[Epidemiology of autoimmune haemolytic anemia in children: French data].
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Acute Disease↗
Biomedical subjects
Y Colin
Publications and source records attributed to Y Colin.
Hydrophobic cluster analysis and modeling of the human Rh protein three-dimensional structures.
Rh (Rhesus) is a major blood group system in man, which is clinically significant in transfusion medicine. Rh antigens are carried by an oligomer of two major erythroid specific polypeptides, the Rh (D and CcEe) proteins and the RhAG glycoprotein, that shared a common predicted structure with 12 transmembrane a-helices (M0 to M11). Non erythroid homologues of these proteins have been identified (RhBG and RhCG), notably in diverse organs specialized in ammonia production and excretion, such as kidney, liver and intestine. Phylogenetic studies and experimental evidence have shown that these proteins belong to the Amt/Mep/Rh protein superfamily of ammonium/methylammonium permease, but another view suggests that Rh proteins might function as CO2 gas channels. Until recently no information on the structure of these proteins were available. However, in the last two years, new insight has been gained into the structural features of Rh proteins (through the determination of the crystal structures of bacterial AmtB and archeaebacterial Amt-1. Here, models of the subunit and oligomeric architecture of human Rh proteins are proposed, based on a refined alignment with and crystal structure of the bacterial ammonia transporter AmtB, a member of the Amt/Mep/Rh superfamily. This alignment was performed considering invariant structural features, which were revealed through Hydrophobic Cluster Analysis, and led to propose alternative predictions for the less conserved regions, particularly in the N-terminal sequences. The Rh models, on which an additional Rh-specific, N-terminal helix M0 was tentatively positioned, were further assessed through the consideration of biochemical and immunochemical data, as well as of stereochemical and topological constraints. These models highlighted some Rh specific features that have not yet been reported. Among these, are the prediction of some critical residues, which may play a role in the channel function, but also in the stability of the subunit structure and oligomeric assembly. These results provide a basis to further understand the structure/function relationships of Rh proteins, and the alterations occurring in variant phenotypes.
Amino Acid Sequence↗
Generation of mice with inactivated Rh or Rhag genes.
Mice carrying inactivated Rh and Rhag genes were generated by insertional targeting. KO animals exhibited normal growth, development and fertility and both types were indistinguishable at a gross phenotypic level from their wild type littermates. Preliminary analysis revealed that red cells from Rh-/- mice lack Rh protein and have a moderate decrease of Rhag protein, whereas those from Rhag-/- mice have a total absence of Rhag and Rh proteins. Studies are in progress to delineate the antigenic, biochemical and functional abnormalities of red cells from these animals as well as the impact on hematological parameters and erythropoiesis.
Animals↗
Functional interaction between Rh proteins and the spectrin-based skeleton in erythroid and epithelial cells.
We summarize the different experimental approaches which provide evidence that direct interaction of Rh and RhAG to ankyrin-R constitutes, together with the AE-1 (Band 3)-ankyrin-protein 4.2 and GPC-protein 4.1-p55 complexes, another major anchoring site between the red cell membrane bilayer and the underlying spectrin-based skeleton. The observations that some residues of the ankyrin binding site are mutated in Rh and RhAG proteins from some weak D and Rh(null) variants, respectively, suggest that the Rh-RhAG/ankyrin-R interaction plays a crucial role in the biosynthesis and/or the stability of the Rh complex in the red cell membrane. Similarly, binding to ankyrin G is required for cell surface expression of the non-erythroid member of the Rh protein family, RhBG, at the basolateral membrane domain of polarized epithelial cells. The next challenge will be to determine whether binding to the membrane skeleton may be critical for the emerging ammonium transport function of Rh proteins in erythroid and non-erythroid cells.
Animals↗
Ammonium transport properties of HEK293 cells expressing RhCG mutants: preliminary analysis of structure/function by site-directed mutagenesis.
We have recently shown by monitoring intracellular pHi with a stopped-flow fluorimeter, that when expressed in HEK293 kidney cells, two Rh glycoproteins, RhBG and RhCG, facilitated NH3 movement across the plasma membrane. Based on the results of 3D structure determination of AmtB, a bacterial member of the Amt/Mep/Rh superfamily, and of homology modeling of the human Rh proteins, we have attempted to determine if some selected residues predicted to be located in the pore or in the vestibule of the channel are essential for NH3 transport. Accordingly, wild type and mutant forms of RhCG were expressed in HEK293 cells and their ammonium function was analyzed with the stopped-flow fluorimeter. Some mutants that were not expressed at a significant level in HEK293 could not be tested for function, but mutations at positions F74, V137 and F235 (equivalent positions in AmtB: I28, L114, F215, respectively) resulted in a severe reduction of NH3 transport.
