Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

[Vascular myeloproliferative neoplasm with normal cell blood count: Exploration and medical management]. / Les syndromes myéloprolifératifs d'expression vasculaire à numération normale : comment les explorer ?

UNLABELLED: Negative BCR ABL myeloproliferative neoplasm (MPN) such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (MFP) are clonal hematological malignancies and may lead to a high risk of venous, arterial or microcirculatory thrombosis. Atypical sites of thrombosis can sometimes reveal the neoplasm disorder. Their diagnoses are a major issue because of the propensity to develop acute myeloid leukemia and/or myelofibrosis. The acquired JAK2V617F variant (Janus kinase 2; 9p24) is a prevalent MPN and also a sensitive marker for PV diagnosis (95% positive mutation), but not specific since found in approximately 50% of patients with ET and MFP. PATIENT AND METHODS: We present a diagnostic and a therapeutic approach based on one patient with microcirculatory ischemic manifestations in the toes, and who had strictly normal cell blood counts and was positive for JAK2V617F mutation: thrombotic risk factor evaluation; bone marrow biopsy; red cell adhesion assays. These experimental assays are promising for the development of new therapeutics in MPN; they assess red cell adherence to the vascular endothelium after the phosphorylation of Lu/BCAM subsequent to a positive JAK2V617F mutation. RESULTS: Compared with controls, our patient exhibited increased Lu/BCAM receptor phosphorylation and red blood cell adhesion. CONCLUSION: This development may lead to improved care for patients with thrombotic manifestations, normal blood cell counts, and a positive JAK2V617F mutation: multidisciplinary management, including regular hematological monitoring, could lead to the introduction of a cytoreductive treatment.

Role of Lu/BCAM glycoproteins in red cell diseases.

Lu/BCAM glycoproteins (gps) are the unique erythroid receptors of laminin alpha5 chain, a major component of the extracellular matrix. They interact with the membrane skeleton by binding directly to spectrin via the Lu/BCAM RK573-574 motif. Lu/BCAM gps are involved in abnormal sickle red blood cell (RBC) adhesion to components of the vascular wall. This adhesion is activated by the phosphorylation of the Lu/BCAM long isoform Lu in a protein kinase A-dependent manner. A similar high adhesion to laminin was also observed with RBCs from Hereditary Spherocytosis (HS) patients suffering from haemolytic anaemia subsequent to spectrin deficiencies. We investigated the molecular mechanisms responsible for the Lu/BCAM-mediated abnormal RBC adhesion to laminin in sickle cell disease (SCD) and HS. We showed that SCD patients treated with hydroxycarbamide (HC) had a diminished RBC adhesion to laminin that was associated with reduced levels of the PKA upstream effector cAMP and a severe decrease in Lu isoform phosphorylation. On the other hand, we showed that increased Lu/BCAM-mediated HS RBC adhesion to laminin was independent of Lu/BCAM phosphorylation. A cellular model expressing the RK573-574AA Lu/BCAM mutant, which is unable to bind to spectrin, showed increased Lu/BCAM detergent extractability and enhanced cell adhesion to laminin. Similar results were obtained with HS RBCs, strongly suggesting that their increased adhesion could result from alteration of the Lu/BCAM-spectrin interaction following the severe spectrin deficiency.

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.

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.

Flow cytometric analysis of the association between blood group-related proteins and the detergent-insoluble material of K562 cells and erythroid precursors.

The linkage between blood group-related cell surface proteins and the detergent-insoluble material (DIM) was estimated by flow cytometry using a panel of specific monoclonal antibodies (mAbs) as a comparison of the antibody-binding capacity of intact and Triton-X100-treated cells. Studies were performed with K562 cells expressing endogenous or recombinant proteins and with human erythroid progenitors during their proliferation and differentiation in vitro. Glycophorin C (GPC) was found to be Triton-insoluble in both cellular models. When expressed (erythroid progenitors), Band 3 remained Triton-insoluble. Glycophorin A (GPA), however, behaved as Triton-soluble or insoluble according to the absence (K562) or the presence (erythroid progenitors) of Band 3 respectively. Comparison of the cellular models regarding the proteins that compose the Rh complex also indicated that Rh(D), RhAG and CD47 were resistant to Triton extraction in cells lacking Band 3. Similarly, RhAG and CD47 remained predominantly Triton-insoluble in K562 cells and early progenitors before Rh and Band 3 expression. Further analysis showed that the Kell protein was DIM-associated. In contrast, CD99 and DARC (Fy) proteins were not, or were very poorly, DIM-associated. Additionally, the adhesion molecules CD44 and Lu were completely or partially resistant to detergent extraction respectively. Deletion of the Lu cytoplasmic tail or its replacement by the cytoplasmic domain of GPC resulted in significant increase or decrease of the Triton solubility of the transfected proteins respectively. These data suggest that Triton insolubility of Lu results in part from direct attachment of its cytoplasmic tail with the cytoskeleton. We assume that this method should provide a useful tool to map interaction sites localized in the cytoplasmic domain of recombinant transmembrane proteins.

Characterization of the laminin binding domains of the Lutheran blood group glycoprotein.

Lutheran (Lu) blood group antigens and the basal cell adhesion molecule antigen reside on two glycoproteins that belong to the Ig superfamily (IgSF) and carry five Ig-like extracellular domains. These glycoproteins act as widely expressed adhesion molecules and represent the unique receptors for laminin-10/11 in erythroid cells. Here, we report the mapping of IgSF domains responsible for binding to laminin. In plasmonic resonance surface experiments, only recombinant Lu proteins containing the N-terminal IgSF domains 1-3 were able to bind laminin-10/11 and to inhibit binding of laminin to Lu-expressing K562 cells. Mutant recombinant proteins containing only IgSF domain 1, domains 1 + 2, domains 1 + 3, domains 2 + 3, domain 3, domain 4, domain 5, and domains 4 + 5 failed to bind laminin as well as a construct containing all of the extracellular domains except domain 3. Altogether, these results indicate that IgSF domains 1-3 are involved in laminin binding and that a specific spatial arrangement of these three first domains is most probably necessary for interaction. Neither the RGD nor the N-glycosylation motifs present in IgSF domain 3 were involved in laminin binding.

Analysis of deletions in three McLeod patients: exclusion of the XS locus from the Xp21.1-Xp21.2 region.

The McLeod syndrome is a rare X-linked recessive disorder characterized by blood group, neuromuscular and haematopoietic abnormalities. It is caused by XK gene defects and may include large deletions in the Xp21 region. Analysis of three unrelated McLeod patients for the presence of the XK, DMD, CYBB, ETX1, RPGR and OTC loci, as well as for the DXS709 marker, revealed deletions from the 39th exon of DMD to the ETX1 locus (patient Be), from the XK to RPGR loci (patient Bi) and from the XK to CYBB loci (patient Lh). All three patients normally expressed the Lutheran (Lu) red cell antigens, thus excluding the interval between the RPGR and DMD genes as site of the XS locus, previously mapped to the Xp21.2-Xq21.1 region and thought to regulate the expression of the LU blood group gene on chromosome 19.

Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.

Characterization of a mouse liminin receptor gene homologous to the human blood group Lutheran gene.

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.

The Lutheran blood group glycoproteins, the erythroid receptors for laminin, are adhesion molecules.

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) Blood 88, 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 vitro erythropoiesis 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.

Organization of the human LU gene and molecular basis of the Lu(a)/Lu(b) blood group polymorphism.

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.