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At least 19 recordsLinked to original sources
PubMed2000

A novel St(a) glycophorin produced via gene conversion of pseudoexon III from glycophorin E to glycophorin A gene.

Stone (St(a)) is a variant antigen carried on human erythrocyte MNSs glycophorins (GPSt(a)) that are genetically associated with splicing mutations in GPA genes or with hybrid formation between GPA and GPB genes. Here we identify the first and rare gene conversion event in which GPE, the third member of the family, recombined with GPA, giving rise to a GPA-E-A hybrid gene encoding the St(a) antigen. Western blot detected expression in the proband of both GPA and GPSt(a) on the plasma membrane. Southern blot showed a new restriction fragment from the GPSt(a) gene, indicating an altered exon III-intron 3 junction. Sequencing of RT-PCR products identified one full-length and two shortened glycophorin cDNAs. The shortened forms were derived from GPSt(a) lacking one (exon III) and two exons (exon III and IV), respectively. To define the molecular basis for exon skipping, the genomic region spanning exon III of the GPSt(a) gene was amplified and sequenced. This revealed transfer from GPE to GPA of a DNA segment containing the pseudoexon III and its silent donor splice site. Thus, the inactivation of GPA exon III by conversion of a silent GPE donor splice site portrays a new molecular mechanism for St(a) antigen expression in human erythrocytes.

Alleles↗
PubMed1989

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.

Blotting, Southern↗
PubMed1993

A point mutation in the GYPC gene results in the expression of the blood group Ana antigen on glycophorin D but not on glycophorin C: further evidence that glycophorin D is a product of the GYPC gene.

Glycophorin C (GPC) and glycophorin D (GPD) are homologous sialoglycoproteins in the human red blood cell membrane. Both are thought to be encoded by the GPC gene (GYPC). We report that the rare blood group antigen, Ana, is expressed on GPD but not on GPC. cDNA was synthesized from total RNA obtained from two unrelated, heterozygous Ana+ blood donors and analyzed by the polymerase chain reaction using primers that spanned sequences encoded by the GYPC gene. The expected 412-bp fragment was generated, and sequencing of the amplified product showed a G-->T substitution at nucleotide 67 of the coding sequence, resulting in the substitution of alanine by serine at amino acid residue 23 of GPC and, presumably, residue 2 of GPD. To explain the expression of Ana on GPD but not on GPC, we postulate that the conformation of the amino acid residues at the N-terminal region of GPD determines the antigenic expression as this conformation would be different from that of the same sequence of amino acids occurring within GPC. Other possible reasons for antigen expression on a shorter protein product but not on the full-length protein product of the same gene are discussed. We extrapolate this reasoning to account for the expression of the common GE2 blood group antigen on GPD but not on GPC.

Amino Acid Sequence↗
PubMed1993

Glycophorin B and glycophorin E genes arose from the glycophorin A ancestral gene via two duplications during primate evolution.

Human glycophorin A, B, and E genes are homologous from the 5'-flanking region to 1 kilobase downstream from the exon encoding the transmembrane region. Analysis of human Alu sequences at the transition site from the homologous to nonhomologous region suggested that the GPA gene most closely resembles the ancestral gene, whereas GPB and GPE genes arose by homologous recombination within the Alu repetitive sequence, and acquired 3' sequences from an unrelated gene (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623; Kudo, S., and Fukuda, M. (1990) J. Biol. Chem. 265, 1102-1110). To understand glycophorin gene evolution in primate phylogeny, transmembrane and Alu regions of several primate genomes were amplified by the polymerase chain reaction and their sequences were analyzed. These studies revealed that the GPA gene was present in all primates studied, and the GPB gene was present in pygmy chimpanzee, chimpanzee, and gorilla, but absent from orangutan and gibbon. GPE gene was present in all species with a GPB gene, but was detected in only 7 out of 16 gorillas. The 24-base pair insertion sequence found in the transmembrane exon of the human GPE gene was shown to be derived from the ancestral GPB gene and was inserted into the ancestral GPE gene prior to gorilla divergence. The recombination site in the GPA gene was confirmed to be within an Alu repetitive sequence. We conclude that GPB and GPE genes arose from an ancestral GPA gene via two gene duplications occurring during primate evolution, prior to gorilla divergence.

Amino Acid Sequence↗
PubMed1989

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid.

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.

Amino Acid Sequence↗
PubMed1985

Glycophorin A on normal and leukemia cells detected by monoclonal antibodies, including a new monoclonal antibody reactive with glycophorins A and B.

