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.

Two new alleles of the RHCE gene in Black individuals: the RHce allele ceMO and the RHcE allele cEMI.

Six unrelated individuals of Afro-Caribbean origin, whose red cells have a marked reduction of the Rhe antigen expression, have been identified. All exhibited the same serological profile with anti-e monoclonal antibodies and lacked expression of the high frequency e-related antigen hrS. Transcripts and genomic analysis showed that these phenotypes resulted from the presence of two new RHCE alleles, ceMO and cEMI. The ceMO allele corresponded to a RHce gene carrying a G667T mutation (exon 5) and was detected at the homozygous state in sample 1 and at the heterozygous state in samples 2-6. The G667T mutation resulted in a Val223Phe substitution on the Rhce polypeptide, in close proximity to Ala226 (e-antigen polymorphism), which might account for the altered expression of e. The ceMO allele is also associated with the lack of expression of the hrS antigen. The absence of the hrS antigen expression may have implications in transfusion as hrS-negative individuals may develop clinically significant antibodies. The cEMI allele corresponded to a silent RHE allele carrying a nine nucleotide deletion within exon 3 and was detected at the heterozygous state in sample 2. This deletion resulted in a shortened polypeptide of 414 residues (instead of 417) that was absent (or severely reduced) at the red cell surface, as the E antigen was undetectable using serology and Western blot analysis with anti-E reagents. In DNA-based polymerase chain reaction genotyping for RHE determination, the cEMI allele provided a false positive result as the cells carrying this allele are serologically phenotyped as E-negative. The incidence of this allele in the Black population is unknown but, as shown already for D genotyping, one must exercise caution when genotyping is performed to detect the e/E polymorphism.

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.

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.

Heterogeneity of blood group RhE variants revealed by serological analysis and molecular alteration of the RHCE gene and transcript.

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.

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification.

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.

A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblot.

We describe the first human monoclonal anti-D (LOR-15C9) which reacts with a D-specific motif exposed either on a native form on intact D-positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR-15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription-translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR-15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314-358). These findings correlate well with the reactivity of LOR-15C9 with erythrocytes of some nonhuman primates (D(gor)-positive gorillas), but not of chimpanzee and Old or New World monkeys. In membrane proteins from partial D(VI) red cells, LOR-15C9 detected two proteins of molecular weight 33 and 21 kD: the presence of the latter was specific for category D(VI) and presumably represented the product of an alternatively spliced RHD(VI) transcript in these cells. This is consistent with the finding that LOR-15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription-translation and lacking amino-acids 163-313 encoded by exons 4-6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D--, using a rabbit anti-Rh polypeptide antibody (MPC8) raised against the C-terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript. This study demonstrates that LOR-15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.

Characterization of the recombination hot spot involved in the genomic rearrangement leading to the hybrid D-CE-D gene in the D(VI) phenotype.

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.

Molecular analysis of blood group Rh transcripts from a rGr variant.

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.

Tentative model for the mapping of D epitopes on the RhD polypeptide.

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.

Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities.

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.

PCR-based determination of Rhc and RhE status of fetuses at risk of Rhc and RhE haemolytic disease.

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.

Rearrangements of the blood group RhD gene associated with the DVI category phenotype.

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.

Molecular characterization of the Rh-like locus and gene transcripts from the rhesus monkey (Macaca mulatta).

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.

Molecular genetic basis of the human Rhesus blood group system.

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.