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Biomedical subjects

M J Tanner

Publications and source records attributed to M J Tanner.

At least 73 records · Page 4Linked to original sources
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Hereditary ovalocytosis with compensated haemolysis.

The clinical and laboratory phenotype of compensated haemolysis in a patient with hereditary ovalocytosis is reported. Clinical presentation was intermittent jaundice and abdominal pain due to pigment gall stones. Haematological analysis revealed an absolute reticulocytosis with an otherwise normal full blood count and biochemical evidence of haemolysis. Variable results were observed with blood grouping reagents. The patient's red cells were stomatocytic ovalocytic, rigid, resistant to malarial parasite invasion, defective in anion transport, and had the characteristic two linked mutations in the red cell band 3 gene.

Adult↗
PubMed1992

A red cell band 3 variant with altered stilbene disulphonate binding is associated with the Diego (Dia) blood group antigen.

1. We have shown that the Dia antigen of the Diego blood group system is associated with the presence of red cell band 3 Memphis, but not all band 3 Memphis samples carry the Dia antigen. 2. The band 3 Memphis associated with the Dia antigen was covalently labelled by 4,4'-di-isothiocyanato-1,2-diphenylethane-2,2'-disulphonic acid (H2DIDS) more readily than was normal band 3 or band 3 Memphis not associated with the Dia antigen. This altered reactivity with H2DIDS has previously been noted for a band 3 Memphis sub-type designated variant 2. 3. This is the first example of a band 3 polymorphism associated with an antigenic change in the extracellular region of the band 3 polypeptide and with altered H2DIDS binding.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.

Animals↗
PubMed1992

Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (rhesus) blood-group antigen expression.

The Rh blood-group antigens are associated with human erythrocyte membrane proteins of approx. 30 kDa (the Rh30 polypeptides). Heterogeneously glycosylated membrane proteins of 50 and 45 kDa (the Rh50 glycoproteins) are coprecipitated with the Rh30 polypeptides on immunoprecipitation with anti-Rh-specific mono- and poly-clonal antibodies. We have isolated cDNA clones representing a member of the Rh50 glycoprotein family (the Rh50A glycoprotein). We used PCR with degenerate primers based on the N-terminal amino acid sequence of the Rh50 glycoproteins and human genomic DNA as a template and cloned and sequenced three types of PCR product of the expected size. Two of these products, Rh50A and Rh50B, gave the same translated amino acid sequence which corresponded to the expected Rh50 glycoprotein sequence but had only 75% DNA sequence similarity. The third product (Rh50C) contained a single base deletion, and the translated amino acid sequence contained an in-frame stop codon. We have isolated cDNA clones containing the full coding sequence of the Rh50A glycoprotein. This sequence predicts that it is a 409-amino acid N-glycosylated membrane protein with up to 12 transmembrane domains. The Rh50A glycoprotein shows clear similarity to the Rh30A protein in both amino acid sequence and predicted topology. Our results are consistent with the Rh30 and Rh50 groups of proteins being different subunits of an oligomeric complex which is likely to have a transport or channel function in the erythrocyte membrane. We mapped the Rh50A gene to human chromosome 6p21-qter, showing that genetic differences in the Rh30 rather than the Rh50 genes specify the major polymorphic forms of the Rh antigens.

Amino Acid Sequence↗
PubMed1992

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.

Amino Acid Sequence↗
PubMed1992

Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells.

Hereditary ovalocytosis is common in some areas of Melanesia and South East Asia where malaria is endemic. These red cells resist invasion by malarial parasites in vitro and ovalocytic individuals are less parasitized than normal. This has been attributed to the greater rigidity of ovalocytic red cells. It has been suggested that South East Asian ovalocytosis results from the heterozygous presence of an altered membrane anion transporter (band 3). We have used the polymerase chain reaction to clone the abnormal band 3 complementary DNA from an ovalocytic of Indian origin and found two changes from the normal protein: a point mutation (Lys 56----Glu) and the deletion of the sequence AFSPQVLAA (residues 400-408), but no evidence for an N-terminal extension. The deletion is also found in the abnormal band 3 of South East Asian ovalocytes and seems to be responsible for the unusual properties of the ovalocytic red cell. We show here that the membrane domain of the abnormal ovalocyte band 3 has a substantially altered structure and that the protein is defective in anion transport activity. The changed transport properties of the red cells may have a role in the reduced parasitaemia of ovalocytic individuals.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.

Adult↗
PubMed1991

Biochemical studies on red blood cells from a patient with the Inab phenotype (decay-accelerating factor deficiency).

A 38-year-old Russian woman (KZ) has been identified as the fourth proposita with the Inab blood group phenotype. Like the first two propositi, she has a chronic intestinal disorder and, as shown for the third proposita, her Inab phenotype is demonstrably inherited. KZ's serum contained anti-IFC, which reacted with a red blood cell (RBC) membrane component with an Mr of 70,000, which is decay accelerating factor (DAF). Her RBCs lacked all Cromer-related blood group antigens and DAF. Her RBCs were no more susceptible than normal control RBCs to lysis in acid lysis or in rabbit or human antibody-initiated complement lysis tests. Northern blots of total RNA isolated from KZ's Epstein-Barr virus-transformed lymphoblasts showed a marked reduction of DAF mRNA when compared with normal. Polymerase chain reaction (PCR) amplification of cDNA confirmed this reduced level of DAF mRNA. Sequencing of the PCR product showed a 44-nucleotide deletion in the mRNA close to the short consensus repeats IIIa/IIIb intron/exon boundary. This deletion results in a change in the reading frame that places a termination codon six amino acids after the deletion. The putative translation product would lack a glycosyl phosphatidyl-inositol linkage site and, therefore, would not be membrane-bound in the RBC.

