RESUMEN
The Rhesus (RH) blood group locus is composed of two related structural genes, D and CcEe, that encode red cell membrane proteins carrying the D, Cc and Ee antigens. As demonstrated previously, the RhD-positive/RhD-negative polymorphism is associated with the presence or the absence of the D gene. Sequence analysis of transcripts and genomic DNA from individuals that belong to different Rh phenotypes were performed to determine the molecular basis of the C/c and E/e polymorphisms. The E and e alleles differ by a single nucleotide resulting in a Pro226Ala substitution, whereas the C and c alleles differ by six nucleotides producing four amino acid substitutions Cys16Trp, Ile60Leu, Ser68Asn and Ser103Pro. With the recent cloning of the RhD gene, these findings provide the molecular genetic basis that determine D, C, c, E and e specificities.
Asunto(s)
Genes , Polimorfismo Genético , Sistema del Grupo Sanguíneo Rh-Hr/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Drosophila melanogaster/genética , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas de Transporte de Catión/química , Glicoproteínas/química , Glicoproteínas de Membrana/química , Proteínas de Transporte de Membrana/química , Sistema del Grupo Sanguíneo Rh-Hr/química , Secuencia de Aminoácidos , Amoníaco/metabolismo , Proteínas Sanguíneas/genética , Proteínas de Escherichia coli/química , Variación Genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/genética , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Mutación Missense , Fenotipo , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The partial D phenotypes correspond to D-positive individuals that may develop anti-D antibodies following immunization by transfusion or pregnancy, since they lack some of the D epitopes that compose the D antigen. When these red cells are tested with a panel of human monoclonal anti-D, different patterns of reactivity are observed and at least nine distinct epitopes termed epD1 to epD9 can be identified. Molecular analysis of partial D variants have shown that the loss of some D epitopes is associated either with intergenic recombination events between the D and CE genes generating hybrid gene structures D-CE-D or CE-D-CE, or with point mutations of the D gene. Based on these findings, a tentative model that correlates critical amino acid positions and D epitope expression on the D protein was proposed. Although recent studies suggest that the D antigen may be composed of as many as 30 epitopes, the relatively simple model presented here may be useful to serologists as a preliminary approach to understanding the basis of D antigenic variation in terms of structure-activity relationship.
Asunto(s)
Modelos Moleculares , Sistema del Grupo Sanguíneo Rh-Hr/genética , Anticuerpos Monoclonales , Epítopos , Humanos , Péptidos , Fenotipo , Sistema del Grupo Sanguíneo Rh-Hr/inmunologíaRESUMEN
The Rh antigens are carried by 30-32 kDa integral proteins of the red cell membrane. The Rh locus is composed of two genes: RhD, which encodes the major D antigen and is present only in Rh-positive genomes, and RhCcEe, which encodes both the Cc and Ee polypeptides, most likely by alternative splicing events. The D and non-D Rh mRNAs have been cloned. Their sequence homology suggest that the two Rh genes have evolved by duplication of a common ancestor.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/química , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Membrana Eritrocítica/química , Genes , Humanos , Datos de Secuencia Molecular , Fenotipo , ARN Mensajero/genética , Sistema del Grupo Sanguíneo Rh-Hr/fisiologíaRESUMEN
In the Caucasian population, the RH locus of RhD-positive individuals is composed of two homologous genes, RHD and RHCE, arranged in tandem but of a single gene, RHCE, in RhD-negative individuals. Many variants recently characterized carry rearranged RH genes, most often by an unidirectional segmental DNA-exchange (gene-conversion) event. In D(VI) variants of type II, RHD is a D-CE-D hybrid gene in which the DNA fragment carrying exons 4-6 has been replaced by the corresponding sequences from the RHCE gene. To identify precisely and characterize the two transition sites, we have studied, by both PCR and sequence analysis, a genomic region between the 3' end of intron 3 and exon 7 in normal RHCE and RHD genes as well as in D(VI) DNA. We show that the D-CE breakpoint is located in intron 3, within a 250-bp fragment comprising an Alu S sequence, and that the CE-D breakpoint lies within a 39-bp fragment in intron 6. This Alu S sequence (and the 100-bp region immediately downstream) most likely defines a recombination hot spot, since there lies also the 5' breakpoint of different rearrangement events leading to D-CE and CE-D transitions in hybrid D(VI),DFR and Dc-,R(N) gene complexes, respectively.