A new hemagglutinating monoclonal antibody, MoAb31, detected glycophorins A and B in Western blots. Results with enzyme-modified erythrocytes indicated the MoAb31 determinants were sialic acid dependent, and resided on glycophorin A on the trypsin-resistant, ficin-sensitive segment, and on glycophorin B on the ficin-sensitive segment. Another new monoclonal antibody, MoAb36, detected the Wrb antigen, located on the non-glycosylated segment of glycophorin A near its insertion into the lipid bilayer. Immunofluorescent staining of normal hematopoietic and leukemia cells with these and other monoclonal antibodies to glycophorin A demonstrated glycophorin A on erythroid cells only. Cytofluorograph analysis showed the majority of cells of the erythroleukemia cell lines K562 and HEL expressed glycophorin A, as indicated by reactivity with the monoclonal glycophorin A antibodies R10, R18, 6A7 and 10F7. However, reactivity with monoclonal antibodies to glycosylated determinants (MoAb31 and R1.3) and to the non-glycosylated segment near the membrane insertion (MoAb36, and R7.1) was reduced or absent. Expression of "missing" glycophorin A antigens on K562 and HEL could not be induced using a variety of chemical and biologically active modifiers. We conclude that glycophorin A of erythroleukemia cell lines K562 and HEL differs from glycophorin A at the surface of normal, mature erythrocytes with respect to reactivity with monoclonal glycophorin A antibodies.

Animals↗
PubMed1988

Alteration of the genes for glycophorin A and B in glycophorin-A-deficient individuals.

Glycophorins A and B are homologous glycoproteins of the red cell membrane which carry the blood-group MN and Ss antigens, respectively, and are encoded by two distinct genes closely linked on chromosome 4, which are probably derived from each other by duplication during evolution. The lack of glycophorin A is associated with the rare phenotype En(a-), indicating individuals who are defective for MN antigens, as well as for the Ena antigens, also located on this glycoprotein. The En(a-) condition is heterogenous and includes two categories of variants exemplified by the Finnish and the English types referred to as En(Fin) and En(UK), respectively. By Southern blot and preliminary genomic clone analyzes we have compared the status of the genes for glycophorins A and B, as well as that of the gene encoding glycophorin C, another unrelated red cell membrane glycoprotein, in the En(a-) variants and in the En(a+) control donors. Our data indicate that the En(Fin) variant is homozygous for a complete deletion of the glycophorin A gene without any detectable abnormality of the genes encoding glycophorins B or C. In the genome of the En(UK) variant, with the presumed genotype Mk/En(UK), and where the Mk condition abolishes the expression of MN and Ss antigens, we have identified several abnormalities of the glycophorin A and B genes, but the glycophorin C gene was unaffected. Our results strongly support the view that in Mk chromosome the glycophorin A and B genes are largely deleted, whereas the En(UK) chromosome probably contains a gene fusion product encoding a hybrid glycoprotein AM-B, composed of the N-terminal portion of a blood group M-type glycophorin A and of the C-terminal portion of glycophorin B. The determination of the 5' and 3' limits of the hybrid gene and elucidation of the mechanism involved will require sequencing of the rearranged DNA of the variant and a full knowledge of the organization of the glycophorin A and B genes.

Blotting, Southern↗
PubMed1988

A family study of multiple mutations of alpha and delta glycophorins (glycophorins A and B).

Glycophorins alpha and delta are the carriers of the antigens of the MNSs blood system; this report documents the presence of three glycophorin mutations in two individuals of a 16 member family. Erythrocytes were examined by serology, sodium dodecyl sulfate electrophoresis, and immunoblotting. The inheritance pattern and immunoblot profile revealed: (1) A variant Dantu glycophorin showed properties consistent with a delta-alpha glycophorin hybrid structure, previously noted in other individuals. The gene responsible for the Dantu glycophorin in this family is linked to a gene coding for an M-specific alpha glycophorin. (2) Another variant glycophorin, Mi-III glycophorin, was first revealed by immunoblotting and subsequently confirmed by erythrocyte antigen typing. This autosomal dominant trait is associated with N blood group activity and the inheritance pattern indicates that it could be a variant of delta glycophorin. (3) In the individuals with both Dantu and Mi-III glycophorins a delta glycophorin deficiency was observed suggesting that a deletion or alteration of delta gene may exist cis to the Dantu gene. Our findings that document clustering of multiple mutations in MNSs gene loci in the propositus family are very unusual as such variants are relatively rare.

