Rh50 Glycoprotein Research Articles

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca(2+)-permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the alpha1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro site-directed mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca(2+) accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca(2+) accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.

Sequence, organization, and evolution of Rh50 glycoprotein genes in nonhuman primates

Sequence, organization, and evolution of Rh50 glycoprotein genes in nonhuman primates

Molecular biology and genetics of the Rh blood group system

The Rh (Rhesus) blood group system is the most complex of the known human blood group polymorphisms. The expression of its antigens is controlled by a two-component genetic system consisting of RH and RHAG loci, which encode Rh30 polypeptides and Rh50 glycoprotein, respectively. Over the past decade, there has been a rapid advance in knowledge of the biochemistry, molecular biology, and genetics of the Rh genes and proteins. The primary structures of D and CcEe antigens have become well understood and the molecular genetic basis of a vast array of phenotype polymorphisms has been delineated. The identification of various molecular defects associated with Rh deficiency syndrome clarifies the nature of the amorph, suppressor, and modifier genes. The observed mutation spectrum defines a basic set of components essential for Rh complex assembly in the erythrocyte membrane. The resulting molecular information, combined with new experimental tools, is helping to dissect the fine structure of Rh antigens in terms of epitope mapping. The discovery of novel Rh homologs in primitive organisms and in nonerythroid tissues opens new avenues of research beyond the scope of erythrocytes and Rh antigens. This review provides an update on the Rh family in antigen expression, phenotype diversity, and disease association.

Time-course expression of polypeptides carrying blood group antigens during human erythroid differentiation.

The time course expression of blood group antigens was examined by flow cytometry using a two-phase liquid culture system that supports the proliferation and maturation of human erythroid progenitors from adult peripheral blood. The progression towards erythroid differentiation was followed by the expression changes of the transferrin receptor (CD71++) and glycophorin A (GPA+). Four main categories of blood group markers were identified: (i) those characterized by an early expression like ABO (A), Kell (K:2) and Rh50 which were detected in the Epo-independent phase 1, (ii) those including GPC (Gerbich, Ge antigens) and Fy6 which were expressed in the late phase 1, (iii) GPA (MN antigens), Wrb (Band 3/GPA interaction), Rh(D, Cc/Ee) and LW which appeared during the Epo-dependent phase 2 and (iv) those like Jk3 and Lub which were expressed in late phase 2. Regarding blood group molecules exhibiting adhesive properties (LW/ICAM-4, Oka and Lu) the most significant event was a sharp decrease of Oka (neurothelin) expression with the concomitant loss of ICAMs expression during the later stage of differentiation. These studies suggest that Oka, ICAMs and LW might contribute to the adhesive interactions involved in the formation of erythroblastic islands and attachment to stroma cells and the extracellular matrix. We also noted an asynchronous expression of the proteins that compose the core of the Rh complex, since Rh50 glycoprotein was expressed earlier than Rh(D, CE) proteins.

Molecular basis for Rh(null) syndrome: identification of three new missense mutations in the Rh50 glycoprotein gene.

Rh(null) is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rh(null) cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rh(null)(HT), RHAG harbored in exon 6 two G-->A transitions, GTT-->ATT and GGA-->AGA, which cause Val(270)-->Ile and Gly(280)-->Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rh(null)(WO), RHAG contained in exon 9 a single G-->T transversion, GGT-->GTT, which caused a Gly(380)-->Val missense change in TM12 segment. The G-->T transversion, which is located at the +1 position of exon 9, had also affected pre-mRNA splicing and caused partial exon skipping. Although both Rh(null) cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein.

Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene

Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene

Expression of the Rh-Related Glycoprotein (Rh50)

The Rh50 glycoprotein is suspected of being involved in Rh antigen expression. We prepared Rh50 cDNA from a human bone marrow library by polymerase chain reaction and then subcloned this cDNA into various vectors. The vector containing Rh50 cDNA produced a 30-kDa nonglycosylated form of Rh50 in a rabbit reticulocyte lysate system and produced partially glycosylated Rh50 (32 kDa) when microsomes were added to the system. COS-1 cells transiently transfected with the vector containing Rh50 cDNA produced partially glycosylated Rh50 (32 kDa) recognized by a Rh50-specific antibody. Surface expression of Rh50 in K562 cells was also detected by flow cytometry using mouse monoclonal antibody (2D10) specific to Rh50. Partially glycosylated Rh50 (32 kDa) was again isolated from the lysates of K562 cells metabolically labeled with [³⁵S]-methionine or [³H]-mannose using anti-Rh50 antisera. These systems (K562 and COS-1 cells) should prove useful for studying the transport of Rh proteins within the cell and the necessary components needed for Rh antigenicity at the cell surface.