Amino Acid Substitution↗
Role of RhAG and AQP1 in NH3 and CO2 gas transport in red cell ghosts: a stopped-flow analysis.
To clarify the potential role Rh/RhAG and AQP1 proteins in erythrocyte gas transport, NH3 and CO2 transport was measured in erythrocyte ghost membrane vesicles from rare human variants (Rh(null), CO(null),) and knockout mice (homozygous AQP1-/-, Rh-/- and Rhag-/-) exhibiting well-characterized protein defects. Transport was measured from intracellular pH (pHi) changes in a stopped-flow fluorimeter. NH3 transport was measured in chloride-free conditions with ghosts exposed to 20 mM inwardly directed gradients of gluconate salts of ammonium, hydrazine and methylammonium at 15 degrees C. Alkalinization rates of control samples were 6.5+/-0.3, 4.03+/-0.17, 0.95+/-0.08 s(-1) for each solute, respectively, but were significantly reduced for Rh(null) and CO(null) samples that are deficient in RhAG and AQP1 proteins, respectively. Alkalinization rates of Rh(null) ghosts were about 60%, 83% and 94% lower than that in control ghosts, respectively, for each solute. In CO(null) ghosts, the lack of AQP1 resulted in about 30% reduction of the alkalinization rates as compared to controls, but the transport selectivity of RhAG for the three solutes was preserved. Similar observations were made with ghosts from KO mice Rhag-/- and AQP1-/-. These results confirm the major contribution of RhAG/Rhag in the NH3 conductance of erythrocytes and suggest that the reduction of transport rates in the absence of AQP1 would be better explained by a direct or indirect effect on RhAG/Rhag-mediated transport. When ghosts were preloaded with carbonic anhydrase and exposed to a 25 mM CO2/HCO3- gradient at 6 degrees C, an extremely rapid kinetics of acidification corresponding to CO2 influx was observed. The rate constants were not significantly different between controls and human variants (125+/-6 s(-1)), or between wild-type and KO mice, suggesting no major role of RhAG or AQP1 in CO2 transport, at least in our experimental conditions.
Ammonia↗
A structural model of a seven-transmembrane helix receptor: the Duffy antigen/receptor for chemokine (DARC).
The Duffy antigen/receptor for chemokine (DARC) is an erythrocyte receptor for malaria parasites (Plasmodium vivax and Plasmodium knowlesi) and for chemokines. In contrast to other chemokine receptors, DARC is a promiscuous receptor that binds chemokines of both CC and CXC classes. The four extracellular domains (ECDs) of DARC are essential for its interaction with chemokines, whilst the first (ECD1) is sufficient for the interaction with malaria erythrocyte-binding protein. In this study, we elaborate and analyze structural models of the DARC. The construction of the 3D models is based on a comparative modeling process and on the use of many procedures to predict transmembrane segments and to detect far homologous proteins with known structures. Threading, ab initio, secondary structure and Protein Blocks approaches are used to build a very large number of models. The conformational exploration of the ECDs is performed with simulated annealing. The second and fourth ECDs are strongly constrained. On the contrary, the ECD1 is highly flexible, but seems composed of three consecutive regions: a small beta-sheet, a linker region and a structured loop. The chosen structural models encompass most of the biochemical features and reflect the known experimental data. They may be used to analyze functional interaction properties.
Amino Acid Sequence↗
Recombinant forms of Gerbich blood group antigens: expression and purification.
Recombinant forms of normal glycophorin C (GPC), carrying the high frequency Gerbich blood group antigens, and its natural deletion mutants of Yus and Ge type (all combined with oligohistidyl tag) were expressed in CHO and COS 7 cells. The stable expression of all recombinant forms of GPC in CHO cells was obtained, but the level of expression was low and detectable only by flow cytometry. The high level of transient expression of GPC recombinant forms in COS 7 cells allowed their purification on Ni-NTA-agarose. The purified recombinant GPC and mutants of Yus and Ge type behaved in SDS-PAGE similarly to normal GPC forms from RBC membranes. The recombinant GPC.Yus and GPC.Ge mutants appeared as diffuse bands, suggesting the similar heterogeneity of glycosylation that was observed in natural GPC.Yus and GPC.Ge glycoproteins. The flow cytometry analysis of the transfected CHO and COS 7 cells showed that binding of anti-GPC monoclonal antibodies to GPC variants was accordant with the known fine specificity of these antibodies. The obtained recombinant forms of GPC carrying common Gerbich antigens may be useful in serology, and also as model molecules for structure-function studies.
