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 alpha(5) 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 laminin 10/11. 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 alpha(4)beta(1), 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 alpha(4)beta(1) and that this adhesion is mediated by endothelial Lu/BCAM gps. Our studies show that Lu/BCAM gps expressed either on erythroid oron 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. (c) 2008 Elsevier Masson SAS. All rights reserved.

AbstractRecent studies shed new lights on the biological function of blood group antigens, such as the adhesion properties of the Lutheran (Lu) bloodgroup antigens carried by the Lu/BCAM glycoproteins. The Lu/BCAM adhesion glycoproteins were Þrst identiÞed as laminin-10/11 erythroidreceptors involved in RBC adhesion to endothelium in sickle cell anemia. Lu/BCAM mediated cell adhesion to laminin is stimulated byepinephrine, a physiological stress mediator, and is dependent of phosphorylation by protein kinase A. More recently, we demonstrated thatconstitutive phosphorylation of Lu/BCAM is also involved in abnormal RBC adhesion to endothelium in patients with polycythemia vera (PV), afrequent myeloproliferative disorders associated with the V617F mutation of the tyrosine kinase JAK2 leading to continuous stimulation oferythropoiesis. This observation suggests that Lu/BCAM could participate to the high incidence of vascular thrombosis that also characterizes PVdisease. In mice, which do not express Lu/BCAM in erytroid tissues, invalidation of theLu/BCAMgene provided evidence that Lu/BCAM gps, aslaminin-a5 receptors, are involved invivo in the maintenance of normal basement membrane organization in different non erythroid tissues sinceup to 90% of the mutant kidney glomeruli exhibited a reduced number of visible capillary lumens and irregular thickening of the glomerularbasement membrane, while intestine exhibited smooth muscle coat thickening and disorganization. All these results further illustrate that minorblood group antigens might have important role under physiological and physiopathological conditions in erythroid and non erythroid tissues aswell.# 2008 Elsevier Masson SAS. All rights reserved.ResumeDes Žtudes rŽcentes ont apportŽ de nouvelles informations sur la fonction des antignes de groupes sanguins, telles que les propriŽtŽsdOadhŽrence des antignes LuthŽran (Lu) portŽs par les glycoprotŽines Lu/BCAM. Ces glycoprotŽines ont dOabord ŽtŽ identiގes comme lesuniques rŽcepteurs Žrythro•des des laminines-10/11 (composants majeurs de la matrice extracellulaire) impliquŽs dans lOadhŽrence anormale desglobules rouges drŽpanocytaires ˆ lOendothŽlium vasculaire. Ces propriŽtŽs dOadhŽrence de Lu/BCAM sont stimulŽes par lOadrŽnaline, unmŽdiateur physiologique de stress induisant la phosphorylation de Lu/BCAM par la protŽine kinase A. Plus rŽcemment, nous avons dŽmontrŽ quela phosphorylation constitutive de Lu/BCAM est Žgalement impliquŽe dans lOadhŽrence anormale ˆ lOendothŽlium des globules rouges de patientsatteint de polyglobulie de Vaquez (polycythemia vera: PV), un dŽsordre myŽloprolifŽratif frŽquent associŽ ˆ la mutation V617F de la tyrosinekinase JAK2 et conduisant ˆ une stimulation continue de lOŽrythropo•se. Ainsi Lu/BCAM participerait ˆ lOincidence ŽlevŽe des Žvnementsthrombotiques qui caractŽrise Žgalement la polyglobulie de Vaquez. Chez la souris, qui nOexprime pas Lu/BCAM dans les cellules Žrythro•des,lOinactivation du gneLu/BCAMa permis de montrer que les protŽines Lu/BCAM, en tant que rŽcepteurs aux laminines-10/11, sont impliquŽesdans le maintien de lOorganisation des membranes basales de nombreux tissus. Ainsi jusquOˆ 90 % des glomŽrules rŽnaux des sourisLu/BCAM

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-alpha 5 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. (C) 2008 Elsevier Masson SAS. All rights reserved.

