonal antibodies submitted to the workshop, 28 antibodies directed to glycophorins A and/or B but without blood group specificity were investigated by a series of methods involving agglutination, flow cytometry with CHO transfected cells expressing glycophorin A, ELISA with a carbohydrate-free peptide (residues 1-72) of glycophorin A, and immunoblotting. These MAbs were subdivided in several groups according to their specificity: N-terminal portion of GPA and GPB; N-terminal trypsin-sensitive portion of GPA; extracellular ficin-sensitive portion of GPA; intracellular domain of GPA; undetermined. Both flow cytometry with transfectant cells and ELISA with the synthetic peptide prove to be of value in order to determine subspecificities within these groups.

Characterization of a human Y chromosome Pvu II repeated sequence.

The human Y chromosome carries numerous copies of a tandemly repeated Pvu II sequence, 2.4 kb long. These sequences are specific to humans, and are present in a much smaller amount in the DNA of females. They are localized on the long arm of the Y chromosome. We have compared this sequence with the Hae III 2.1 kb Y-specific repeated sequence, already described.

Post-transcriptional regulation of the cell surface expression of glycophorins A, B, and E.

GYPA, GYPB, and GYPE represent a small gene family localized on chromosome 4q28-q31 that encodes the major red cell membrane glycophorins, GPA and GPB, and a new but as yet uncharacterized glycoprotein, GPE. There are 3-4 times more copies of GPA as compared with GPB on human erythrocytes (10(6) versus 2 x 10(5) copies/cell), whereas GPE is absent or poorly represented. Whether these quantitative differences reflect a transcriptional or post-transcriptional regulation was investigated. We found the functional activities of the glycophorin promoters to be similar, as shown by DNase I footprinting, gel retardation, methylation interference, and deletion analysis. Run-on analysis indicated that the transcription rate of each glycophorin gene in K562 cells was also very similar. However, large differences in mRNA decay were found in actinomycin-treated K562 cells. GPA transcripts were very stable (at least 24 h), whereas GPB transcripts were severely reduced after 17 h. The GPE transcripts were barely detectable and disappeared completely after 1 h. These results suggest that a difference in stability of the GPA, GPB, and GPE transcripts rather than a transcriptional regulation may predominantly account for the different levels of glycophorin expression on erythrocytes.

Comparison of two methods of high-molecular-weight DNA isolation from human leucocytes.

Among the different methods for isolation of high-molecular-weight DNA from leucocytes, two of them were retained as being the most interesting and were compared. This choice was based on three criteria: the quality of the DNA obtained (high-molecular-weight DNA, RNA, and protein free), the efficiency of the method (in terms of the yield of DNA obtained), and the ease and length of the method. The first method described was chosen as a reference (100% efficiency); in comparison, the second one had a 70% efficiency, but was shorter and easier to handle.

ABH and Lewis glycosyltransferases in human red cells, lymphocytes and platelets.

The alpha-O-N-acetylgalactosaminyl-, alpha-3-D-galactosyl- and alpha-2-L-fucosyltransferases, the direct products of the respective A, B and H blood group genes have been identified in red cells, lymphocytes and platelets homogenates from secretor and non-secretor individuals of appropriate ABO group. The H enzyme was not detectable in red cell membranes from Bombay individuals. Since the uptake of A antigen from the plasma has been shown at least for red cells [10] and lymphocytes [25], the present results suggest that the ABH determinants of the blood cells may arise both from intrinsic and extrinsic origin, but the relative contribution of each mechanism is not known. In contrast, the Lewis enzyme was absent or inactive in all the investigated blood cell samples, which can be considered as an additional proof that the Lewis antigens of these cells are entirely derived from the plasma.

Erythroid-specific activity of the glycophorin B promoter requires GATA-1 mediated displacement of a repressor.

