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D J Anstee

Publications and source records attributed to D J Anstee.

At least 55 records · Page 3Linked 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

Monoclonal antibodies against Kell glycoprotein: serology, immunochemistry and quantification of antigen sites.

Monoclonal antibodies BRIC 18, BRIC 68, BRIC 107 and BRIC 203 recognize high-frequency epitopes absent from erythrocytes expressing the Ko phenotype. BRIC 107 has anti-k (K2)-like specificity. BRIC 203 has a unique specificity denoted anti-Kpbc. All four monoclonal antibodies identify an M(r) 95,600 erythrocyte membrane protein by immunoprecipitation from radio-iodinated erythrocytes. In quantitative binding studies using IgG it is estimated that there are from 2000 (BRIC 18) to 4000 (BRIC 68) copies of the Kell glycoprotein per erythrocyte. Using Fab fragments the estimates are in the range 4000 (BRIC 18) to 18,000 (BRIC 68) copies. In competitive binding assays the four epitopes defined by the BRIC monoclonal antibodies fall into two non-overlapping groups. The first group comprises BRIC 18, BRIC 68, BRIC 203 and an antibody (6-22) with anti-K14 specificity. The second group contains BRIC 107 and two further anti-k-like monoclonal antibodies (BS45 and OSK5). The results suggest that the polymorphisms encoded at the K/k and Kpa/Kpb/Kpc loci may be located in two spatially distinct regions of the Kell glycoprotein(s).

Animals↗
PubMed1993

Use of the MAIEA technique to confirm the relationship between the Cromer antigens and decay-accelerating factor and to assign provisionally antigens to the short-consensus repeats.

The MAIEA (monoclonal-antibody-specific immobilisation of erythrocyte antigens) assay has recently been developed for the assignment of red cell antigens, recognised by human alloantisera, to particular membrane components of the red cell membrane. This technique detects trimolecular complexes formed by the reaction of a human antibody and a mouse antibody with a particular red cell protein. A positive reaction, in an ELISA-type detection procedure, occurs if the epitopes to the human and mouse antibodies are present on the same membrane component but at different regions. In this report, we show how the MAIEA assay can be used to confirm the relationship between Cromer system antigens and the complement-regulatory protein, decay-accelerating factor (DAF, CD 55). In addition, the location of the antigens along the protein is postulated by using three anti-DAF monoclonal antibodies with specificities to different regions of DAF. Tca and Esa are assigned provisionally to the first short-consensus repeat (SCR), UMC to the second SCR, Dra to the third SCR and Cra, WESa and WESb to the fourth SCR or to the serine/threonine rich region of the DAF protein.

Antibodies, Monoclonal↗
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

Mapping of epitopes, glycosylation sites, and complement regulatory domains in human decay accelerating factor.

Decay accelerating factor (DAF, CD55) is a glycophospholipid-anchored membrane protein that protects cells from complement-mediated damage by inhibiting the formation and accelerating the decay of C3/C5 convertases. DAF deletion mutants lacking each of the four short consensus repeats (SCR) or the serine/threonine-rich region (S/T) were created by site-directed mutagenesis. These deletion mutants were expressed by stable transfection in Chinese hamster ovary cells for the purpose of mapping important structural and functional sites in DAF. The epitopes on DAF for 16 murine mAb were mapped by immunoprecipitation studies as follows: SCR1, 6; SCR2, 3; SCR3, 3; SCR4, 3; S/T, 1. Testing of 13 mAb showed complete blocking of DAF function only by 1C6 and 1H4, both directed at SCR3. The single N-linked glycosylation site was confirmed at a location between SCR1 and SCR2, and the multiple O-linked oligosaccharides were localized to the S/T region. Functional activity of DAF mutants was assessed by the ability of these transfected constructs to protect Chinese hamster ovary cells from cytotoxicity induced by rabbit antibody plus human complement. Removal of SCR1 had no effect on DAF function, but individual deletion of SCR2, SCR3, or SCR4 totally abolished DAF function. Surprisingly, deletion of the S/T region totally abrogated DAF function, but this could be restored by a fusion construct placing the four SCR domains of DAF onto the HLA-B44 molecule, implying that the O-glycosylated S/T region serves as an important but nonspecific spacer projecting the DAF functional domains above the plasma membrane. Overall, the creation of DAF deletion mutants has elucidated important structure-function relations in the DAF molecule.

Animals↗
PubMed1992

Evidence that the antigens of the Yt blood group system are located on human erythrocyte acetylcholinesterase.

The Yt blood group system comprises two antigens, Yta and Ytb. Human anti-Yta and human anti-Ytb immune precipitate a component of the same apparent molecular weight as acetylcholinesterase from radioiodinated erythrocytes of appropriate Yt phenotype. Immune precipitates obtained with anti-Yta and anti-Ytb contained acetylcholinesterase activity. In contrast, immune precipitates obtained with human anti-Gya and murine monoclonal anti-CD55, which identify other glycosylphosphatidylinositol-linked erythrocyte surface proteins, did not have acetylcholinesterase activity. Quantitative binding assays using murine monoclonal antiacetylcholinesterase antibodies (AE-1 and AE-2) gave 3,000 to 5,000 binding sites/cell for IgG and 7,000 to 10,000 sites/cell for Fab fragments. Endo F digestion of immune precipitates obtained with AE-1 and anti-Yta indicated that approximately 10% of the enzyme comprises N-glycans. These results indicate that the Yt antigens define an inherited polymorphism on erythrocyte acetylcholinesterase and that the recent assignment of the Yt blood group locus to the long arm of chromosome 7 (Zelinski et al, Genomics 11:165, 1991) provisionally identifies the position of the acetylcholinesterase gene.