Adult↗
PubMed1991

The abundance and organization of polypeptides associated with antigens of the Rh blood group system.

Twelve murine monoclonal antibodies, which react with human red cells of common Rh phenotype but give weak or negative reactions with Rh null erythrocytes, were used in quantitative binding assays and competitive binding assays to investigate the abundance and organization of polypeptides involved in the expression of antigens of the Rh blood group system. Antibodies of the R6A-type (R6A, BRIC-69, BRIC-207) and the 2D10-type (MB-2D10, LA18.18, LA23.40) recognize related structures and 100,000-200,000 molecules of each antibody bind maximally to erythrocytes of common Rh phenotype. Antibodies of the BRIC-125 type (BRICs 32, 122, 125, 126, 168, 211) recognize structures that are unrelated to those recognized by R6A-type and 2D10-type antibodies and between 10,000 and 50,000 antibody molecules bind maximally to erythrocytes of the common Rh phenotype. The binding of antibodies of the R6A-type and the 2D10-type, but not of antibodies of the BRIC-125-type could be partially inhibited by human anti-D antibodies (polyclonal and monoclonal) and a murine anti-e-like antibody. These results are consistent with evidence (Moore & Green 1987; Avent et al., 1988b) that the Rh blood group antigens are associated with a complex that comprises two groups of related polypeptides of M(r) 30,000 and M(r) 35,000-100,000, respectively, and suggest that there are 1-2 x 10(5) copies of this complex per erythrocyte. The polypeptide recognized by antibodies of the BRIC-125 type is likely to be associated with this complex.

Animals↗
PubMed1990

The membrane domain of the human erythrocyte anion transport protein. Epitope mapping of a monoclonal antibody defines the location of a cytoplasmic loop near the C-terminus of the protein.

We have used synthetic peptides to study the location of the amino acid sequences in the human erythrocyte anion transport protein (band 3) which are recognized by four murine monoclonal antibodies, BRIC 130, 132, 154 and 155. These antibodies are known to react with epitopes in the protein which are on the cytoplasmic side of the membrane. The results suggest that the amino acid residues important for the reaction of BRIC 130 and BRIC 154/155 are located within amino acids 899-908 and 895-901 respectively in the cytoplasmic tail of the protein. The BRIC 132 epitope is located within amino acid residues 813-824. This is part of a surface loop in the protein which probably extends from residue 814 to residue 832 and is located on the cytoplasmic side of the membrane. These results provide direct evidence for the topographical location of a sequence in a poorly understood region of the protein.

Amino Acid Sequence↗
PubMed1990

cDNA cloning of a 30 kDa erythrocyte membrane protein associated with Rh (Rhesus)-blood-group-antigen expression.

The Rh-blood-group antigens (often described as Rhesus antigens) are associated with erythrocyte membrane proteins of approx. 30 kDa. We have determined the N-terminal 54 amino acid residues of the 30 kDa Rh D polypeptide (D30 polypeptide). We used primers based on these sequence data and the polymerase chain reaction (PCR) on human reticulocyte cDNA and genomic DNA to clone two types of PCR product of identical size. The two PCR products had related translated amino acid sequences between the 3' ends of the primers, one of which was identical with that found for the D30 polypeptide. We designate the two related mRNA species which gave rise to the PCR products as Rh30A and Rh30B, the latter corresponding to the D30 polypeptide. We have isolated cDNA clones for the Rh30A protein which encode a hydrophobic membrane protein of 417 amino acids. The Rh30A protein has the same N-terminal 41 amino acids as the D30 polypeptide, but beyond this point the sequence differs, but is clearly related. The Rh30A protein probably corresponds to the R6A32 polypeptide, another member of the Rh 30 kDa family of proteins, which may carry the C/c and/or E/e antigens. Hydropathy analysis suggests that the Rh30A protein has up to 12 transmembrane domains. Three of these domains are bordered by a novel cysteine-containing motif, which might signal substitutions at these cysteine residues. Information which supplements this paper (amino-acid-sequence-analysis histograms) is reported in Supplementary Publication SUP 50160 (4 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1990) 265, 5.

Amino Acid Sequence↗
PubMed1990

Preferential expression of the complement regulatory protein decay accelerating factor at the fetomaternal interface during human pregnancy.

The expression of decay accelerating factor (DAF) was investigated in human fetal and extra-fetal tissues using a panel of mAb directed against different epitopes on the DAF molecule. By immunostaining, extensive reactivity was observed on the placental trophoblast epithelium and this was confined exclusively to sites of direct contact with maternal blood and tissues at the fetomaternal interface. Within the fetus, by contrast, very little staining was observed especially on hemopoietic cell populations in the developing liver. The antibodies identified a component of 70,000 m.w., comigrating on SDS-PAGE with red cell DAF, on isolated trophoblast membranes and an apparently quantitative increase in the expression of DAF was observed during placental development. Northern analysis using a DAF cDNA clone revealed 1.5-, 1.6-, and 2.2-kb mRNA transcripts typical of DAF in mRNA prepared from whole term placentae and from purified trophoblast cells. DAF appears to be preferentially expressed at the fetomaternal interface during development and may function specifically to inhibit amplification convertases formed at this site either directly or indirectly as a result of maternal complement activation. This molecule may play an important role in protecting the semiallogeneic human conceptus from maternal C-mediated attack.

Antibodies, Monoclonal↗
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↗