The use of monoclonal antibodies to quantify the levels of sialoglycoproteins α and δ and variant sialoglycoproteins in human erythrocyte membranes

Merry, Anthony H.; Hodson, Christopher T. C.; Thomson, Eileen E.; Mallinson, Gary; Anstee, David J.

doi:10.1042/bj2330093

Cited by 77 publications

(45 citation statements)

References 21 publications

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“…Measurements of the hybrid protein on cells from four of these individuals with "Leporetype" hybrids showed that the hybrid protein was expressed at about 25% of normal for one GPA allele (28,31). This corresponds to the labeling characteristics of the small discrete population of variant cells commented on earlier, that lying below the MN peak that was observed consistently in all seven BS samples in the present study (Fig.…”

Section: Discussionsupporting

confidence: 86%

“…While doublets of one-copy variants could produce the peak at the intensity expected for two-copy variants, visual observation of the cells sorted from these regions confirmed that two-copy variants are single cells with brighter fluorescence than one-copy variants. Since it has been shown that individuals who have inherited a defect in one GPA allele express the remaining allele at a one-copy level (28,31,34), it is probable that these two variant-cell types are the progeny of two different populations of erythroid precursor cells having one or two copies of the remaining allele.…”

Section: Resultsmentioning

confidence: 99%

“…The region around the GPA gene may be unusually susceptible to unequal recombination in view of many reports that have described individuals who heritably express an altered glycophorin protein apparently resulting from an unequal meiotic cross-over event between the GPA gene and the closely linked and partially homologous glycophorin B (GPB) gene (28,31,42,43). Measurements of the hybrid protein on cells from four of these individuals with "Leporetype" hybrids showed that the hybrid protein was expressed at about 25% of normal for one GPA allele (28,31).…”

Section: Discussionmentioning

confidence: 99%

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Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome.

Langlois

Bigbee

Jensen

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

177

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The glycophorin A assay was used to estimate the frequency of mutations that accumulate in vivo in somatic cells of persons with Bloom's syndrome (BS). This assay measures the frequency in persons of blood type MN of variant erythrocytes that lack the expression of one allelic form of glycophorin A, presumably due to mutational or recombinational events in erythroid precursor cells. Samples of blood from persons with BS showed dramatic 50-to 100-fold increases in the frequency of variants of three types, those with a hemizygous phenotype, those with a homozygous phenotype, and those with what appears to be partial loss of the expression of one locus. The high frequency of homozygous variants, genetic evidence for altered allelic segregation of a specific biochemical locus, provides evidence for increased somatic crossing-over in vivo in BS. An increased generation of functional hemizygosity and homozygosity in their somatic cells may play an important role in the extreme cancer risk of persons with BS.Bloom's syndrome (BS) (1) is a rare autosomal recessive genetic disorder of growth that greatly increases the affected individual's chance of developing cancer. In the 130 affected individuals in the Bloom's Syndrome Registry, 57 malignant neoplasms have been detected, at a mean age at diagnosis of 24.7 years (2). A wide spectrum of tumor types has been observed in affected individuals, including lymphoid and myeloid leukemias, lymphomas, squamous cell carcinomas, and adenocarcinomas (3).In addition, a distinctive array of cytological abnormalities in a variety of cell types, signifying a remarkable degree of genomic instability, is a constant feature of BS (4, 5). These include high spontaneous frequencies of chromosome breaks and rearrangements (5-8), sister-chromatid exchanges (SCEs) (5, 9-11), micronucleated cells (12, 13), and cells with specific-locus mutations (14-17). A unique cellular abnormality in untreated BS cells in vitro is a dramatic increase over normal in the frequency of a certain type of quadriradial configuration (Qr) in metaphase cells (5,18). The Qrs of BS are the microscopically visible consequence of interchanges that, earlier in the cell cycle, occurred between homologous chromosomes at apparently homologous sites; they are interpreted as cytological evidence for an increased frequency of somatic crossing-over in BS (18)(19)(20)(21). Heterozygous carriers of the BS mutation (bl/+) are clinically and cytologically normal (22). Although the primary genetic defect responsible for BS may not have been defined, DNA ligase I activity has been reported to be decreased (23)(24)(25), and this very possibly is responsible for the genomic instability of BS. The complex cytogenetic abnormalities readily demonstrable in BS cells in vitro raise the possibility that somatic cell mutation (point mutation, chromosome segmental rearrangement, and somatic recombination) is also substantially increased in vivo in BS, and this could explain the extreme cancer risk.In the present study we have used the ...

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Section: Discussionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome.

