Ubc9 interacts with Lu/BCAM adhesion glycoproteins and regulates their stability at the membrane of polarized MDCK cells.

Lu (Lutheran) blood group and BCAM (basal cell adhesion molecule) antigens both reside on two gp (glycoprotein) isoforms, Lu and Lu(v13), that differ by the size of their cytoplasmic tail. They are receptors of laminin-10/11 and are expressed in RBCs (red blood cells), epithelial cells of multiple tissues and vascular endothelial cells. To gain more insights into the biological function of Lu/BCAM gps, we looked for potential partners of their cytoplasmic tail. We isolated Ubc9 (ubiquitin-conjugating enzyme 9) protein by screening a human kidney library using the yeast two-hybrid system. Lu/Ubc9 interaction was validated by GST (glutathione S-transferase) pull-down and co-immunoprecipitation experiments. Endogenous Ubc9 formed a complex with endogenous or recombinant Lu gp in A498 and MDCK (Madin-Darby canine kidney) epithelial cells respectively. Replacement of Lys(585) by alanine in the Lu gp abolished in vitro and ex vivo interactions of Lu gp with Ubc9 protein. Lu K585A mutant transfected in MDCK cells exhibited a normal basolateral membrane expression but was overexpressed at the surface of polarized MDCK cells as compared with wild-type Lu. Pulse-chase experiments showed extended half-life of Lu K585A gp at the plasma membrane, suggesting an impaired endocytosis of this mutant leading to protein accumulation at the membrane. Furthermore, we showed that the ability of MDCK-Lu K585A cells to spread on immobilized laminin was dramatically decreased. Our results support a physiological role for the direct interaction between Lu gp and Ubc9 protein and reveal a role for this enzyme in regulating the stability of Lu gp at the cell membrane.

Human Rhesus B and Rhesus C glycoproteins: properties of facilitated ammonium transport in recombinant kidney cells.

The mammalian Rh (Rhesus) protein family belongs to the Amt/Mep (ammonia transporter/methylammonium permease)/Rh superfamily of ammonium transporters. Whereas RhCE, RhD and RhAG are erythroid specific, RhBG and RhCG are expressed in key organs associated with ammonium transport and metabolism. We have investigated the ammonium transport function of human RhBG and RhCG by comparing intracellular pH variation in wild-type and transfected HEK-293 (human embryonic kidney) cells and MDCK (Madin-Darby canine kidney) cells in the presence of ammonium (NH4+/NH3) gradients. Stopped-flow spectrofluorimetry analysis, using BCECF [2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein] as a pH-sensitive probe, revealed that all cells submitted to inwardly or outwardly directed ammonium gradients exhibited rapid alkalinization or acidification phases respectively, which account for ammonium movements in transfected and native cells. However, as compared with wild-type cells known to have high NH3 lipid permeability, RhBG- and RhCG-expressing cells exhibited ammonium transport characterized by: (i) a five to six times greater kinetic rate-constant; (ii) a weak temperature-dependence; and (iii) reversible inhibition by mercuric chloride (IC50: 52 microM). Similarly, when subjected to a methylammonium gradient, RhBG- and RhCG-expressing cells exhibited kinetic rate constants greater than those of native cells. However, these constants were five times higher for RhBG as compared with RhCG, suggesting a difference in substrate accessibility. These results, indicating that RhBG and RhCG facilitate rapid and low-energy-dependent bi-directional ammonium movement across the plasma membrane, favour the hypothesis that these Rh glycoproteins, together with their erythroid homologue RhAG [Ripoche, Bertrand, Gane, Birkenmeier, Colin and Cartron (2005) Proc. Natl. Acad. Sci. U.S.A. 101, 17222-17227] constitute a family of NH3 channels in mammalian cells.

Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

Increased adhesion to endothelial cells of erythrocytes from patients with polycythemia vera is mediated by laminin alpha5 chain and Lu/BCAM.

