scholarly journals A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

1984 ◽  
Vol 73 (6) ◽  
pp. 1688-1695 ◽  
Author(s):  
J Lawler ◽  
S C Liu ◽  
J Palek ◽  
J Prchal
Keyword(s):  
Alpha Subunit ◽  
Molecular Defect ◽  
Hereditary Elliptocytosis ◽  
Self Association

scholarly journals Molecular heterogeneity of hereditary pyropoikilocytosis: identification of a second variant of the spectrin alpha-subunit

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1182-1189
Author(s):  
J Lawler ◽  
J Palek ◽  
SC Liu ◽  
J Prchal ◽  
WM Butler
Keyword(s):  
Alpha Subunit ◽  
Tryptic Digestion ◽  
Beta Subunits ◽  
Molecular Defect ◽  
Molecular Heterogeneity ◽  
Mechanical Instability ◽  
Concomitant Increase ◽  
Red Cell Membrane ◽  
Tryptic Fragment ◽  
Self Association

In hereditary pyropoikilocytosis (HPP), the red cell membrane skeletons exhibit a mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To determine the underlying molecular defect, we have subjected HPP spectrin to limited tryptic digestion, followed by one- and two-dimensional separations of the peptides. Two of the HPP kindreds exhibited a marked decrease in 80,000- dalton peptide (previously identified as the spectrin dimer-dimer contact domain of the alpha-subunit) and a concomitant increase of the 74,000-dalton polypeptide (presumably derived from the 80,000-dalton domain) and a decrease in a 22,000-dalton polypeptide. We now report tryptic digests of two other HPP kindred that are characterized by a decrease or complete absence of the 80,000-dalton tryptic fragment, with a concomitant increase in fragments at 46,000 and 17,000 daltons. The 46,000-dalton fragment separated into multiple spots on isoelectric focusing, ranging in isoelectric point from 5.25 to 5.35, and the 17,000-dalton fragment focused to a single spot at 5.4. Minor fragments at 56,000 and 22,000 daltons were also decreased, while a 38,000-dalton fragment increased. Limited tryptic digestion of the separated alpha- and beta-subunits revealed that the 74,000-dalton fragment in the first group of patients and the 46,000-dalton fragment in the second group of patients were derived from the alpha-subunit. Both subtypes exhibited a similar defect of spectrin self-association, with 30%-38% of spectrin dimers in O degrees C extracts. The results indicate that at least two distinct forms of structurally defective spectrin may give rise to the clinical presentation of HPP.

scholarly journals Molecular heterogeneity of hereditary pyropoikilocytosis: identification of a second variant of the spectrin alpha-subunit

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1182-1189 ◽  
Author(s):  
J Lawler ◽  
J Palek ◽  
SC Liu ◽  
J Prchal ◽  
WM Butler
Keyword(s):  
Alpha Subunit ◽  
Tryptic Digestion ◽  
Beta Subunits ◽  
Molecular Defect ◽  
Molecular Heterogeneity ◽  
Mechanical Instability ◽  
Concomitant Increase ◽  
Red Cell Membrane ◽  
Tryptic Fragment ◽  
Self Association

Abstract In hereditary pyropoikilocytosis (HPP), the red cell membrane skeletons exhibit a mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To determine the underlying molecular defect, we have subjected HPP spectrin to limited tryptic digestion, followed by one- and two-dimensional separations of the peptides. Two of the HPP kindreds exhibited a marked decrease in 80,000- dalton peptide (previously identified as the spectrin dimer-dimer contact domain of the alpha-subunit) and a concomitant increase of the 74,000-dalton polypeptide (presumably derived from the 80,000-dalton domain) and a decrease in a 22,000-dalton polypeptide. We now report tryptic digests of two other HPP kindred that are characterized by a decrease or complete absence of the 80,000-dalton tryptic fragment, with a concomitant increase in fragments at 46,000 and 17,000 daltons. The 46,000-dalton fragment separated into multiple spots on isoelectric focusing, ranging in isoelectric point from 5.25 to 5.35, and the 17,000-dalton fragment focused to a single spot at 5.4. Minor fragments at 56,000 and 22,000 daltons were also decreased, while a 38,000-dalton fragment increased. Limited tryptic digestion of the separated alpha- and beta-subunits revealed that the 74,000-dalton fragment in the first group of patients and the 46,000-dalton fragment in the second group of patients were derived from the alpha-subunit. Both subtypes exhibited a similar defect of spectrin self-association, with 30%-38% of spectrin dimers in O degrees C extracts. The results indicate that at least two distinct forms of structurally defective spectrin may give rise to the clinical presentation of HPP.