Lutheran (Lu) blood group and basal cell adhesion molecule (B-CAM) antigens reside on two glycoprotein (gp) isoforms Lu and Lu(v13) that belong to the Ig superfamily and differ only by the size of their cytoplasmic tail. Lu/B-CAM gps have been recognized as laminin alpha 5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha 5 when intracellular cAMP is up-regulated by physiological stimuli such as epinephrine and that this signaling pathway is protein kinase A- and Lu/B-CAM-dependent. In this study, we analyzed the relationship between the phosphorylation status of Lu/B-CAM gps and their adhesion function to laminin alpha 5. We showed that Lu isoform was phosphorylated in sickle red cells as well as in erythroleukemic K562 and epithelial Madin-Darby canine kidney cells and that this phosphorylation is enhanced by different stimuli of the PKA pathway. Lu gp is phosphorylated by glycogen synthase kinase 3 beta, casein kinase II, and PKA at serines 596, 598, and 621, respectively. Alanine substitutions of serines 596 and 598 abolished phosphorylation by glycogen synthase kinase 3 beta and casein kinase II, respectively, but had no effect on adhesion of K562 cells to laminin under flow conditions. Conversely, mutation of serine 621 prevented phosphorylation by PKA and dramatically reduced cell adhesion. Furthermore, stimulation of K562 cells by epinephrine increased Lu gp phosphorylation by PKA and enhanced adhesion to laminin. It is postulated that modulation of the phosphorylation state of Lu gp might be a critical factor for the sickle red cells adhesiveness to laminin alpha 5 in sickle cell disease.

Lutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B-CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent-insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione-S-transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site-directed mutagenesis mapped a major interaction site with spectrin to the RK573-574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.