Female↗
PubMed1987

Delta glycophorin (glycophorin B) gene deletion in two individuals homozygous for the S--s--U-- blood group phenotype.

Blood cells from two unrelated individuals whose erythrocytes exhibit respectively N S-s-U- and MN S-s-U- blood group phenotypes were examined by immunoblotting, periodic acid-Schiff (PAS) staining, and Southern blotting. Protein bands characteristic of delta glycophorin (glycophorin B) were absent from the immunoblots of whole erythrocyte lysates when probed with polyclonal glycophorin antisera and from isolated erythrocyte membranes stained with PAS reagents. Genomic DNA from the two individuals' leukocytes was digested with a panel of restriction enzymes and probed with alpha M glycophorin cDNA obtained from human K562 leukemic cell line. The EcoRI, PstI, and KpnI restriction site patterns were identical to those of S+s+U+ controls in fragment numbers and relative size but differed from controls in band intensities. Restriction mapping with HindIII, PvuII, SacI, MspI, and BamHI revealed that S-s-U- individuals lack fragments that are reproducibly observed in S+s+U+ controls, and most likely encode delta glycophorin. Using truncated 5' and 3' cDNA segments as probes and comparing, in control individuals, hybridization intensities of fragments with amino acid sequence homologies, we have inferred the assignment of restriction fragments to the alpha and delta glycophorin genes. Our results suggest that the absence of delta glycophorin in the two S-s-U- individuals is a result of deletion of the entire delta glycophorin gene. This is the first report of a glycophorin gene deletion.

Chromosome Deletion↗
PubMed1989

Amino acid sequence of monkey erythrocyte glycophorin MK. Its amino acid sequence has a striking homology with that of human glycophorin A.

A major sialoglycoprotein, glycophorin MK, was isolated from monkey erythrocyte membranes by extraction with lithium diiodosalicylate and partition in aqueous phenol. Chemical analysis of glycophorin MK revealed that the glycophorin consists of 51% protein and 49% carbohydrate by weight, and contains no N-glycosidic oligosaccharide units. Only N-glycolylneuraminic acid (Neu5Gc) was detected as sialic acid. The amino acid sequence of glycophorin MK was determined, which demonstrated that the glycophorin consists of 144 amino acid residues and 18 oligosaccharide units linked O-glycosidically to the peptide backbone through serine or threonine residues. The molecular weight was estimated to be about 35,000 based on the amino acid residues and carbohydrate content. By comparison of the amino acid sequence with those of human, equine and porcine glycophorins, a striking sequence homology was observed between monkey and human glycophorin. Glycophorin MK demonstrated both M and N blood group activities.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A.

Here we describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector lambda gt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH2-terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. We used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. We established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes.

Amino Acid Sequence↗
PubMed1985

The influence of lipid composition and lectin-glycophorin interaction on the rotational diffusion of glycophorin in vesicles, as measured by time-resolved phosphorescence depolarization.

The rotational mobility of glycophorin in various lipid vesicles was studied, using time-resolved measurements of the depolarization of laser flash excited phosphorescence of glycophorin labelled with the triplet probe erythrosin. With the exception of dimyristoylphosphatidylcholine at the phase transition no phosphorescence depolarization decays were observed in the 1-300 microseconds time interval following the laser flash. Instead, a constant anisotropy level was observed, with two distinct values depending on the experimental system. In liquid-crystalline bilayers of dioleoylphosphatidylcholine, bovine brain phosphatidylserine and dimyristolyphosphatidylcholine, the anisotropy was 0.01. This was increased to 0.03 upon addition of wheat germ agglutinin which aggregates glycophorin. In the case of gel state dimyristoylphosphatidylcholine and liquid-crystalline dioleoylphosphatidylethanolamine the anisotropy also amounted to 0.03. Experiments with glycerol to vary the viscosity of the medium, and theoretical considerations, exclude the possibility that these different anisotropy levels are related to differences in motional properties of the entire protein/lipid vesicles. These results strongly suggest that the anisotropy level of 0.03 corresponds to slowly rotating glycophorin (rotational relaxation time greater than 0.3 ms) while the anisotropy level of 0.01 corresponds to fast rotating glycophorin (rotational correlation time less than 1 microseconds). The difference in glycophorin mobility is discussed in terms of aggregation state of the protein, lipid composition of the vesicle bilayer and membrane viscosity. The observed differences in rotational mobility of glycophorin in glycophorin/dioleoylphosphatidylcholine vesicles, glycophorin/bovine heart phosphatidylserine vesicles as compared to glycophorin/dioleoylphosphatidylethanolamine vesicles are not in quantitative agreement with the relative size of the intramembrane particles in these systems as revealed by freeze-fracture electron microscopy.