Langlois

Bigbee

Jensen

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

177

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“…Antibody 10F7 binds the common variants of human glycophorin A (huGYPA) (Fig. 1C), which is restricted to the RBC lineage and expressed at ∼800,000 copies on mature RBCs (20,21). Several characteristics of huGYPA make it a desirable receptor to target: (i) it has a small extracellular domain (18), such that an scFv-EPO fusion protein likely can bind simultaneously to both huGYPA and EPO-R; (ii) sequences of the 10F7 V regions are available (GenBank AAK85 297.1); and (iii) loss of huGYPA is phenotypically silent (22), so binding to huGYPA is unlikely to cause side effects.…”

Section: Resultsmentioning

confidence: 99%

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

Burrill

Vernet

Collins

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.protein engineering | drug targeting | platelet | scFv

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“…The amount of M1.V glycoprotein present on red cells from M1.V heterozygotes has been estimated as 98000 copies using murine monoclonal antibodies against the extracellular domain of GPA [38]. This should be compared to the 300000 and 40 000 copies/haploid genome determined simultaneously for GPA and GPB, respectively.…”

Section: Gpb Gene Statusmentioning

confidence: 99%

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi.V) human erythrocytes

Vignal

Rahuel

Maliki

et al. 1989

European Journal of Biochemistry

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In the Miltenberger class V (M1.V) condition, red cells lack glycophorin A (GPA) and glycophorin B (GPB) but carry instead an unusual glycoprotein thought to be a hybrid molecule produced by the unequal crossingover between the closely linked genes encoding for GPA and GPB. By Western blot analysis with rabbit anti-CPA antibodies specific for discrete domains of GPA, it was found that the Mi.V glycoprotein (donor F. M.) contains approximately 60 amino acid residues of GPA at its N-terminus.As a preliminary approach to the molecular analysis of this variant the restriction maps of the GPA and GPB genes were established by Southern blot analysis of genomic DNA and from genomic clones isolated from a human leukocyte library constructed in AEMBL4. The GPA and GPB genes cover about 30 kb of DNA and are organized into seven exons (A-l -A-7) and five cxons (B-1 -B-5), respectively. In addition to the normal genes, a third gene (named inv), closely resembling the GPA and GPB genes, was also identified. In the homozygous Mi.V individual the normal GPA and GPB genes were absent, but an unusual form of gene structure was detected by Southern blot analysis. The Mi.V glycoprotein gene was composed of exon B-I of the GPB gene followed by exons A-2 and A-3 of the GPA gene and the exons B-3, B-4 and B-5 of the GPB gene. Exon B-1 can be distinguished from exon A-1 of GPA since it is located within a different restriction fragment, but both encode the same amino acid sequence (N-terminal region of the signal peptides). Using the polymerase chain reaction, the junction between exon A-3 and exon B-3 was confirmed by amplification of the DNA region where the putative crossingover has occurred and it was deduced that the Mi.V glycoprotein is a hybrid molecule composed of amino acid residues 1 -58 from GPA fused to amino acid residues 27 -72 of GPB. In addition, the finding that part of the signal peptide and the 5'-untranslated region are derived from CPB suggests that the genetic background of the M1.V variant is rather complex and may involve a cascade of recombination or gene conversion events.Glycophorin A (GPA) and glycophorin B (GPB) are the major glycoproteins of the human erythrocyte membrane which carry the MN and the Ss blood group antigens, respectively (for reviews see [l, 21). These glycoproteins are highly homologous at both the amino acid and nucleotide levels [3 -61. They are encoded by two closely linked genes in position q28-q31 on the long arm of chromosome 4 [7] and are probably derived from a common ancestral gene by duplication PI. ~-homozygotes by the lack of GPA and GPB expression on red cells and the presence of an unusual glycoprotein exhibiting properties common to GPA and GPB [15]. Accordingly, it has been proposed that the Mi.V glycoprotein is a hybrid molecule composed of the N-terminal domain of GPA and of the C-terminal domain of GPB.In order to understand the genetic basis for the origin and expression of this glycophorin variant we have performed a detailed study of the Mi.V condition based on immu...

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The use of monoclonal antibodies to quantify the levels of sialoglycoproteins α and δ and variant sialoglycoproteins in human erythrocyte membranes

Cited by 77 publications

References 21 publications

Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome.

Evidence for increased in vivo mutation and somatic recombination in Bloom's syndrome.

Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

Molecular analysis of glycophorin A and B gene structure and expression in homozygous Miltenberger class V (Mi.V) human erythrocytes

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