British Journal of HaematologyVolume 126, Issue 1 Free Access Papers to be published in forthcoming issues. Toward more effective antifungal therapy: the prospects of combination therapy. Myeloma cells can directly contribute to the pool of RANKL in bone bypassing the classic stromal and osteoblast pathway of osteoclast stimulation. Haematogones in the peripheral blood of adults: a four-colour flow cytometry study of 102 patients. Plasma levels of von Willebrand factor regulate ADAMTS-13, its major cleaving protease. Synthesis of osteoprotegerin and RANKL by megakaryocytes is modulated by oestrogen. Direct interaction between the Lu/B-CAM adhesion glycoproteins and erythroid spectrin. Applications of murine and humanized chimeric monoclonal antibodies for red cell phenotyping. Impact of pre-analytical handling on bone marrow mRNA gene expression. Identification of protein Sα gene mutations including four novel mutations in eight unrelated patients with protein S deficiency D. P. Kontoyiannis, D. P. KontoyiannisSearch for more papers by this authorR. E. Lewis, R. E. LewisSearch for more papers by this authorF. P. L. Lai, F. P. L. LaiSearch for more papers by this authorM. Cole-Sinclair, M. Cole-SinclairSearch for more papers by this authorW.-J. Cheng, W.-J. ChengSearch for more papers by this authorJ. M. W. Quinn, J. M. W. QuinnSearch for more papers by this authorM. T. Gillespie, M. T. GillespieSearch for more papers by this authorJ. W. Sentry, J. W. SentrySearch for more papers by this authorH.-G. Schneider, H.-G. SchneiderSearch for more papers by this authorS. H. Kroft, S. H. KroftSearch for more papers by this authorS. L. Asplund, S. L. AsplundSearch for more papers by this authorR. W. McKenna, R. W. McKennaSearch for more papers by this authorN. J. Karandikar, N. J. KarandikarSearch for more papers by this authorP. M. Mannucci, P. M. MannucciSearch for more papers by this authorC. Capoferri, C. CapoferriSearch for more papers by this authorM. T. Canciani, M. T. CancianiSearch for more papers by this authorS. Bord, S. BordSearch for more papers by this authorE. Frith, E. FrithSearch for more papers by this authorD. C. Ireland, D. C. IrelandSearch for more papers by this authorM. A. Scott, M. A. ScottSearch for more papers by this authorJ. I. O. Craig, J. I. O. CraigSearch for more papers by this authorJ. E Compston, J. E CompstonSearch for more papers by this authorY. Kroviarski, Y. KroviarskiSearch for more papers by this authorW. El Nemer, W. El NemerSearch for more papers by this authorP. Gane, P. GaneSearch for more papers by this authorC. Rahuel, C. RahuelSearch for more papers by this authorE. Gauthier, E. GauthierSearch for more papers by this authorM. C. Lecomte, M. C. LecomteSearch for more papers by this authorJ. P. Cartron, J. P. CartronSearch for more papers by this authorY. Colin, Y. ColinSearch for more papers by this authorC. Le Van Kim, C. Le Van KimSearch for more papers by this authorE. Lee, E. LeeSearch for more papers by this authorG. Burgess, G. BurgessSearch for more papers by this authorG. R. Halverson, G. R. HalversonSearch for more papers by this authorT. J. Huang, T. J. HuangSearch for more papers by this authorM. E. Reid, M. E. ReidSearch for more papers by this authorS. Breit, S. BreitSearch for more papers by this authorM. Nees, M. NeesSearch for more papers by this authorU. Schaefer, U. SchaeferSearch for more papers by this authorM. Pfoersich, M. PfoersichSearch for more papers by this authorC. Hagemeier, C. HagemeierSearch for more papers by this authorM. Muckenthaler, M. MuckenthalerSearch for more papers by this authorA. E. Kulozik, A. E. KulozikSearch for more papers by this authorH. Okada, H. OkadaSearch for more papers by this authorA. Takagi, A. TakagiSearch for more papers by this authorT. Murate, T. MurateSearch for more papers by this authorT. Adachi, T. AdachiSearch for more papers by this authorK. Yamamoto, K. YamamotoSearch for more papers by this authorT. Matsushita, T. MatsushitaSearch for more papers by this authorJ. Takamatsu, J. TakamatsuSearch for more papers by this authorK. Sugita, K. SugitaSearch for more papers by this authorM. Sugimoto, M. SugimotoSearch for more papers by this authorA. Yoshioka, A. YoshiokaSearch for more papers by this authorT. Yamazaki, T. YamazakiSearch for more papers by this authorH. Saito, H. SaitoSearch for more papers by this authorT. Kojima, T. KojimaSearch for more papers by this author D. P. Kontoyiannis, D. P. KontoyiannisSearch for more papers by this authorR. E. Lewis, R. E. LewisSearch for more papers by this authorF. P. L. Lai, F. P. L. LaiSearch for more papers by this authorM. Cole-Sinclair, M. Cole-SinclairSearch for more papers by this authorW.-J. Cheng, W.-J. ChengSearch for more papers by this authorJ. M. W. Quinn, J. M. W. QuinnSearch for more papers by this authorM. T. Gillespie, M. T. GillespieSearch for more papers by this authorJ. W. Sentry, J. W. SentrySearch for more papers by this authorH.-G. Schneider, H.-G. SchneiderSearch for more papers by this authorS. H. Kroft, S. H. KroftSearch for more papers by this authorS. L. Asplund, S. L. AsplundSearch for more papers by this authorR. W. McKenna, R. W. McKennaSearch for more papers by this authorN. J. Karandikar, N. J. KarandikarSearch for more papers by this authorP. M. Mannucci, P. M. MannucciSearch for more papers by this authorC. Capoferri, C. CapoferriSearch for more papers by this authorM. T. Canciani, M. T. CancianiSearch for more papers by this authorS. Bord, S. BordSearch for more papers by this authorE. Frith, E. FrithSearch for more papers by this authorD. C. Ireland, D. C. IrelandSearch for more papers by this authorM. A. Scott, M. A. ScottSearch for more papers by this authorJ. I. O. Craig, J. I. O. CraigSearch for more papers by this authorJ. E Compston, J. E CompstonSearch for more papers by this authorY. Kroviarski, Y. KroviarskiSearch for more papers by this authorW. El Nemer, W. El NemerSearch for more papers by this authorP. Gane, P. GaneSearch for more papers by this authorC. Rahuel, C. RahuelSearch for more papers by this authorE. Gauthier, E. GauthierSearch for more papers by this authorM. C. Lecomte, M. C. LecomteSearch for more papers by this authorJ. P. Cartron, J. P. CartronSearch for more papers by this authorY. Colin, Y. ColinSearch for more papers by this authorC. Le Van Kim, C. Le Van KimSearch for more papers by this authorE. Lee, E. LeeSearch for more papers by this authorG. Burgess, G. BurgessSearch for more papers by this authorG. R. Halverson, G. R. HalversonSearch for more papers by this authorT. J. Huang, T. J. HuangSearch for more papers by this authorM. E. Reid, M. E. ReidSearch for more papers by this authorS. Breit, S. BreitSearch for more papers by this authorM. Nees, M. NeesSearch for more papers by this authorU. Schaefer, U. SchaeferSearch for more papers by this authorM. Pfoersich, M. PfoersichSearch for more papers by this authorC. Hagemeier, C. HagemeierSearch for more papers by this authorM. Muckenthaler, M. MuckenthalerSearch for more papers by this authorA. E. Kulozik, A. E. KulozikSearch for more papers by this authorH. Okada, H. OkadaSearch for more papers by this authorA. Takagi, A. TakagiSearch for more papers by this authorT. Murate, T. MurateSearch for more papers by this authorT. Adachi, T. AdachiSearch for more papers by this authorK. Yamamoto, K. YamamotoSearch for more papers by this authorT. Matsushita, T. MatsushitaSearch for more papers by this authorJ. Takamatsu, J. TakamatsuSearch for more papers by this authorK. Sugita, K. SugitaSearch for more papers by this authorM. Sugimoto, M. SugimotoSearch for more papers by this authorA. Yoshioka, A. YoshiokaSearch for more papers by this authorT. Yamazaki, T. YamazakiSearch for more papers by this authorH. Saito, H. SaitoSearch for more papers by this authorT. Kojima, T. KojimaSearch for more papers by this author First published: 14 June 2004 https://doi.org/10.1111/j.1365-2141.2004.005016.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume126, Issue1July 2004 RelatedInformation