Animals↗
PubMed1991

Presence of influenza virus-reactive glycophorins other than glycophorin A in human erythrocyte membranes.

The reactivities against seven hemagglutinins including influenza A and B viruses of six sialoglycoprotein (glycophorin) fractions, M-Frs.1-3 and N-Frs.1-3, separated from human OMs and ONs erythrocyte membranes by a combination of the LIS-phenol method and gel filtration were studied. The serological results show that M-Fr.1 and N-Fr.1 were glycophorins AM and AN, respectively, the reactivities against influenza A and B viruses of both M-Fr.2 and N-Fr.2 which contained glycophorins B and C were remarkably higher than that of glycophorin A and the reactivities against influenza viruses of both M-Fr.3 and N-Fr.3 which contained glycophorins B and D were considerably lower than that of glycophorin A.

Amino Acids↗
PubMed1993

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots. Identification of glycophorin and band 3 variant forms.

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with 125I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1994

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.

Animals↗
PubMed1988

Characterization of cDNA clones for human glycophorin A. Use for gene localization and for analysis of normal of glycophorin-A-deficient (Finnish type) genomic DNA.

Glycophorin A is the major membrane sialoglycoprotein of human erythrocytes and represents a typical example of a transmembrane glycoprotein. The functional role of this cell-surface component is not known but it represents a receptor for viruses, bacteria and parasites like Plasmodium falciparum. 1. Two cDNA clones encoding glycophorin A have been characterized from human fetal cDNA libraries. The longer cDNA extended from the coding region of glycophorin A (residues 4-131) to the 3' untranslated region which included two polyadenylation signals and a poly(A) tail. 2. The structural gene for glycophorin A is located on chromosome 4, q28-q31 as shown by in situ hybridization, thus confirming the previous localization by genetic linkage analysis. 3. Three distinct mRNA species (1.0 kb, 1.7 kb and 2.2 kb) have been identified in erythroid spleen. Northern blot analyses with a probe directed against the 3' untranslated region of the mRNAs indicated that all these species share a homologous 3' non-coding region and that the first polyadenylation signal downstream the stop codon is not used. 4. Preliminary studies by Southern blot analysis of the genomic DNA from normal En(a+) and rare En(a-) donors suggest that the glycophorin A gene has a complex organization and is largely deleted in donors of the En(a-) phenotype (Finnish type) who lack glycophorin A on their red cells.

Base Sequence↗
PubMed1985

Erythrocyte membrane rigidity induced by glycophorin A-ligand interaction. Evidence for a ligand-induced association between glycophorin A and skeletal proteins.

Erythrocyte skeletal proteins are known to play an important role in determining membrane deformability. In order to see whether transmembrane proteins also influence deformability and, if so, whether this influence is mediated by an interaction with the membrane skeleton, we examined the effect on deformability of ligands specific for transmembrane proteins. We found membrane deformability markedly reduced in erythrocytes that were pretreated with glycophorin A-specific ligands. In contrast, ligands specific for band 3 and A and B blood group antigens had no effect. The increase in membrane rigidity appeared to depend upon a transmembrane event and not upon a rigidity-inducing lattice on the outside surface of the cell in that a monovalent Fab of antiglycophorin IgG caused decreased deformability. We therefore looked for a ligand-induced association of glycophorin and the skeletal proteins and found, in Triton X-100-insoluble residues, a partitioning of glycophorin with the skeletal proteins only after preincubation with a ligand specific for glycophorin. We then studied cells and resealed membranes with skeletal protein abnormalities. In spectrin-deficient and protein 4.1-deficient erythrocytes and in 2,3-diphosphoglycerate-treated resealed membranes, the antiglycophorin IgG was only one-third as effective in decreasing deformability as it was in normal cells. Thus, normal skeletal proteins appear to be essential for liganded glycophorin to affect membrane deformability maximally. Taken together, these observations indicate that there is a ligand-induced interaction between glycophorin A and skeletal proteins and that this interaction can directly influence membrane deformability.

Antigen-Antibody Complex↗