Deficiency of protein 4.2 in erythrocytes from a patient with a Coombs negative hemolytic anemia. Evidence for a role of protein 4.2 in stabilizing ankyrin on the membrane.

Rybicki, Anne C.; Heath, RH; Wolf, Janina; Lubin, Bertram H.; Schwartz, Robert S.

doi:10.1172/jci113400

Cited by 89 publications

(46 citation statements)

References 26 publications

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“…In all cases, a mild hemolytic anemia with an intact red cell membrane skeleton, similar to our ld/ld mice, is observed (32)(33)(34)(35). It is possible that ankyrin binds to cdb3 at a second, low-affinity site (36).…”

Section: Hematologic Indices Of Wild-type (؉/؉) Heterozygous (؉/Ldsupporting

confidence: 61%

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

Stefanovic

Markham

Parry

et al. 2007

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

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The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin-actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a ␤-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein-band 3 interaction. To evaluate the importance of the ankyrin-band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa ␤-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin-band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin-actin-based membrane cytoskeleton to the plasma membrane.cytoskeleton ͉ membrane ͉ transgenic ͉ spherocytosis

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Section: Hematologic Indices Of Wild-type (؉/؉) Heterozygous (؉/Ldsupporting

confidence: 61%

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

Stefanovic

Markham

Parry

et al. 2007

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

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“…Affinity-purified rabbit anti-human P4.2 IgG prepared by Rybicki et al (8) was used to screen a cDNA expression library in Agtll constructed from human reticulocyte mRNA, kindly provided by J. G. Conboy and Y. W. Kan (5). Immunoscreening of the Agtll expression library was performed according to Huynh et al (12), except that positive clones were identified with goat anti-rabbit IgG conjugated with horseradish peroxidase (BioRad).…”

Section: Methodsmentioning

confidence: 99%

“…5'-End Extension of cDNA. Three oligonucleotides were prepared in a technique based on the polymerase chain reaction (PCR) to synthesize the missing 5' sequence of the partial cDNA clone: pl was composed of nucleotides (nt) [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] of clone 7 (c.7); p2 was complementary to nt 36-52 of c.7 plus an EcoRI restriction site at its 5' end; p3 was composed of the EcoRI polylinker and the poly(dC) originally used for the first-strand cDNA synthesis when the library was constructed. The sequences of pl, p2, and p3 were, respectively, 5'-dTGAGGATGCTGTGTTCC-3', 5'-dTCGAAlJCGTACTC-CATGCGCTGAG-3', and 5'-dGCGGLAAlTCCCCCCCCCC-CCCC-3', with the EcoRI sites underlined.…”

Section: Methodsmentioning

confidence: 99%

“…P4.2 has an apparent molecular mass of -72 kDa and associates with the cytoplasmic domain of the anion exchanger, band 3 (7). Recent evidence suggests that P4.2 interacts with ankyrin and may function to stabilize ankyrin in the membrane (8). Individuals whose RBCs are severely deficient in P4.2 experience various levels of anemia, further indicating an important functional role for this protein (8).…”

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confidence: 99%

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Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

Sung¹,

Chien²,

Chang³

et al. 1990

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

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Protein 4.2 (P4.2) comprises -5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4-and 2.5-kb cDNAs predict proteins of -77 and =80 kDa, respectively, and the authenticity was confimed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4. Subcloning and Sequence Analysis. cDNA inserts from positive phage clones were subcloned into pBS(+) plasmids (Stratagene). Unidirectional deletion clones were generated by using BAL-31 exonuclease (13), and cDNA fragments were sequenced with T3 and T7 primers by the dideoxynucleotide chain-termination method (14). Sequence analysis and GenBank data base searches were performed by IBI Pustell sequence analysis software (International Biotechnologies). 5'-End Extension of cDNA. Three oligonucleotides were prepared in a technique based on the polymerase chain reaction (PCR) to synthesize the missing 5' sequence of the partial cDNA clone: pl was composed of nucleotides (nt) 7-23 of clone 7 (c.7); p2 was complementary to nt 36-52 of c.7 plus an EcoRI restriction site at its 5' end; p3 was composed of the EcoRI polylinker and the poly(dC) originally used for the first-strand cDNA synthesis when the library was constructed. The sequences of pl, p2, and p3 were, respectively, 5'-dTGAGGATGCTGTGTTCC-3', 5'-dTCGAAlJCGTACTC-CATGCGCTGAG-3', and 5'-dGCGGLAAlTCCCCCCCCCC-CCCC-3', with the EcoRI sites underlined. p2 and p3 were used as PCR primers, and the reticulocyte cDNA library (5 ,u with 106 phages per pl) was used as a template. The reaction product was electrophoresed and stained with ethidium bromide. The major band was excised and subcloned into pGEM 3zf plasmids (Promega). From >500 transformants, 24 colonies were randomly chosen to make minipreparations of plasmid DNA, of which 80%o were positive when hybridized with 32P-labeled pl. tTo whom reprint requests should be addressed. IThe sequences reported in this paper have been deposited in the GenBank data base (accession nos. M30646 and M30647). 955The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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“…[14][15][16] To date, nine mutations in EPB42 have been associated with hereditary spherocytosis, 6,[17][18][19][20][21][22] some of these mutations influence protein 4.2 stability and band 3-ankyrin-1 binding. 8,23 Protein 4.2 deficiency in humans causes a severe reduction in the marker of self, 24 CD47 (by approximately 80%), 6,25,26 suggesting an association between protein 4.2 and CD47.…”

Section: Introductionmentioning

confidence: 99%

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

Akker¹,

Satchwell²,

Pellegrin³

et al. 2010

Haematologica

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The online version of this article has a Supplementary Appendix. BackgroundProtein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. Design and MethodsWe used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. ResultsWe report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship. ConclusionsWe have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.

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Deficiency of protein 4.2 in erythrocytes from a patient with a Coombs negative hemolytic anemia. Evidence for a role of protein 4.2 in stabilizing ankyrin on the membrane.

Cited by 89 publications

References 26 publications

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2)

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