Patients with polycythemia vera (PV) have a JAK2 (a cytosolic tyrosine kinase) mutation and an increased risk of vascular thrombosis related to red blood cell (RBC) mass and platelet activation. We investigated functional RBC abnormalities that could be involved in thrombosis. RBC adhesion to human umbilical vein endothelial cells (HUVECs) was measured by a radiometric technique and in a flow system by video microscopy, and adhesion molecule expression was determined using specific antibodies (against CD36, CD49d, ICAM-4, Lu/BCAM, CD147, and CD47) and flow cytometry in a group of 38 patients with PV and a group of 36 healthy volunteers. Adhesion of PV RBCs was 3.7-fold higher than that of normal RBCs (P < .001). Adhesion was inhibited when PV RBCs were incubated with anti-Lutheran blood group/basal cell adhesion molecule (Lu/BCAM) or when HUVECs were treated with anti-laminin alpha(5) and to a lesser extent with anti-alpha(3) integrin. Lu/BCAM was constitutively phosphorylated in PV RBCs. Transfection of K562 cells with JAK2 617V>F resulted in increased expression and phosphorylation of Lu/BCAM. Phosphorylation of Lu/BCAM increases RBC adhesion. Our results indicate that JAK2 mutation might be linked to Lu/BCAM modification and increased RBC adhesiveness, which may be a factor favoring thrombosis in PV.

Improved in vitro biocompatibility of bicarbonate-buffered peritoneal dialysis fluid.

BACKGROUND: Conventional peritoneal dialysis fluids (PDFs) have been shown to damage the mesothelial layer and are associated with the development of peritoneal fibrosis and neoangiogenesis. New-generation PDFs have therefore been developed with physiological pH and reduced levels of glucose degradation products (GDPs), precursors of advanced glycation end products (AGEs). In this work, we evaluated and compared the improved biocompatibility of two new-generation PDFs (Balance and bicaVera) using mesothelial cell biology; we also compared them to a standard PDF (stay.safe) (all PDFs by Fresenius Medical Care, Fresnes, France). METHODS: stay.safe, Balance, and bicaVera were tested for their effect on human peritoneal mesothelial cell (HPMC) viability by measuring cell proliferation and apoptosis, and oncosis induction. The formation of AGEs was evaluated by immunoassay. Transforming growth factor beta-1 and vascular endothelial growth factor (VEGF) were immunoassayed in HPMC supernatants exposed to the above PDFs. RESULTS: At 15 g/L glucose concentration, HPMC exposure to bicaVera resulted in higher cell proliferation compared to Balance (p < 0.001) and stay.safe (p < 0.001). Compared to the lactate-buffered PDFs (Balance and stay.safe), oncosis was significantly lower in cells exposed to bicaVera (p < 0.05). bicaVera, containing lower amounts of GDPs, generated less AGE formation (p < 0.05) and VEGF production (p < 0.05) than either Balance or stay.safe. CONCLUSIONS: New-generation PDFs with physiological pH and lower GDP levels, especially if bicarbonate-buffered (bicaVera), have fewer in vitro toxic effects on mesothelial cells and may contribute to peritoneal preservation, thus improving long-term treatment of PD patients.

Phage-derived monoclonal anti-Lu.

BACKGROUND: Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost. STUDY DESIGN AND METHODS: A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further. RESULTS: Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins. CONCLUSION: With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.

The Kell protein of the common K2 phenotype is a catalytically active metalloprotease, whereas the rare Kell K1 antigen is inactive. Identification of novel substrates for the Kell protein.

The Kell blood group is a highly polymorphic system containing over 20 different antigens borne by the protein Kell, a 93-kDa type II glycoprotein that displays high sequence homology with members of the M13 family of zinc-dependent metalloproteases whose prototypical member is neprilysin. Kell K1 is an antigen expressed in 9% of the Caucasian population, characterized by a point mutation (T193M) of the Kell K2 antigen, and located within a putative N-glycosylation consensus sequence. Recently, a recombinant, non-physiological, soluble form of Kell was shown to cleave Big ET-3 to produce the mature vasoconstrictive peptide. To better characterize the enzymatic activity of the Kell protein and the possible differences introduced by antigenic point mutations affecting post-translational processing, the membrane-bound forms of the Kell K1 and Kell K2 antigens were expressed either in K562 cells, an erythroid cell line, or in HEK293 cells, a non-erythroid system, and their pharmacological profiles and enzymatic specificities toward synthetic and natural peptides were evaluated. Results presented herein reveal that the two antigens possess considerable differences in their enzymatic activities, although not in their trafficking pattern. Indeed, although both antigens are expressed at the cell surface, Kell K1 protein is shown to be inactive, whereas the Kell K2 antigen binds neprilysin inhibitory compounds such as phosphoramidon and thiorphan with high affinity, cleaves the precursors of the endothelin peptides, and inactivates members of the tachykinin family with enzymatic properties resembling those of other members of the M13 family of metalloproteases to which it belongs.