Rh mod Syndrome: A Family Study of the Translation-Initiator Mutation in the Rh50 Glycoprotein Gene

Rhmod syndrome is a rare genetic disorder thought to result from mutations at a "modifier" but not at the suppressor underlying the regulator type of Rhnull disease. We studied this disorder in a Jewish family with a consanguineous background and analyzed RH and RHAG, the two loci that control Rh-antigen expression and Rh-complex assembly. Despite the presence of a d (D-negative) haplotype, no other gross alteration was found at RH, and cDNA sequencing showed a normal structure for D, Ce, and ce Rh transcripts in family members. However, analysis of RHAG transcript, which encodes Rh50 glycoprotein, identified a single G-->T transversion in the initiation codon, causing a missense amino acid change (ATG[Met]-->ATT[Ile]). This point mutation also occurred in the genomic region spanning exon 1 of RHAG, and its genotypic status in the mother and two children was confirmed by analysis of single-strand conformation polymorphism. Although blood typing showed a very weak expression of Rh antigens, immunoblotting barely detected the Rh proteins in the Rhmod membrane. In vitro transcription-coupled translation assays showed that the initiator mutants of Rhmod-but not those of the wild type-could be translated from ATG codons downstream. Our findings point to incomplete penetrance of the Rhmod mutation, in the form of "leaky" translation, leading to some posttranslational defects affecting the structure, interaction, and processing of Rh50 glycoprotein.

The human Rh50 glycoprotein gene—Structural organization and associated splicing defect resulting in Rh null disease : Huang C-H. J Biol Chem 273:2207–2213, 1998

The human Rh50 glycoprotein gene—Structural organization and associated splicing defect resulting in Rh null disease : Huang C-H. J Biol Chem 273:2207–2213, 1998

Rh50 Glycoprotein Gene and Rhnull Disease: A Silent Splice Donor Is trans to a Gly279→Glu Missense Mutation in the Conserved Transmembrane Segment

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at theRH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G→A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy intrans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical fromCaenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.© 1998 by The American Society of Hematology.

Rh50 Glycoprotein Gene and Rhnull Disease: A Silent Splice Donor Is trans to a Gly279→Glu Missense Mutation in the Conserved Transmembrane Segment

Rhnull disease includes the amorph and regulator types that are thought to result from homozygous mutations at theRH30 and RH50 loci, respectively. Here we report an unusual regulator Rhnull where two G→A nucleotide (nt) transitions occurred in trans, targeting different regions of the two copies of Rh50 gene. The nt 836 G→A mutation was a missense change located in exon 6; it converted Gly into Glu at position 279, a central amino acid of the transmembrane segment 9 (TM9). While cDNA analysis showed expression of the 836A(Glu279) allele only, genomic studies showed the presence of both 836A(Glu279) and 836G(Gly279) alleles. A detailed analysis of gene organization led to the identification in the Rh50(836G) allele of a defective donor splice site, caused by a G→A mutation in the invariant GT element of intron 1. This is the first known example of such mutations that has apparently abolished the functional splicing of a pre-mRNA encoding a multipass integral membrane protein. With a silent phenotypic copy intrans, the negatively charged Glu279 residue may disrupt TM9 and impair the interaction of the missense protein with Rh30 polypeptides. To evaluate the significance of the mutation, we took a comparative genomic approach and identified Rh50 homologues in different species. We found that Gly279 is a conserved residue and its adjacent amino acid sequence is identical fromCaenorhabditis elegans to human. These findings provide new insight into the diversity of Rhnull disease and suggest that the C-terminal region of Rh50 may also participate in protein-protein interactions involving Rh complex formation.© 1998 by The American Society of Hematology.

A splicing mutation of the RHAG gene associated with the Rhnull phenotype.