The Kell blood-group antigen was originally reported to be a protein expressed in erythroid tissue only. Transcriptional analysis of the KEL promoter activity in human erythroleukaemia K562 and epithelial HeLa cells by electrophoretic mobility-shift and supershift assays, chloramphenicol acetyltransferase assays, co-transfection studies and site-directed mutagenesis provided the following results: (i) the KEL promoter exhibits a strong transcriptional activity in K562 cells and, unexpectedly, a basal non-erythroid activity in HeLa cells, (ii) up-regulation of the 5' distal promoter activity occurs only in the erythroid context, and (iii) two motifs localized in the exon 1 region, which bind the Sp1/Sp3 and the human GATA-1/Ku70/80 factors, were required for down-regulation of the promoter activity, but inhibition of the promoter activity by the repressing factors in HeLa cells was incomplete. KEL expression in HeLa cells was performed further by primer-extension analysis, which revealed the presence of a low amount of Kell transcript correlating with basal expression of the Kell protein in these cells, as shown by immunopurification and Western-blot analysis. DNA sequencing of the transcript revealed a sequence identical to that obtained from erythroid tissue. In human tissues, KEL expression was investigated by dot-blot analysis and revealed high levels of Kell mRNAs, particularly in brain tissues, testis and lymphoid tissues. Moreover, most tissues analysed exhibited low levels of Kell transcripts. The Kell protein was also detected by immunohistochemistry in the Sertoli cells of the testis and in lymphoid tissues like spleen and tonsil, specifically localized in the follicular dendritic cells. Altogether, the results indicated that KEL expression is not restricted to erythroid tissue.

Previous studies performed on the glycophorin B (GPB) expression demonstrated that this gene is expressed in erythroid cells only and that the ubiquitous factor Ku70 is involved in the process. Here, we investigated the contribution of the -70 E-box sequence toward the GPB promoter expression. We found that the E-box bound two factors, the USF1/USF2 protein and an unidentified ubiquitous protein which was named factor U. Site-directed mutagenesis performed on the -70 E-box showed that the USF factor had an activating effect in CAT assays. Conversely, mutation of the -70 E-box that impaired the binding of factor U led to a positive CAT activity in nonerythroid cells and thus to the loss of the erythroid-specific expression of the GPB gene. This indicates that, in addition to the Ku70 factor, the extinction of the GPB promoter expression in nonerythroid cells depends also on the repressing effect of the factor U.