Protein kinase A-dependent phosphorylation of Lutheran/basal cell adhesion molecule glycoprotein regulates cell adhesion to laminin alpha5.

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 alpha5 receptors on red blood cells and epithelial cells in multiple tissues. It has been shown that sickle red cells exhibit enhanced adhesion to laminin alpha5 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 alpha5. 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 alpha5 in sickle cell disease.

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells.

Rhesus (Rh) antigens are carried by a membrane complex that includes Rh proteins (D and CcEe), Rh-associated glycoproteins (RhAG), and accessory chains (LW and CD47) associated by noncovalent bonds. In heterologous expression systems, RhAG and its kidney orthologs function as ammonium transporters. In red blood cells (RBCs), it is generally accepted that NH(3) permeates by membrane lipid diffusion. We have revisited these issues by studying RBC and ghosts from human and mouse genetic variants with defects of proteins that comprise the Rh complex. In both normal and mutant cells, stopped-flow analyses of intracellular pH changes in the presence of inwardly directed methylammonium (CH(3)NH(+)(3)+CH(3)NH(2)) or ammonium (NH(+)(4)+NH(3)) gradients showed a rapid alkalinization phase. Cells from human and mouse variants exhibited a decrease in their kinetic rate constants that was strictly correlated to the degree of reduction of their RhAG/Rhag expression level. Rate constants were not affected by a reduction of Rh, CD47, or LW. CH(3)NH(2)/NH(3) transport was characterized by (i) a sensitivity to mercurials that is reversible by 2-mercaptoethanol and (ii) a reduction of alkalinization rate constants after bromelain digestion, which cleaves RhAG. The results show that RhAG facilitates CH(3)NH(2)/NH(3) movement across the RBC membrane and represents a potential example of a gas channel in mammalian cells. In RBCs, RhAG may transport NH(3) to detoxifying organs, like kidney and liver, and together with nonerythroid tissue orthologs may contribute to the regulation of the systemic acid-base balance.

Integrin receptor specificity for human red cell ICAM-4 ligand. Critical residues for alphaIIbeta3 binding.

The red cell intercellular adhesion molecule-4 (ICAM-4) binds to different members of the integrin receptor families. To better define the ICAM-4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that alphaLbeta2, alphaMbeta2, and alphaIIbbeta3 (activated) bind specifically and dose dependently to the recombinant ICAM-4-Fc protein. We also show that cell surface ICAM-4 interacts with the cell surface alphaVbeta3 integrin. In addition, using a alpha4beta1 cell transfectant and beta2 integrin-deficient LAD cells, we show here that ICAM-4 failed to interact with alpha4beta1 even after alpha4beta1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+). ICAM-4 amino acids that are critical for alphaIIbbeta3 and alphaVbeta3 interaction were identified by domain deletion analysis, site-directed mutagenesis and synthetic peptide inhibition. Our results provide evidence that the beta3 integrin binding sites encompass the first and second Ig-like domains of ICAM-4. However, while the alphaIIbbeta3 contact site comprises the ABED face of domain D1 with an extension in the C'-E loop of domain D2, the alphaVbeta3 contact site comprises residues on both faces of D1 and in the C'-E loop of D2. These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM-4, and that ICAM-4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells.

The triggering of human peritoneal mesothelial cell apoptosis and oncosis by glucose and glycoxydation products.