Rhnull is a syndrome serologically characterized by the deficiency of all Rh antigens on human red blood cells. Rhnull is divided into two types: regulator and amorph. Recently, Cherif-Zahar et al. proposed that the RHAG gene encoding the Rh50 glycoprotein is a candidate for inducing regulator type Rhnull. We investigated both the RH and RHAG genes in an Rhnull individual. The reticulocytes from the propositus had RHD, RHcE, and RHCe transcripts without any mutation. However, the sequence analysis of RHAG cDNA showed a deletion of 122 bp from nucleotide 946 to 1067. This deletion was revealed to be due to a homozygous splicing mutation, which is a single base substitution at the consensus sequence of the splicing acceptor site (AG-->AT). The mutation appeared to break the 'GT-AG' splicing rule and to cause 122 bp exon skipping accompanied by a frameshift. This study confirms that the RHAG gene is the most likely candidate for the 'regulator' gene of Rhnull cases.

Identification of 5′ Flanking Sequence ofRH50Gene and the Core Region for Erythroid-Specific Expression

The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes,RHCEandRHD.Rh50 glycoprotein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression ofRH50gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5′ flanking region ofRH50gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence ofRHgenes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5′ flanking sequence. The core promoter ofRH50gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Krüppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms forRHvsRH50genes.

The Human Rh50 Glycoprotein Gene: STRUCTURAL ORGANIZATION AND ASSOCIATED SPLICING DEFECT RESULTING IN Rhnull DISEASE

The Rh (Rhesus) protein family comprises Rh50 glycoprotein and Rh30 polypeptides, which form a complex essential for Rh antigen expression and erythrocyte membrane integrity. This article describes the structural organization of Rh50 gene and identification of its associated splicing defect causing Rhnull disease. The Rh50 gene, which maps at chromosome 6p11-21.1, has an exon/intron structure nearly identical to Rh30 genes, which map at 1p34-36. Of the 10 exons assigned, conservation of size and sequence is confined mainly to the region from exons 2 to 9, suggesting that RH50 and RH30 were formed as two separate genetic loci from a common ancestor via a transchromosomal insertion event. The available information on the structure of RH50 facilitated search for candidate mutations underlying the Rh deficiency syndrome, an autosomal recessive disorder characterized by mild to moderate chronic hemolytic anemia and spherostomatocytosis. In one patient with the Rhnull disease of regulator type, a shortened Rh50 transcript lacking the sequence of exon 7 was detected, while no abnormality was found in transcripts encoding Rh30 polypeptides and Rh-related CD47 glycoprotein. Amplification and sequencing of the genomic region spanning exon 7 revealed a G-->A transition in the invariant GT motif of the donor splice site in both Rh50 alleles. This splicing mutation caused not only a total skipping of exon 7 but also a frameshift and premature chain termination. Thus, the deduced translation product contained 351 instead of 409 amino acids, with an entirely different C-terminal sequence following Thr315. These results identify the donor splicing defect, for the first time, as a loss-of-function mutation at the RH50 locus and pinpoint the importance of the C-terminal region of Rh50 in Rh complex formation via protein-protein interactions.

Molecular cloning and primary structure of a Rhesus (Rh)-like protein from the marine sponge Geodia cydonium.

In humans, the 30,000 M(r) Rhesus (Rh) polypeptide D (RhD) is a dominant antigen (Ag) of the Rh blood group system. To date, an Rh-like protein has been found in chimpanzees, gorillas, gibbons, and rhesus monkeys. Related to the 30,000 M(r) Rh Ag protein are two polypeptides of 50,000 M(r), the human 50,000 M(r) Rh Ag and the RhD-like protein from Caenorhabditis elegans. The function of all these proteins is not sufficiently known. Here we characterize a cDNA clone (GCRH) encoding a putative 57,000 M(r) polypeptide from the marine sponge Geodia cydonium, which shares sequence similarity both to the RhD Ag and the Rh50 glycoprotein. The sponge Rh-like protein comprises 523 aa residues; hydropathy analysis hints at the presence of ten transmembrane domains. An N-terminal hydrophobic cleavage signal sequence is missing, suggesting that the first membrane-spanning domain of the sponge Rh-like protein acts as a signal-anchor sequence. The sponge Rh-like protein, like the human Rh50, lacks the CLP motif which is characteristic of the 30,000 M(r) RhD. In addition, the hydropathy profile of the sponge Rh-like protein is of a similar size and shape as that of human Rh50. This data indicates that the RhD and its structurally related Rh50 glycoprotein, which are highly immunogenic in humans, share a common ancestral molecule with the G. cydonium Rh-like protein.

Molecular insights into the Rh protein family and associated antigens.