We have performed a detailed analysis of the cis-acting sequences involved in the erythroid-specific expression of the human glycophorin B (GPB) promoter and found that this promoter could be divided into two regions. The proximal region, -1 to -60, contains a GATA binding sequence around -37 and an SP1 binding sequence around -50. This region is active in erythroid and non-erythroid cells. The distal region, -60 to -95, contains two overlapping protein binding sites around -75, one for hGATA-1 and one for ubiquitous proteins. This distal region completely represses the activity of the proximal promoter in non-erythroid cells and defines the -95 GPB construct as a GPB promoter that displays erythroid specificity. Using site directed mutagenesis, we show that the -37 GATA and the -50 SP1 binding sites are necessary for efficient activity of the -95 GPB construct. Mutations that impair the -75 GATA-1 binding result in extinction of the -95 GPB construct activity if the -75 ubiquitous binding site is not altered, or in loss of erythroid specificity if the -75 ubiquitous binding site is also mutated. Using a cotransfection assay, we found that hGATA-1 can efficiently activate transcription of the -95 GPB construct in non-erythroid cells. This transactivation is abolished by mutations that impair either the -37 GATA-1 or the -50 SP1 binding. Mutations that impair the -75 GATA-1 binding and still allow the -75 ubiquitous binding also abolish the transactivation of the -95 GPB construct, indicating that hGATA-1 can remove repression of the GPB promoter by displacement of the ubiquitous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Erythrocyte glycophorin B deficiency may occur by two distinct gene alterations.

The genomic DNA from rare persons whose erythrocytes are deficient in glycophorin B (GPB) (S-s-U- phenotype), was examined by Southern hybridizations using glycophorin B probes and was subdivided into two main categories. In the type I variant (Fav., M.H., S.K.), we found that the S-s-U- condition is generated by a large gene deletion extending from exons B2 to B4 of glycophorin B gene. Conversely, in the type II variant (Del.), the entire gene is present, and its promoter is almost similar to common Glycophorin A (GPA) and GPB as well as to type I promoters, except for four-point mutations, which do not occur in potential cis-acting elements. We concluded that the same phenotypic glycophorin B deficiency may occur by different gene alterations, including either a gene deletion or a mutation that might alter transcription or translation of the gene.

A unique gene encodes spliceoforms of the B-cell adhesion molecule cell surface glycoprotein of epithelial cancer and of the Lutheran blood group glycoprotein.

Two new members of the Ig superfamily, the Lutheran (Lu) blood group glycoprotein and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen, have been recently cloned from human placenta and colon cancer HT29 cell line, respectively. Although amino acid sequences deduced from cDNA analysis suggested that B-CAM should represent an abridged form of the Lu glycoprotein lacking the last 40 amino acids of the putative cytoplasmic tail, the relationship between the genes encoding these polypeptides has not been determined. In the present report, we showed by Southern blot analysis that the Lu and B-CAM cDNAs derived from a unique LU gene which exhibited an HindIII RFLP associated with the Lua/Lub blood group polymorphism. Accordingly, in situ hybridization of the Lu cDNA probe confirmed the localization of the Lutheran blood group locus to chromosome 19 q13.2-13.3, as previously shown for a B-CAM DNA probe. Sequence comparison between cDNA and genomic PCR fragments indicated that the Lu and B-CAM transcripts previously isolated are generated through the alternative use of internal splice donor and acceptor sites within an exon located at the 3' end of the LU gene. These spliceoforms corresponded to 2.5 kb and 4.0 kb mRNA species detectable by Northern blot in all tissues and cell lines in which the LU gene is expressed; their primary structures are consistent with the presence of both the Lu and B-CAM antigens on two glycoprotein isoforms. However, the 4.0 kb transcript was very poorly expressed as compared to the 2.5 kb species except in the colon carcinoma HT29 cell line, suggesting a differential regulation of the Lu/B-CAM messenger RNA in some tumor tissues.

The repressor which binds the -75 GATA motif of the GPB promoter contains Ku70 as the DNA binding subunit.