BACKGROUND: Peritoneal dialysis fluids (PDFs) have been shown to alter mesothelial cell functions. To further determine the mechanisms involved, we investigated the effects of glucose, glucose degradation products (GDPs) and advanced glycation end products (AGEs) on the inhibition of human peritoneal mesothelial cell (HPMC) proliferation and the induction of apoptosis and oncosis. METHODS: Four PDF solutions, heat-sterilized dextrose-lactate, filtered dextrose-lactate and heat-sterilized dextrose-bicarbonate-lactate, each containing 15 or 45 g/l glucose, and heat-sterilized icodextrin-lactate, containing 75 g/l icodextrin, were tested. In addition, we analysed the independent and synergistic effects of two glucose compounds, i.e. 3-deoxyglucosone (3-DG), a major GDP, and Nepsilon-(carboxymethyl)-lysine (CML), a high-affinity AGE receptor (RAGE) ligand on HPMC viability. Cell proliferation was measured by methyl-[(3)H]thymidine incorporation. Oncosis was quantified by nuclear propidium iodide (PI) DNA-intercalating capability, and apoptosis by the decrease in mitochondrial transmembrane potential ( triangle up psim). RESULTS: It was found that heat-sterilized dextrose-lactate inhibited HPMC proliferation to a greater extent than filtered dextrose-lactate, heat-sterilized dextrose-bicarbonate-lactate, or heat-sterilized icodextrin-lactate (P<0.001). Compared to filtered dextrose-lactate, heat-sterilized dextrose-lactate induced a significantly greater degree of apoptosis (P<0.05) and oncosis (P<0.01). Glucose-induced cell death and antiproliferative activity were significantly potentiated by the action of 3-DG or CML-albumin. By blocking the AGE-RAGE interaction recombinant soluble-RAGE reduced the PDF-induced inhibitory effect on cell proliferation (P<0.001) and apoptosis (P<0.05). CONCLUSION: Heat-sterilized PDFs that contain high glucose concentrations and GDPs, which are AGE precursors, reduce cell proliferation, induce mesothelial cell apoptosis and oncosis, and may be involved in peritoneal damage. PDFs containing lower glucose derivative products are more biocompatible.

Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line.

Nonsteroidal anti-inflammatory drugs, which inhibit cyclooxygenase (COX) activity, are powerful antineoplastic agents that exert their antiproliferative and proapoptotic effects on cancer cells by COX-dependent and/or COX-independent pathways. Celecoxib, a COX-2-specific inhibitor, has been shown to reduce the number of adenomatous colorectal polyps in patients with familial adenomatous polyposis. Here, we show that celecoxib induces apoptosis in the colon cancer cell line HT-29 by inhibiting the 3-phosphoinositide-dependent kinase 1 (PDK1) activity. This effect was correlated with inhibition of the phosphorylation of the PDK1 downstream substrate Akt/protein kinase B (PKB) on two regulatory sites, Thr(308) and Ser(473). However, expression of a constitutive active form of Akt/PKB (myristoylated PKB) has a low protective effect toward celecoxib-induced cell death. In contrast, overexpression of constitutive active mutant of PDK1 (PDK1(A280V)) was as potent as the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, to impair celecoxib-induced apoptosis. By contrast, cells expressing a kinase-defective mutant of PDK1 (PDK1(K114G)) remained sensitive to celecoxib. Furthermore, in vitro measurement reveals that celecoxib was a potential inhibitor of PDK1 activity with an IC(50) = 3.5 microm. These data indicate that inhibition of PDK1 signaling is involved in the proapoptotic effect of celecoxib in HT-29 cells.

AQP3 deficiency in humans and the molecular basis of a novel blood group system, GIL.

AQP3 is a water and glycerol channel present on human erythrocytes and in various tissues. By protein and molecular biology analysis, two unrelated probands who developed alloantibodies to the high frequency antigen GIL were found to be AQP3-deficient. The defect is caused by homozygous mutation affecting the 5' donor splice site of intron 5 of the AQP3 gene. This mutation causes the skipping of exon 5 and generates a frameshift and premature stop codon. Functional studies by 90 degrees light scattering using a stopped-flow spectrometer revealed the absence of facilitated glycerol transport across red cell membranes from the probands, but the water and urea transports were normal. Expression studies into COS-7 cells followed by flow cytometry analysis showed that only cells transfected with AQP3 cDNA strongly reacted with anti-GIL antibodies. These findings represent the first reported cases of AQP3 deficiency in humans and provide the molecular basis of a new blood group system, GIL, encoded by the AQP3 protein.

Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation.