Asunto(s)
Reordenamiento Génico , Recombinación Genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Bases , Exones/genética , Variación Genética , Genoma Humano , Humanos , Intrones/genética , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.
Asunto(s)
Empalme del ARN , ARN Mensajero/genética , ARN/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Secuencia de Bases , Médula Ósea/química , ADN/química , ADN/genética , ADN/aislamiento & purificación , Eritroblastos/química , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN/químicaRESUMEN
After anti-RhD, anti-Rhc is the most important red cell alloantibody which can cause haemolytic disease of the newborn (HDN) when the mother is Rhc-negative and the fetus Rhc-positive. We report here the development of polymerase chain reaction (PCR) assays which detect the presence of the Rhc alleles in amniotic cells by the use of allele-specific primers (ASP). It is expected that such determination will help in the management of pregnancies at risk of Rhc haemolytic disease. In the course of this study we have similarly performed PCR-ASP experiments to detect fetal RHE alleles since, in rare cases, anti-RhE can also cause HDN.
Asunto(s)
Eritroblastosis Fetal/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Prenatal/métodos , Sistema del Grupo Sanguíneo Rh-Hr/genética , Líquido Amniótico/citología , Secuencia de Bases , ADN/análisis , Eritroblastosis Fetal/sangre , Eritroblastosis Fetal/genética , Humanos , Recién Nacido , Isoanticuerpos/sangre , Isoanticuerpos/genética , Datos de Secuencia Molecular , Isoinmunización Rh/sangre , Isoinmunización Rh/diagnóstico , Isoinmunización Rh/genética , Sistema del Grupo Sanguíneo Rh-Hr/sangre , Factores de RiesgoRESUMEN
Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.
Asunto(s)
Fragmentos de Péptidos/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Membrana Eritrocítica/química , Variación Genética , Humanos , Immunoblotting , Proteínas de la Membrana/sangre , Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Ratones , Sistema del Grupo Sanguíneo Rh-Hr/genéticaRESUMEN
The RH (rhesus) blood group locus from RhD-positive donors is composed of two homologous structural genes, one of which encodes the Cc and Ee polypeptides, whereas the other, which is missing in the RhD-negative condition, encodes the D protein that carries the major antigen of the RH system. Recently, different splicing isoforms transcribed from the CcEe gene were isolated. We report now the characterization of two other Rh clones, RhII and RhXIII, generated by alternative choices for poly(A) addition sites that were identified as the RhD gene transcripts. That these cDNAs represented the RhD messenger and that the previously described Rh clones were derived from the CcEe gene was demonstrated by amplification of RhII/XIII sequences only from D-positive genomes and by cloning and sequencing of D- and CcEe-specific gene fragments. The predicted translation product of the RhD mRNA is a 417-amino acid protein (M(r) = 45,500) that exhibited a similar membrane organization with 13 bilayer-spanning domains compared with the polypeptide encoded by the CcEe gene. The D and Cc/Ee polypeptides differ by 36 amino acid substitutions (8.4% divergence), but the NH2- and COOH-terminal regions of the two proteins are well conserved. Similarly, five of the six cysteine residues of the Cc/Ee proteins were conserved in the D protein, including the unique exofacial cysteine, which is critical for antigenic reactivity. The sequence homology between the Cc/Ee and D proteins supports the concept that the genes encoding these polypeptides have evolved by duplication of a common ancestor gene.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Médula Ósea/fisiología , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Sondas de ADN , Genes , Biblioteca Genómica , Humanos , Sustancias Macromoleculares , Modelos Estructurales , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Conformación Proteica , ARN Mensajero/genéticaRESUMEN
The human Rh blood group locus consists of two structurally related genes (D and CcEe) in Rh-positive haplotypes but a single gene (CcEe) in Rh-negative haplotypes. The genome of rhesus monkeys (Macaca mulatta), while not expressing any of the human Rh D, C, c, E, or e specificities, carries a Rh-like locus strongly related to the human Rh locus. Southern blot analysis suggested the presence of only one Rh-like gene with an additional truncated fragment corresponding to the 5' region. RNA preparations from M. mulatta bone marrow cells contained Rh-like species of 1.7 kb. Two allelic Rh-like transcripts were amplified by PCR and sequenced. The predicted translation product of the first transcript was a 417-amino-acid protein closely similar to the human Rh counterpart. The predicted product of the second transcript consisted of a 361-amino-acid polypeptide truncated in the NH2 terminal region and differing from the former by a few substitutions. The macaque Rh-like protein sequences differed from those of human D and Cc/Ee polypeptides by 22-25%, whereas the degree of identity between the human proteins was 91.5%. Implications of these results in the analysis of the evolutionary pathway of the Rh locus are discussed.
