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M J Tanner

Publications and source records attributed to M J Tanner.

At least 19 recordsLinked to original sources
PubMed1995

Perturbation of red blood cell membrane rigidity by extracellular ligands.

It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and univalent anti-band 3 antibodies implies the existence of an overriding effect on rigidity, resulting from the bifunctionality of the intact antigen. Freeze-fracture electron microscopy shows that the anti-band 3 promotes the formation of small clusters of intra-membrane proteins. Extracellular ligands may in general act by promoting strong or transient interactions between integral membrane proteins, thereby impeding local distortion of the membrane skeletal network in response to shear.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1995

Co-expressed complementary fragments of the human red cell anion exchanger (band 3, AE1) generate stilbene disulfonate-sensitive anion transport.

We have constructed cDNA clones encoding various portions of the human red cell anion transporter (band 3), a well characterized integral membrane protein with up to 14 transmembrane segments. The biosynthesis, stability, cell surface expression, and functionality of these band 3 fragments were investigated by expression from the cRNAs into microsomal membranes using the reticulocyte cell-free translation system and in Xenopus oocytes. Co-expression of the pairs of recombinants encoding the first 8 and last 6 transmembrane spans (8 + 6) or the first 12 and last 2 spans (12 + 2) of band 3 generated stilbene disulfonate-sensitive anion transport in oocytes. When the pairs of fragments 8 + 6 or 12 + 2 were co-expressed with glycophorin A (GPA), translocation to the plasma membrane of the fragment corresponding to the first 12 or the first 8 transmembrane spans was greater than in the absence of GPA. Only the fragment encoding the first 12 transmembrane spans showed GPA-dependent translocation when expressed in the absence of its complementary fragment. A truncated form of band 3 encoding all 14 transmembrane spans but lacking the carboxyl-terminal 30 amino acids of the cytoplasmic tail did not induce anion transport activity in oocytes and was not translocated to the plasma membrane but appeared to be degraded in oocytes. Our results suggest that there is no single signal for the insertion of the different transmembrane spans of band 3 into membranes and that the integrity of the loops between transmembrane spans 8-9 or 12-13 is not essential for anion transport function. Our data also suggest that a region of transmembrane spans 9-12 of band 3 is involved in the process by which GPA facilitates the translocation of band 3 to the surface.

Amino Acid Sequence↗
PubMed1995

Identification of the membrane attachment sites for protein 4.1 in the human erythrocyte.

The nature of the membrane attachment site(s) for protein 4.1 in the human erythrocyte membrane has yet to be fully elucidated. In this paper we show that the major attachment site is glycophorin (GP) C/D, and that purified protein 4.1 can bind to two distinct sites on glycophorin C/D. One of these interactions is direct, involving residues 82-98 on glycophorin C (61-77 on glycophorin D), while the other interaction is mediated by p55. We have localized the binding site for p55 on glycophorin C to residues 112-128 (glycophorin D91-107). We also provide evidence that band 3 is an additional, minor, protein 4.1 binding site. The binding sites for band 3, glycophorin C/D, and p55 are all located within the 30-kDa domain of protein 4.1. We estimate that the relative utilization of the three sites in normal membranes comprises 40% to p55, 40% to GPC/D, and 20% to band 3. The same region of protein 4.1 binds GPC/D and band 3, while the p55 binding site is distinct. The interactions involving protein 4.1 with p55 and p55 with GPC/D are of high affinity (nM), while those involving GPC/D and band 3 are 100-fold lower (microM). These results suggest that the most significant interactions between protein 4.1 and the membrane are those involving p55.

Binding Sites↗
PubMed1995

Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: a site of interaction between band 3 and glycophorin A under certain conditions.

The Wright (Wr) blood group antigens, Wra and Wrb, have been suggested to be determined by alleles of the same gene. The Wrb antigen appears to involve both red blood cell (RBC) band 3 and glycophorin A (GPA). We have examined the cDNA sequences of the band 3 and GPA of one of the two known Wr(a+b-) individuals. We show that this individual is homozygous for the mutation Glu658-->Lys in band 3, but has normal GPA. Putative heterozygotes with Wr(a+b+) RBCs have both Glu and Lys at residue 658 of band 3, whereas the common Wr(a-b+) RBC phenotype only have band 3 with Glu658. The Wra and Wrb antigens are determined by the amino acid at residue 658 of band 3 and are antithetical. Examination of the amino acid sequence and Wrb antigen expression of GPA-related hybrid glycophorins suggests that Arg61 of GPA interacts with Glu658 of band 3 to form the Wrb antigen. We suggest that the interaction is stabilized by the presence of anti-Wrb antibodies and that this site of association between GPA and band 3 may be responsible for the previously reported ability of anti-GPA antibodies to decrease the deformability of RBCs.

Alleles↗
PubMed1995

A point mutation in the protein 4.2 gene (allele 4.2 Tozeur) associated with hereditary haemolytic anaemia.

A recessively transmitted haemolytic anaemia associated with the lack of protein 4.2 was found in a Tunisian kindred. Trace amounts of this protein (72 kD component) became visible using high-sensitivity Western blots. Band 3 and ankyrin genes were excluded as candidate genes by linkage studies, and nucleotide sequencing of band 3 cytoplasmic domain cDNA revealed no alteration. In contrast, protein 4.2 gene contained in the homozygous state a mutation at position 310: CGA-->CAA (Arg-->Gln). This mutation defining allele 4.2 Tozeur was co-inherited with the disease. The mRNA encoding the variant protein was normal in size and approximately normal in amount. Recombinant protein 4.2 Tozeur bound normally to red cell IOVs but disclosed an increased susceptibility to proteolysis in vitro. We infer that the nearly total absence of protein 4.2 in the patients results from imbalance between destruction and synthesis of mutated protein 4.2 prior to its binding to the membrane.