The human Lutheran (Lu) blood group antigens are carried by two glycoproteins (gps) that belong to the immunoglobulin (Ig) superfamily. These gps represent adhesion molecules that function as the unique erythroid receptors for laminin. We report here the cloning and functional expression of the orthologous mouse Lu mRNA as well as the genomic organization of the mouse Lu gene. The deduced human and mouse Lu gps share 72.5% identity and similar organization of the Ig-like domains. As in the human, the mouse Lu gene is organized in 15 exons. The proximal promoter showed consensus CACC-binding sites whereas the distal promoter exhibits a GATA-1-binding site and multiple E boxes. Like the human gene, the mouse Lu gene is also widely expressed among tissues but is transcribed as a unique 2.4-kb mRNA species. Expression of the mouse Lu mRNA is upregulated upon dimethyl sulfoxide-induced erythroid differentiation of murine erythroleukemia cells (MEL). During mouse embryonic development, the Lu transcript is detected as early as day 7 of gestation. Analysis of transfected human erythroleukemia K562 cells indicated that the adhesive properties of the Lu gps to laminin are conserved between human and mouse.

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.

1999 ABSTRACT 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 iden-tified 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

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitroerythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

The Lutheran (Lu) blood group antigens and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen are carried by recently cloned integral glycoproteins that belong to the Ig superfamily. We have previously shown that the Lu and B-CAM antigens are encoded by the same gene, LU, and that alternative splicing of the primary transcript most likely accounts for the presence of both antigens on two isoforms that differ by the length of their cytoplasmic tails. In the present report, we isolated the human LU gene by cloning a 20-kb HindIII fragment from Lu(a - b+) genomic DNA. The LU gene is organized into 15 exons distributed over 12.5 kb. Alternative splicing of intron 13 generates the 2.5- and 4.0-kb transcript spliceoforms encoding the long tail and the short tail Lu polypeptides, respectively. Sequencing of the major mRNA species (2.5 kb) amplified from human bone marrow, kidney, placenta, and skeletal muscle did not suggest the presence of tissue-specific Lu glycoprotein isoforms. The same transcription initiation point, located 22 bp upstream from the initiation codon, was characterized in several tissues. In agreement with the wide tissue distribution of the Lu messengers, the GC-rich proximal 5' flanking region of the LU gene does not contain TATA or CAAT boxes, but includes several potential binding sites for the ubiquitous Sp1 transcription factor. In addition, the distal 5' region, encompassing nucleotides -673 to -764, contains clustered binding sequences for the GATA, CACCC, and Ets transcription factors. Analysis of the coding sequences amplified from genomic DNA of Lu(a + b-) or Lu(a - b+) donors showed a single nucleotide change in exon 3 (A229G) that correlates with an Aci I restriction site polymorphism and results in a His77Arg amino-acid substitution. Polymerase chain reaction/restriction fragment length polymorphism analysis indicated that the A229G mutation is associated with the Lu(a)/Lu(b) blood group polymorphism. When expressed in Chinese hamster ovary (CHO) cells, Lu cDNAs carrying the A229 or the G229 produced cell surface proteins that reacted with anti-Lu(a) or anti-Lu(b) antibodies, respectively, showing that these nucleotides specify the Lu(a) and Lu(b) alleles of the Lutheran blood group locus. CHO cells expressing recombinant short-tail or long-tail Lu glycoproteins reacted as well with anti-Lu as with anti-B-CAM antibodies, providing the definitive proof that the Lu blood group and B-CAM antigens are carried by the same molecules.

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.

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.

Les glycophorines sont des glycoprotéines intégrales riches en acides sialiques de la membrane des érythrocytes humains qui portent des antigènes de groupes sanguins et se présentent comme des ligands pour des virus, des bactéries et des parasites. Ces molécules servent de modèles pour l'étude des protéines membranaires et de marqueurs pour l'étude de la différenciation normale et pathologique du tissu érythroïde. Les protéines et les gènes codant pour les glycophorines A, B, C, D et E ont été bien caractérisées et deux groupes de molécules peuvent être individualisés.

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.

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.