Amino Acid Sequence↗
Structural characterization of the epitope recognized by the new anti-Fy6 monoclonal antibody NaM 185-2C3.
The epitope recognized by a new anti-Fy6 monoclonal antibody (MoAb) (clone name: NaM185-2C3) was characterized using peptides synthesized on pins (Epitope scanning kit). The clone was obtained from splenocytes of mice immunized with CHO cells expressing the recombinant Duffy glycoprotein. NaM185-2C3 recognized a linear epitope, the essential portion of which was pentapeptide Phe-Glu-Asp-Val-Trp comprising amino acid residues 22-26 of the main (336aa) isoform of the Duffy antigen. All the amino acid residues of the epitope, except Asp, were essential for the antibody-binding, because they could not be replaced by any or most other amino acid residues. The Asp residue could be replaced by most other amino acid residues and its replacement by some amino acid residues gave a distinct increase in the antibody-binding. The MoAb NaM185-2C3, similarly as other anti-Fy6 antibodies, inhibits interleukin (IL)-8-binding to the Duffy antigen. A part of the results was presented at ISBT meeting (Blanchard et al., 1998, Vox Sanguinis, 74, S1, Abstract no. 71).
Amino Acid Sequence↗
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.
Amino Acid Motifs↗
Structural and functional diversity of blood group antigens.
Biochemical and molecular genetic studies have revealed that blood group antigens are present on cell surface molecules of wide structural diversity, including carbohydrate epitopes on glycoproteins and/or glycolipids, and peptide antigens on proteins inserted within the membrane via single or multi-pass transmembrane domains, or via glycosylphosphatidylinositol linkages. These studies have also shown that some blood group antigens are carried by complexes consisting of several membrane components which may be lacking or severely deficient in rare blood group 'null' phenotypes. In addition, although all blood group antigens are serologically detectable on red blood cells (RBCs), most of them are also expressed in non-erythroid tissues, raising further questions on their physiological function under normal and pathological conditions. In addition to their structural diversity, blood group antigens also possess wide functional diversity, and can be schematically subdivided into five classes: i) transporters and channels; ii) receptors for ligands, viruses, bacteria and parasites; iii) adhesion molecules; iv) enzymes; and v) structural proteins. The purpose of this review is to summarize recent findings on these molecules, and in particular to illustrate the existing structure-function relationships.
Animals↗
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.
Anion Exchange Protein 1, Erythrocyte↗
Expression of chemokines and chemokine receptors during human renal transplant rejection.
Infiltration of renal allografts by leukocytes is a hallmark of acute transplant rejection. Chemokines attract leukocytes bearing specific chemokine receptors, and the specific leukocyte chemokine receptor phenotype is associated with types of immune responses, ie, T helper subtype 1 (Th1; CXC chemokine receptor 3 [CXCR3], CC chemokine receptor 5 [CCR5]) versus Th2 (CCR3, CCR4, CCR8). We studied the expression of the chemokine monocyte chemoattractant protein-1 and the chemokine receptors CCR2B and CXCR4 messenger RNA (mRNA) by in situ hybridization, as well as the chemokine receptors Duffy antigen receptor for chemokines (DARC) and CCR5 protein by immunohistochemistry in renal biopsy specimens with acute cellular rejection (n = 12) and acute vascular rejection (n = 8), transplant nephrectomy specimens (n = 6), and normal areas of tumor nephrectomy specimens (n = 5). CC chemokines and CC chemokine receptor mRNA expression were evaluated by ribonuclease protection assay in specimens from four transplant nephrectomies and one tumor nephrectomy. Upregulation of mRNAs for the chemokines, interferon-inducible protein-10 (IP-10); regulated on activation normal T-cell expressed and secreted; macrophage inflammatory protein-1alpha (MIP-1alpha); MIP-1beta; and lymphotactin, as well as the chemokine receptors, CCR2 and CCR5, were documented during allograft rejection. CCR1 mRNA was detectable in both allografts and controls, but CCR3 and CCR8 were absent. The number of CXCR4, CCR5, and CCR2B mRNAs expressing leukocytes and DARC-positive vessels increased during rejection episodes. CXCR4 mRNA was the most widely expressed. Leukocytes in diffuse interstitial infiltrates were mainly CCR5 positive, but in areas in which leukocytes formed nodular aggregates of infiltrating cells, the number of CCR5-positive cells was low. Instead, leukocytes in these nodular aggregates mainly expressed CXCR4. DARC was expressed on peritubular capillaries, where it was upregulated in areas of interstitial infiltration. Induction of chemokines during renal allograft rejection is accompanied by infiltration of leukocytes bearing the respective chemokine receptors. The upregulation of the CXCR3 ligand IP-10, as well as CCR5 and its ligands, in the absence of CCR3 and CCR8 is indicative that renal allograft rejection is primarily the result of a Th1-type immune response.