Glycophorin B (GPB) is an abundant cell surface glycoprotein which is only expressed in human erythroid cells. Previous functional analysis demonstrated that the repression of the GPB promoter is determined by the binding of a ubiquitous factor which recognizes a GATA motif centered at position -75. In erythroid cells this ubiquitous factor is displaced by the binding of the erythroid-specific factor hGATA1. Here, we have identified the Ku70 protein as a candidate GPB repressor DNA binding subunit through the screening of a human HeLa expression library using the -75 GATA sequence as bait (one-hybrid method). Electrophoretic mobility shift assays demonstrated that the ubiquitous factor that binds the -75 GATA sequence was the Ku70-Ku80 (Ku) heterodimer. Co-transfection experiments demonstrated that overexpression of Ku70 in the K562 erythroleukeamic cell line resulted in transcriptional repression of the chloramphenicol acetyltransferase reporter gene when placed under the control of the wild-type GPB promoter. Conversely, no repression was observed when a mutation that abolished the binding of Ku was introduced in the GPB promoter construct. Altogether, these results indicate that Ku binds in vivo to the -75 WGATAR motif and is involved in negative regulation of the GPB promoter. These findings suggest that, besides its role in many functions, Ku is also involved in transcriptional regulation of erythroid genes.

DNA restriction polymorphism of alpha-fetoprotein gene after digestion by MspI endonuclease.

Hybridization of alpha-fetoprotein clones cDNA with human DNAs digested by seven restriction endonucleases reveals one polymorphism. This polymorphism, detected after restriction by MspI, is at low frequency (f = 0.02) and is validated by family analysis. It corresponds to an intronic sequence, for a methylated site external to the probes utilized.

Acquired B antigen: further studies using synthetic oligosaccharides.

The reactivity of acquired-B red cells with various antisera has been investigated by agglutination inhibition assays using four trisaccharides obtained by chemical synthesis. Two of these had the structure GalNAc alpha 1-3 (LFuc alpha 1-2). Gal and Gal alpha 1-3 (LFuc alpha 1-2) Gal which are characteristic of the A and B determinants respectively and were indeed strong inhibitors of human anti-A and -B antibodies. The other two sugars denoted B-OAc and B-NH2 are derivatives of the B-trisaccharide by substitution of the hydroxyl group on carbon-2 of the alpha-galactose residue with the O-acetyl group (B-OAc) or the amino group (B-NH2) respectively. We have shown that both B and B-NH2 trisaccharides inhibited strongly the agglutination of acquired B red cells by the anti-B reagents (crude or affinity purified) whereas sera containing "anti-acquired B" agglutinins were specifically inhibited by B-NH2 but not by the A, B or B-OAc structures. We have also shown that the agglutination of Tk-activated erythrocytes by the BS-II lectin is specifically inhibited by N-acetylglucosamine but not by B, B-OAc or B-NH2 structures. These results and the observation that anti-acquired B agglutinins cannot be adsorbed on Tk red cells suggest that (i) the "B-like" and the "acquired-B" determinants share a common structure best represented by B-NH2 and (ii) Tk and "acquired-B" antigens are not identical.

Level of the A, B and H blood group glycosyltransferases in red cell membranes from patients with malignant hemopathies.

Eight patients (4 suffering from acute myeloid leukemia) exhibiting a loss of ABO red cell antigens, as seen by a mixed-field reaction pattern in agglutination tests, were selected and examined for the level of the A, -B, -H blood group glycosyltransferases within membranes prepared from erythrocyte subpopulations (A or B positive and A or B negative red cells). A or B enzyme activities were largely decreased in membranes which had lost A or B antigens (A or B negative subpopulations) but were within normal level in membrane from cells which had not lost A or B antigens (A or B positive subpopulations). The H enzyme level which was frequently low in the serum was within normal limits in the membrane preparations examined. Since A or B negative subpopulations were normally glycosylated in vitro into A or B reactive structures, the results demonstrate that loss of A or B antigens is related to some alteration of the blood group gene products rather than to significant abnormalities of the membrane precursors.

DNA polymorphism of the RC8 sequence on the short arm of the X chromosome in a French population.

Restriction fragment length polymorphism of the RC8 probe, which is loosely linked to the Duchenne muscular dystrophy locus, was studied in a French population. Among 22 females, 18.1% were found to be heterozygous for the two frequent B1 and B2 alleles, and a rare allele was found in 1 woman with a corresponding variant band at 3 kb. Among 18 males, 6 were found to have the B2 allele. The B2 gene frequencies were 0.09 and 0.33 in males and females, respectively. This difference was statistically significant (p less than 0.05), but may nevertheless be fortuitous. There was no significant gene frequency difference between the English and French populations.