Several studies suggest that the Rh complex represents a major interaction site between the membrane lipid bilayer and the red cell skeleton, but little is known about the molecular basis of this interaction. We report here that ankyrin-R is capable of interacting directly with the C-terminal cytoplasmic domain of Rh and RhAG polypeptides. We first show that the primary defect of ankyrin-R in normoblastosis (nb/nb) spherocytosis mutant mice is associated with a sharp reduction of RhAG and Rh polypeptides. Secondly, our flow cytometric analysis of the Triton X-100 extractability of recombinant fusion proteins expressed in erythroleukemic cell lines suggests that the C-terminal cytoplasmic domains of Rh and RhAG are sufficient to mediate interaction with the erythroid membrane skeleton. Using the yeast two-hybrid system, we demonstrate a direct interaction between the cytoplasmic tails of Rh and RhAG and the second repeat domain (D2) of ankyrin-R. This finding is supported by the demonstration that the substitution of Asp-399 in the cytoplasmic tail of RhAG, a mutation associated with the deficiency of the Rh complex in one Rhnull patient, totally impaired interaction with domain D2 of ankyrin-R. These results identify the Rh/RhAG-ankyrin complex as a new interaction site between the red cell membrane and the spectrin-based skeleton, the disruption of which might result in the stomato-spherocytosis typical of Rhnull red cells.

Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin.

ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.

Erythroid expression and oligomeric state of the AQP3 protein.

Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono- and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one.

Cell-surface expression of RhD blood group polypeptide is posttranscriptionally regulated by the RhAG glycoprotein.

In most cases, the lack of Rh in Rh(null) red cells is associated with RHAG gene mutations. We explored the role of RhAG in the surface expression of Rh. Nonerythroid HEK293 cells, which lack Rh and RhAG, or erythroid K562 cells, which endogenously express RhAG but not Rh, were transfected with RhD and/or RhAG cDNAs using cytomegalovirus (CMV) promoter-based expression vectors. In HEK293 cells, a low but significant expression of RhD was obtained only when RhAG was expressed at a high level. In K562 cells, as expected from the opposite effects of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) on erythroid and CMV promoters, the levels of endogenous RhAG and recombinant RhD transcripts were substantially decreased and enhanced upon TPA treatment of RhD-transfected cells (K562/RhD), respectively. However, flow cytometry and fluorescence microscopy analysis revealed a decreased cell-surface expression of both RhAG and RhD proteins. Conversely, TPA treatment of RhAG-transfected cells increased both the transcript and surface expression levels of RhAG. When K562/RhD cells were cotransfected by the RhAG cDNA, the TPA-mediated induction of recombinant RhAG and RhD transcription was associated with an increased membrane expression of both RhAG and RhD proteins. These results demonstrate the role of RhAG as a strictly required posttranscriptional factor regulating Rh membrane expression. In addition, because the postulated 2:2 stoichiometry between Rh and RhAG observed in the native red cell membrane could not be obtained in cotransfected K562 cells, our study also suggests that as yet unidentified protein(s) might be involved for optimal membrane expression of Rh.

Sequence, evolution and ligand binding properties of mammalian Duffy antigen/receptor for chemokines.

The Duffy antigen/ receptor for chemokine, DARC, acts as a widely expressed promiscuous chemokine receptor and as the erythrocyte receptor for Plasmodium vivax. To gain insight into the evolution and structure/function relations of DARC, we analyzed the binding of anti-human Fy monoclonal antibodies (mAbs) and human chemokines to red blood cells (RBCs) from 11 nonhuman primates and two nonprimate mammals, and we elucidated the structures of the DARC genes from gorilla, gibbon, baboon, marmoset, tamarin, night monkey and cattle. CXCL-8 and CCL-5 chemokine binding analysis indicated that the promiscuous binding profile characteristic of DARC is conserved across species. Among three mAbs that detected the Fy6 epitope by flow cytometric analysis of human and chimpanzee RBCs, only one reacted with night monkey and squirrel monkey. Only chimpanzee RBCs bound a significant amount of the anti-Fy3 mAb. Fy3 was also poorly detected on RBCs from gorilla, baboon and rhesus monkey, but not from new world monkeys. Alignment of DARC homologous sequences allowed us to construct a phylogenetic tree in which all branchings were in accordance with current knowledge of primate phylogeny. Although DARC was expected to be under strong internal and external selection pressure, in order to maintain chemokine binding and avoid Plasmodium vivax binding, respectively, our present study did not provide arguments in favor of a selection pressure on the extracellular domains involved in ligand specificity. The amino acid variability of DARC-like polypeptides was found to be well correlated with the hydrophilicity indexes, with the highest divergence on the amino-terminal extracellular domain. Analysis of the deduced amino acid sequences highlighted the conservation of some amino acid residues, which should prove to be critical for the structural and functional properties of DARC.

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.