Anemia, Hemolytic↗
PubMed1994

Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes.

The human erythrocyte blood group system Cromer consists of high-incidence and low-incidence antigens that reside on decay-accelerating factor (DAF; CD55), a glycosyl-phosphatidylinositol-anchored membrane protein that regulates complement activation on cell surfaces. In the Cromer phenotypes Dr(a-) and Inab there is reduced or absent expression of DAF, respectively. This study investigated the molecular basis of the reduced DAF expression by polymerase chain reaction amplification of genomic DNA and RNA/cDNA obtained from Epstein-Barr virus-transformed lymphoblastoid cell lines. Sequence analysis of the Inab propositus showed a single nucleotide substitution in exon 2 of the DAF gene and at the corresponding position in the cDNA, G314-->A resulting in Trp53-->Stop. This truncation near the amino terminus explains the complete absence of surface DAF in the Inab phenotype. A similar analysis was performed for two Dr(a-) individuals, including KZ, who was previously reported to be Inab phenotype but is now shown by immunochemical and serologic methods to be Dr(a-) phenotype. A single nucleotide change was found in exon 5 of the DAF gene, C649-->T resulting in Ser165-->Leu, which we had previously shown to lead to loss of the Dra epitope. However, two species of cDNA were found, one encoding full-length DAF with the single amino acid change and the more abundant species having a 44-nucleotide deletion. The 44 nucleotide deletion includes the single polymorphic site, which creates a cryptic branch point in the Dr(a-) allele that leads to use of a downstream cryptic acceptor splice site. This shifts the reading frame and leads to a premature stop codon that precludes membrane anchoring. Thus, the single point mutation in the Dr(a-) phenotype results in a novel use of alternative splicing and provides a molecular explanation for both the antigenicity and the reduced DAF expression seen in this phenotype.

Amino Acid Sequence↗
PubMed1994

Regulation of System B0 amino-acid-transport activity in the renal epithelial cell line NBL-1 and concomitant changes in SAAT1 hybridizing transcripts.

alpha-(Methylamino)isobutyric acid (MeAIB) insensitive Na(+)-dependent alanine transport activity in the bovine kidney cell line NBL-1 was increased upon amino acid starvation (> or = 20% over control levels). When L-phenylalanine (3 mM) was included in the starvation medium the increase was further enhanced (> or = 85% over control levels). In cells grown in control medium the Vmax, for MeAIB-insensitive Na+/alanine co-transport was found to be 6.0 +/- 0.7 nmol/3 min per mg (Km 41 +/- 12 microM) and for L-phenylalanine-treated amino-acid-starved cells the Vmax. was 21 +/- 5 nmol/3 min per mg (Km 92 +/- 40 microM). The increase in Vmax. was prevented by cycloheximide. Substrate specificity analysis identified the L-phenylalanine-induced transport system as System B0. [35S]Methionine labelling of cells during the amino acid starvation/phenylalanine treatments resulted in the differential labelling of a protein of 78 kDa. Northern-blot analysis using a SAAT1-specific probe revealed the presence of a new transcript (3.2 kb) in RNA extracted from cells incubated in amino acid starvation medium with L-phenylalanine included. The present findings suggest a novel means of control for System B0 by the use of physiological stress. It is also proposed that SAAT1 and System-B0 transcripts have considerable sequence similarity.

Alanine↗
PubMed1994

Studies on the glycoprotein associated with Rh (rhesus) blood group antigen expression in the human red blood cell membrane.

The blood group Rh antigens are associated with non-glycosylated 30-kDa erythrocyte membrane proteins (the Rh30 polypeptides) and the Rh glycoprotein. We used antipeptide antibodies to study the Rh glycoprotein in human erythrocyte membranes. The Rh glycoprotein was present in Rhnull U+ve cells. However, the N-glycan chain of the Rh glycoprotein in Rhnull U+ve cells was smaller than in normal cells. In contrast, the N-glycan chain of the Rh glycoprotein was larger than normal in glycophorin B-deficient red cells. We suggest that this observation reflects a lower rate of movement of newly synthesized Rh glycoprotein through intracellular membranes to the cell surface in the absence of glycophorin B, and that in normal red cells glycophorin B facilitates the movement of the Rh protein complex to the cell surface. Our results provide evidence for the intracellular interaction of at least three proteins, the Rh glycoprotein, Rh30 polypeptides, and glycophorin B during the biosynthesis and cell surface expression of the Rh complex. These observations are likely to be important for the successful design of expression systems for the blood group Rh antigens.

Animals↗
PubMed1993

The expression of the abnormal human red cell anion transporter from South-East Asian ovalocytes (band 3 SAO) in Xenopus oocytes.

South-East Asian ovalocytosis (SAO) is caused by the heterozygous presence of a variant form of the human erythrocyte anion transporter (band 3; AE1). The expression of band 3 SAO has been studied in Xenopus oocytes. Band 3 SAO is not functional as an anion transporter but is inserted stably into the plasma membrane of oocytes. Band 3 SAO translocation to the cell surface does not require co-expression of normal band 3. Co-expression of glycophorin A (GPA) increases the rate of translocation of band 3 SAO to the oocyte membrane but is not essential for this process. We suggest that the increased tendency of band 3 SAO to form oligomers may facilitate its translocation to the cell surface.

Animals↗
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↗