The Rh (Rhesus) protein family is expressed mainly in cells of erythroid lineage and, currently, is thought to comprise Rh30 (RhD and RhCE) polypeptides and Rh50 glycoprotein. As multipass integral membrane proteins, Rh30 and Rh50 are closely related by clear sequence homology and similar membrane topology, functioning respectively as the carrier and coexpressor of the Rh blood group antigens. The past year has seen a further accumulation of evidence concerning the molecular basis of Rh antigen expression, a major expansion of the catalogue of Rh allelic polymorphism, and significant progress in defining the genetic defects underlying the Rh deficiency syndrome. Now the gene structure for many Rh variants has been determined and some information obtained on the mechanisms of Rh genetic diversity and on the factors affecting the formation and surface presentation of discrete antigenic epitopes. The identification of various mutations in the Rh50 gene associated with the Rhnull and Rhmod phenotypes establishes RH50 as a suppressor for the RH locus and Rh50 protein as a critical component of the Rh complex. These new advances have broadened our understanding of the molecular biology of the Rh protein family and provided insight into the functional role of the Rh complex in maintaining the architecture of the erythrocyte membrane.

Defining the Rh blood group antigens: Biochemistry and molecular genetics

The Rh blood group antigens (D, Cc and Ee series) are carried by a family of non glycosylated hydrophobic transmembrane proteins of 30–32 kDa which are missing from the red cells of rare Rh null individuals that express several membrane defects. The structure of these proteins has been deduced from cDNA cloning and studies have shown that the Rh proteins are erythroid specific and share no sequence homology with any known protein. The RhD and non-D proteins exhibit 92% sequence identity and their predicted membrane topology is similar as most of the molecules appear to reside between the leaflets of the phospholipid bilayer with only short hydrophilic loops connecting the twelve putative transmembrane helices. The RHD and RHCE genes encoding the Rh proteins (D and Cc/Ee, respectively) are organized in tandem on chromosome 1p34-p36 and most likely derived by duplication of a common ancestral gene. This concept is supported by the identification of RH-like genes in non human primates. The human RH locus is best described as a two-gene model in which all RhD-positive and most RhD-negative haplotypes are composed of two ( RHD and RHCE) or only one ( RHCE) structural genes, respectively. The RHD gene encodes the D protein and the RHCE gene encodes the C/c and E/e proteins presumably by alternative splicing of a pre messenger RNA. The correlation between the blood group D epitopes and the amino acid polymorphism of the Rh proteins is not yet established, but amino acid polymorphisms at positions 103 and 226 determine the molecular basis for the C/c (Ser → Pro) and E/e (Pro → Ala) specificities, respectively. Most variants analyzed so far are caused by gene conversion which appears as the principal mechanism responsible for polymorphism and gene diversity in the RH system. However, gene deletions have also been found in some occasions. To date, all Rh null phenotypes investigated most likely result from transcriptional regulatory mechanisms that are not yet understood. Rh null individuals suffer a clinical syndrome of varying severity and their red cells are characterized by morphological and functional abnormalities of cation transport and phospholipid asymmetry. In addition, several membrane components including the Rh proteins and other glycoproteins recently characterized (Rh50 glycoprotein, CD47, glycophorin B, Duffy, LW) are absent or severely decreased on these cells. These findings suggest that the Rh proteins are assembled into a multimeric complex with these glycoproteins and further studies should clarify the role in biosynthesis and the potential function of each component in this complex.

4 Biochemical aspects of the blood group Rh (Rhesus) antigens

Despite their importance in clinical haematology, the details of the structures and possible functions of the proteins associated with Rh antigen expression have only recently begun to emerge. The antigens are carried by a multimeric complex between a M(r) 30,000 polypeptide which is not glycosylated (the Rh30 polypeptide), and a heavily glycosylated glycoprotein (the Rh50 glycoprotein). The N-terminal amino acid sequences of the two types of proteins were determined and used to isolated cDNA clones. The Rh30 and Rh50 proteins are both very hydrophobic membrane proteins, each containing up to 12 membrane spans. The two proteins are homologous in sequence and clearly belong to the same family. They are erythroid-specific and not related to any other known family of proteins. The Rh30 polypeptides are the genetic determinants of Rh blood group antigen activity. One polypeptide (Rh30A) is probably associated with CcEe antigen activity, while another (Rh30B) is responsible for the D antigen. The proteins have structures typical of transporters but their functions are still unclear. A number of other red cell membrane proteins (LW, CD47, glycophorin B and Fy) show alterations in red cells lacking Rh antigens (Rhnull). These proteins may have a role in the biosynthesis or function of the Rh30 and Rh50 proteins.