Asunto(s)
Alelos , Macaca mulatta/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , ADN/análisis , ADN Complementario , Humanos , Macaca mulatta/sangre , Datos de Secuencia Molecular , Polimorfismo de Longitud del Fragmento de Restricción , ARN Mensajero/análisis , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Transcripción GenéticaRESUMEN
The Rh blood group antigens are encoded by two highly related genes, RHD and RHCE, and the sequence of the common alleles (D, Ce, CE, ce, and cE) of these genes has been previously elucidated. In this report, Rh transcripts and gene fragments have been amplified using polymerase chain reaction from the blood of donors with the CW+ andCX+ phenotypes. Sequence analysis indicated that the expression of the CW (Rh8) and CX (Rh9) antigens are associated with point mutations in the RHCE gene, which provides the definitive evidence that the CW and CX specificities are encoded by the same gene as the Cc and Ee antigens. As compared with the common (CW- and CX-) transcripts of the RHCE gene, the CW+ and CX+ cDNAs exhibited A122G and G106A transitions that resulted in Gln41Arg and Ala36Thr amino acid substitutions in the CW+ and CX+ polypeptides, respectively. Therefore, although the CW and CX specificities behave serologically as if they were allelic, they cannot not be considered, stricto sensu, as the products of antithetical allelic forms of the RHCE gene. Based on the CW-/CW+ nucleotide polymorphism, a polymerase chain reaction assay useful for diagnosis purposes has been developed that detects the presence of the CW+ allele by the use of an allele-specific primer.
Asunto(s)
Sistema del Grupo Sanguíneo Rh-Hr/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN/química , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I-III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10-50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D(IVb) variant), method III (D(VI) and DFR variants) and method IV (D(VI) variants), but not method I. Weak D (D(u)) was correctly detected as D-positive by all methods, but most cases of Rh(null) appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C- or E-allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.
Asunto(s)
Reacción en Cadena de la Polimerasa , Isoinmunización Rh/diagnóstico , Globulina Inmune rho(D)/genética , Southern Blotting , ADN/análisis , ADN/genética , Amplificación de Genes , Genotipo , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normas , Diagnóstico Prenatal/métodos , Diagnóstico Prenatal/normas , Sensibilidad y EspecificidadRESUMEN
The Rh blood group antigens D, Cc and Ee are encoded by two related genes, RHD and RHCE. The RhG antigen (Rh12) is associated with the expression of RhC and/or RhD, except in rare variant red cells. Here we have determined the molecular basis of G expression in the absence of D and C in the rGr phenotype. Nucleotide sequence analysis revealed that the rG allele resulted either from a segmental DNA exchange between part of exon 2 of the RHce gene and the equivalent region of the RHCE or RHD genes or from a crossing over between positions nt150 and nt178 of the RHce and RHCe genes. The predicted protein encoded by the hybrid rG gene (c-C-e or c-D-e) carries Ile60, Ser68 and Ser103 (as C and D polypeptides); any of these positions appear to be critical in the formation of the G antigen. In addition, Cys16 was found to be important in the phenotypic expression of C.