Acetylcholinesterase↗
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

New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes.

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.

Antibodies, Monoclonal↗
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↗
PubMed1991

New monoclonal antibodies in CD44 and CD58: their use to quantify CD44 and CD58 on normal human erythrocytes and to compare the distribution of CD44 and CD58 in human tissues.

The cell-surface glycoproteins CD44 and CD58 are involved in cell adhesion reactions. In this paper 12 monoclonal antibodies in CD44 and two in CD58 are described. Competitive binding assays using CD44 antibodies identified three distinct epitope groups. Antibodies in Group 1 and, with one exception (BRIC 214), antibodies in group 2, but not antibodies in Group 3, recognized epitopes that are sensitive to reduction and to trypsin or chymotrypsin treatment of intact erythrocytes, and so these epitopes probably reside on the N-terminal disulphide-bonded domain of CD44. Antibodies in CD44 did not inhibit the binding of CD58 antibodies to erythrocytes or vice versa. Quantitative binding studies using radioiodinated IgG measured 1888-5592 copies of CD44 and 1772-3290 copies of CD58 on normal erythrocytes. Similar measurements with radioiodinated Fab fragments gave values of 6508-10,450 (CD44) and 3457-7622 (CD58). Immunocytochemical studies indicated that CD44 is much more widely expressed in non-haemopoietic tissues than CD58. Comparison with previously described CD44 antibodies suggests that antibodies in our Group 1 encompass Hermes 2 and that those in Group 2 encompass Hermes 1. All the CD44 antibodies gave weakened reactions with Lu(a-b-) erythrocytes of the In(Lu) type by one or more methods. BRIC 214 and antibodies in epitope Group 3 were used to demonstrate that CD44 on these variant cells gives membrane-bound trypsin and chymotrypsin cleavage fragments of similar molecular weight to those obtained with normal erythrocytes.

Antibodies, Monoclonal↗
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↗
PubMed1990

Blood group-active surface molecules of the human red blood cell.

The surface of the human red blood cell is dominated by a small number of abundant blood group active proteins. The major proteins are the anion transport protein (band 3) which has AB(H) activity, and Glycophorin A which has MN activity. Band 3 and Glycophorin A are of equal abundance in the normal red cell membrane (approximately 10(6) copies of each) and the two proteins may associate together as a complex. The glucose transporter (band 4.5) had AB(H) activity and there are about 5 x 10(5) copies/red cell. Several polypeptides associate together to form the Rh complex. The major components of this complex (abundance 1-2 x 10(5) copies/red cell) are polypeptides of Mr 30,000, polypeptides of Mr 45,000-100,000 and Glycophorin B. The antigens of the Rh blood group system appear to be associated with the polypeptides of Mr 30,000 and those of Mr 45,000-100,000 (the latter also express AB(H) activity). Glycophorin B expresses the blood group 'N' antigen and the Ss antigens. Glycophorins C and D carry the Gerbich antigens and, together, these polypeptides comprise approximately 10(5) copies/red cell. The complete protein sequence of all the above-mentioned proteins is known, except for the Mr 30,000 and Mr 45,000-100,000 polypeptides of the Rh complex for which only partial sequences are available, and Glycophorin D, the sequence of which can be inferred from that of Glycophorin C. Several of the minor blood group active proteins at the red cell surface (abundance less than 1.2 x 10(4)/red cell) have been the subject of recent studies. The polypeptide expressing Cromer-related blood group antigens has been identified as decay-accelerating factor and that carrying the Ina/Inb antigens as CD44. The protein sequence of both of these proteins has been deduced form nucleotide sequencing. The polypeptides expressing Kell antigens, Lutheran antigens, Fy antigens, and LW antigens have also been identified and partially characterised.

Antigens, Surface↗
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↗
PubMed1989

Rearrangements of the red-cell membrane glycophorin C (sialoglycoprotein beta) gene. A further study of alterations in the glycophorin C gene.

We have cloned portions of the glycophorin C (sialoglycoprotein beta) gene from individuals with red cells of normal, Gerbich and Yus phenotypes. The clones contain up to three exons of the glycophorin C gene (designated exons 2, 3 and 4). Analysis by restriction mapping and DNA sequencing confirmed that the deletions causing the Gerbich and Yus phenotypes are located entirely within the glycophorin C gene. Sequencing of the normal gene showed that not only do exon 2 and exon 3 have related DNA sequences, but also that both the 5' and 3' flanking intronic DNA sequences are almost identical. The two variant genes each lack a different exon: the Yus type gene lacks exon 2, whereas the Gerbich-type gene lacks exon 3. We suggest that the observed deletions are due to recombination between the regions of homologous intronic repeats. We also provide evidence that an unequal cross-over mechanism may be responsible for a number of observed glycophorin C gene rearrangements, including an insertion mutation in Lewis II (Lsa)-type red cells that has not previously been reported.

Base Sequence↗