Antigens, Protozoan↗
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.
Alleles↗
The Duffy antigen receptor for chemokines is up-regulated during acute renal transplant rejection and crescentic glomerulonephritis.
BACKGROUND: Recruitment of leukocytes during immune responses requires the coordinate expression of adhesion molecules in concert with chemokines and their receptors. The Duffy antigen receptor for chemokines (DARC) binds multiple chemokines and is expressed on postcapillary venules in the normal kidney. The chemokine receptor CCR5, which shares the ligand regulated upon activation, normal T-cell expressed and secreted (RANTES) with DARC, is expressed by infiltrating T cells in the renal interstitium. As DARC might be involved in the attraction of CCR5-positive cells, we studied the distribution of DARC and CCR5 in two forms of cell-mediated renal injury: renal allograft rejection and crescentic glomerulonephritis (cGN). METHODS: A total of 87 renal specimens, including 12 pretransplant biopsies, 47 transplant biopsies (Banff 1, N = 10; Banff 2, N = 19; and various other lesions N = 18), and 28 biopsies from patients with cGN, was analyzed. Immunohistochemistry for CCR5 and DARC was performed on serial sections of formalin-fixed and paraffin-embedded tissue. RESULTS: Compared with pretransplant biopsies, the mean number of DARC-positive interstitial venules was significantly increased during both transplant rejection and cGN. This was accompanied by an infiltration of CCR5-positive leukocytes. During transplant rejection, the number and distribution of CCR5-positive cells correlated with DARC-positive venules. Infiltrating CCR5-positive leukocytes were found mainly in the interstitium, often clustering around Bowman's capsules in biopsies from cGN. The number of glomerular CCR5 positive cells is low, but they are common in a subset of crescents. CONCLUSIONS: We hypothesize that the increased number of DARC-positive venules in areas of interstitial injury and the colocalization with CCR5-positive infiltrating leukocytes may indicate a role for endothelial DARC expression during leukocyte adhesion and interstitial infiltration.
Acute Disease↗
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.
Amino Acid Sequence↗
Cloning, expression, and chromosomal mapping of a human ATPase II gene, member of the third subfamily of P-type ATPases and orthologous to the presumed bovine and murine aminophospholipid translocase.
Recently, a P-type ATPase was cloned from bovine chromaffin granules (b-ATPase II) and a mouse teratocarcinoma cell line (m-ATPase II) and was shown to be homologous to the Saccharomyces cerevisiae DRS2 gene, the inactivation of which resulted in defective transport of phosphatidylserine. Here, we report the cloning from a human skeletal muscle cDNA library of a human ATPase II (h-ATPase II), orthologous to the presumed bovine and mouse aminophospholipid translocase (95.3 and 95.9% amino acid identity, respectively). Compared with the bovine and mouse counterparts, the cloned h-ATPase II polypeptide exhibits a similar membrane topology, but contains 15 additional amino acids (1163 vs 1148) located in the second intracytoplasmic loop, near the DKTGTLT-phosphorylation site. However, RT-PCR analysis performed with RNA from different human tissues and cell lines revealed that the coding sequence for these 15 residues is sometimes present and sometimes absent, most likely as a result of a tissue-specific alternative splicing event. The h-ATPase II gene, which was mapped to chromosome 4p14-p12, is expressed as a 9.5-kb RNA species in a large variety of tissues, but was not detected in liver, testis, and placenta, nor in the erythroleukemic cell line K562.
Adenosine Triphosphatases↗
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.
Amino Acid Sequence↗