Asunto(s)
Reordenamiento Génico , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Bases , Southern Blotting , Heterocigoto , Humanos , Datos de Secuencia Molecular , FenotipoRESUMEN
After testing red cells from 12 RhE variants with a panel of anti-E monoclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes designated epE1, epE2, epE3 and epE4. The variants were classified into four categories (cat EI to EIV) which carried epE1 and epE2, epE1 and epE4, epE1, epE3 and epE4, and all four epitopes, respectively. Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and EIII displayed three distinct genetic alterations. Cat EI variants exhibited a point mutation (T500A) in exon 4 of the RHCE gene that resulted in a Met167Lys substitution in the third extracellular loop of the RhcE protein. Cat EII variant carried a hybrid gene structure characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant was also associated with a weak expression of the RhC antigen. In cat EIII variants there was a partial DNA exchange of exon 5 sequences (nt 697 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE-D-cE protein carrying the Glu233 and Val238 substitutions. The serological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and proline at position 226 is necessary but not sufficient for the full expression of the E antigen, (ii) the lack of RhE epitope(s) is associated with heterogenous molecular alterations of the RHCE gene, and (iii) amino-acids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.
Asunto(s)
Heterogeneidad Genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Transcripción Genética , Anticuerpos Monoclonales , ADN Complementario/genética , Epítopos/genética , Humanos , Mutación Puntual , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/genética , ATPasas Transportadoras de Calcio/genética , Mapeo Cromosómico , Proteínas de Transferencia de Fosfolípidos , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/química , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Southern Blotting , ATPasas Transportadoras de Calcio/química , Proteínas Portadoras/química , Proteínas Portadoras/genética , Bovinos , Cromosomas Humanos Par 4/genética , Clonación Molecular , Exones/genética , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Músculo Esquelético , Sistemas de Lectura Abierta/genética , Fosforilación , ARN Mensajero/análisis , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The Rh (Rhesus) blood group antigens, D, Cc, and Ee, are carried by three unglycosylated membrane proteins of the human erythrocytes encoded by two highly related genes, D and CcEe. The major antigen, D, is a mosaic composed of at least nine determinants (epD1 through epD9). The lack of expression of some of these D epitopes at the surface of variant red blood cells defines the so-called D category phenotypes. In this report, we have determined the molecular basis of the DVI category phenotype characterized by the lack of epitopes D1, D2, D5, D6/7, and D8. Southern blot analysis and mRNA sequencing showed that the DVI phenotype is associated with two types of rearrangement of the D gene. Of 10 DVI genomes investigated, 8 exhibited a segmental DNA replacement (gene conversion) between the D fragment encompassing exons 4, 5, and 6 and the equivalent region of the CcEe gene. In the two other variants, these three exons are deleted. In both cases, the genomic rearrangement did not alter the reading frame of the variant RhD transcripts that are translated in 417 and 266 amino acid polypeptides, respectively. A heterogeneity of category DVI samples based on variable reactivity of the red blood cells with anti-D antibodies was previously found to be associated with the CDVIe or cDVIE haplotypes. Interestingly, our present results indicated that this serologic subdivision of the DVI category is correlated to two types of genomic rearrangements of the D gene.
Asunto(s)
Reordenamiento Génico , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , ADN/sangre , ADN/genética , Cartilla de ADN , Membrana Eritrocítica/metabolismo , Exones , Humanos , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
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.
Asunto(s)
Antígenos de Protozoos , Detergentes/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células K562/metabolismo , Proteínas de la Membrana/metabolismo , Octoxinol/metabolismo , Proteínas Protozoarias , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Proteínas Sanguíneas/metabolismo , Antígeno CD47 , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/metabolismo , Citoesqueleto/metabolismo , Sistema del Grupo Sanguíneo Duffy , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Glicoforinas/metabolismo , Humanos , Sistema del Grupo Sanguíneo Lutheran , Proteínas de Neoplasias/metabolismo , Receptores de Superficie Celular/metabolismo , Sistema del Grupo Sanguíneo Rh-Hr , SolubilidadRESUMEN
Another example of rare red cells that failed to react with all anti-Rh and anti-LW antibodies was discovered in a Spanish woman suffering from a severe hemolytic anemia typical of the Rhnull syndrome. Family study and Rh blood typings demonstrated clearly that the proposita was homozygous for a silent Rh gene complex (Rhnull of the amorph type) that she inherited from her parents who are first cousins. Western blot analysis carried out with glycosylation-independent antibodies directed against the Rh polypeptide and the LW glycoprotein, respectively, confirmed that these protein components were absent from the red cells of the proposita. In addition, the patient was typed U-positive, again in agreement with the presence on her red cells of 45-75 kDa glycoproteins detected with the murine monoclonal antibody 2D10.