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.

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.

Transcript analysis of D category phenotypes predicts hybrid Rh D-CE-D proteins associated with alteration of D epitopes.

The RH blood group locus from RhD-positive donors is composed of two closely related genes, RHCE and RHD, encoding the Cc/Ee and D antigens, respectively. The major Rh antigen, D, is serologically defined as a mosaic of at least nine determinants (epD1 to epD9), and the lack of expression of some of these D epitopes at the surface of variant red blood cells defines the D category phenotypes. In this report, we have analyzed the Rh transcripts from reticulocytes of different D category phenotypes (DIVa, DIVb, DVa, and DFR). Although Southern blot analysis did not sow obvious deletions within the RHD gene, sequence analysis of the RhD transcripts indicated that, in all cases studied, the lack of D epitopes is associated with substitutions, in the deduced polypeptides, of amino acids specific of the RhD protein by those encoded at the equivalent position by the RHCE gene. These results strongly suggested that the D category phenotypes resulted from segmental DNA replacement between RHD-specific fragments and their equivalents in the RHCE gene. The regions involved in the DIVa, DIVb, DVa, and DFR phenotypes were shown to encompass all or part of the exons 3 and 7, exons 7 to 9, exon 5, and exon 4, respectively. All protein variants encoded by these rearranged RH genes represent new CE-D-CE hybrid molecules that retain only some of the nine D epitopes. Because segmental DNA replacements have been previously identified in other Rh variant genomes, we postulate that such genomic rearrangements between different regions of the RHCE and RHD genes should be one of the most frequent events involved in the extreme polymorphism of the RH blood group system.

Molecular analysis of the structure and expression of the RH locus in individuals with D--, Dc-, and DCw- gene complexes.

Rh blood group antigens of the D, C/c, and E/e series are carried by at least three red cell membrane polypeptides encoded by two highly related genes, RHD and RHCE. Homozygous individuals carrying the D--, Dc-, and DCw- gene complexes are characterized by a total or partial lack of expression of the RHCE-encoded antigens. Analysis of the molecular genetic basis of these rare conditions indicates that complete or partial expression defect of Cc/Ee antigens result from different alterations at the RH locus, but not from gross deletions. No rearrangement or mutation of the RHCE gene could be detected in donors homozygous for the D-- complex, suggesting that the lack of the Cc and Ee antigens might result from a reduced transcriptional activity of the RHCE gene. The Dc- and DCw- gene complexes, however, exhibited an important rearrangement of the RHCE gene. Instead of the normal RHCE gene, both variants carried a hybrid RHCE-D-CE gene in which exons 4 to 9 (Dc- complex) and 2 (or 3) to 9 (DCw- complex) of the RHCE gene, respectively, have been substituted by the equivalent region of the RHD gene. These gene conversion events provide an explanation for the well-described abnormal antigen profiles associated with the Dc- and DCw- complexes, like the increased expression of RhD, the reduced expression of RhC/c or RhCw, and the absence of RhE/e.

Structure and expression of the RH locus in the Rh-deficiency syndrome.

Red blood cell deficiency of Rh proteins is associated with morphologic and functional abnormalities of erythrocytes and with a chronic hemolytic anemia of varying severity. Rh-deficiency may be the result of homozygosity either for a silent allele at the RH locus (Rhnull amorph type) or for a recessive inhibitor gene(s) at an autosomal locus unlinked to RH locus (Rhnull regulator and Rhmod). In this report, we investigated the RH locus structure of Rh-deficient individuals by Southern analysis using cDNA and exon-specific probes deduced from the recent cloning of Rh genes (CcEe and D). As expected from family studies indicating that Rhmod and Rhnull regulator individuals are unable to express Rh antigens but are able to convey functional Rh genes from one generation to another, no alteration of the Rh genes was detected in these variants. Although Rhnull of the amorph type arose by inheritance of a pair of silent alleles at the RH locus, the general organization of the unique CcEe gene in the genome of the particular individual under examination was apparently normal and indistinguishable from a Rh-negative chromosome. More surprisingly, no mutation could be detected by sequencing the polymerase chain reaction (PCR)-amplified reticulocyte mRNAs, suggesting that the RH locus of this patient might be altered in its transcriptional activity. Through hybridization with exon-specific probes, we were also able to determine the zygosity for the D gene in DNA samples from individuals of known genotypes; using this approach, we found that Rhnull regulator variants could be either of the DD, Dd, or dd genotypes. These findings suggest that the postulated inhibitor gene(s) can negatively suppress the RH locus expression from chromosomes carrying either one or two of the Rh genes.

Klebsiella plasmid K21 is not involved in the aetiology of ankylosing spondylitis.

The possibility that a plasmid carried by Klebsiella pneumoniae plays a role in the pathogenesis of ankylosing spondylitis was explored. K. pneumoniae K21 contains a congruent to 25-kb plasmid, but this plasmid is not present in lymphocyte DNAs of ankylosing spondylitis HLA-B27 patients, as demonstrated by molecular hybridization experiments.

[Cold lymphocytotoxins in various pathological situations]. / Lymphocytotoxines froides dans diverses situations pathologiques

Cold lymphocytotoxins have been described extensively in many situations. In the present work, this kind of antibody has been found less frequently than previously in some diseases like infectious mononucleosis, rheumatoïd arthritis, or kidney transplanted patients. These discrepancies may be due to technical considerations. In contrast, cold lymphocytotoxins, in the present work, have been frequently detected in auto-immune hemolytic anemias with cold agglutinins, and in patients having immuno-deficiencies.