scholarly journals Abnormal spectrin in hereditary elliptocytosis

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 141-151
Author(s):  
SL Marchesi ◽  
WJ Knowles ◽  
JS Morrow ◽  
M Bologna ◽  
VT Marchesi
Keyword(s):  
Hemolytic Anemia ◽  
Alpha Subunit ◽  
Tryptic Digestion ◽  
Clinical Expression ◽  
Hereditary Elliptocytosis ◽  
Oligomer Formation ◽  
Conformational Alteration ◽  
Self Association

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.

scholarly journals Spectrin beta Tandil, a novel shortened beta-chain variant associated with hereditary elliptocytosis is due to a deletional frameshift mutation in the beta-spectrin gene

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 1066-1073 ◽  
Author(s):  
M Garbarz ◽  
L Boulanger ◽  
S Pedroni ◽  
MC Lecomte ◽  
H Gautero ◽  
...  
Keyword(s):  
Frameshift Mutation ◽  
Stop Codon ◽  
Molecular Defect ◽  
Reading Frame ◽  
Hereditary Elliptocytosis ◽  
C Terminus ◽  
The Family ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Self Association

Abstract An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3′ ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

scholarly journals Abnormal spectrin in hereditary elliptocytosis

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 141-151 ◽  
Author(s):  
SL Marchesi ◽  
WJ Knowles ◽  
JS Morrow ◽  
M Bologna ◽  
VT Marchesi
Keyword(s):  
Hemolytic Anemia ◽  
Alpha Subunit ◽  
Tryptic Digestion ◽  
Clinical Expression ◽  
Hereditary Elliptocytosis ◽  
Oligomer Formation ◽  
Conformational Alteration ◽  
Self Association

Abstract An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.

scholarly journals Spectrin beta Tandil, a novel shortened beta-chain variant associated with hereditary elliptocytosis is due to a deletional frameshift mutation in the beta-spectrin gene

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 1066-1073
Author(s):  
M Garbarz ◽  
L Boulanger ◽  
S Pedroni ◽  
MC Lecomte ◽  
H Gautero ◽  
...  
Keyword(s):  
Frameshift Mutation ◽  
Stop Codon ◽  
Molecular Defect ◽  
Reading Frame ◽  
Hereditary Elliptocytosis ◽  
C Terminus ◽  
The Family ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Self Association

An Argentinian family with hereditary elliptocytosis (HE) associated with a shortened beta-spectrin (Sp) chain was studied. As with most of the other shortened Sp beta-chains that have been described, this variant, called SpTandil, has impaired ability to participate in Sp dimer self-association, has lost its ability to become phosphorylated, and is associated with the presence of increased amounts of the alpha I 74-Kd fragment after partial tryptic digestion of Sp. The 3′ ends of the beta-Sp gene of affected patients were analyzed. cDNA was prepared by reverse transcription of peripheral blood mRNA and amplified by the polymerase chain reaction (PCR) using primers corresponding to sequences 400 bp apart on the cDNA, spanning the last three exons (X, Y, Z) of the beta-Sp gene. Agarose gel electrophoresis of the cDNA amplification showed the presence of one band, the size of which was apparently the same as the band amplified from mRNA of a normal control. cDNA from one HE patient was subcloned and sequenced. Several clones showed the presence of a 7-bp deletion at codon 2041 in exon X. Genomic DNA of all the affected members of the family were amplified by PCR using primers flanking the deletion and corresponding to sequences 128 bp apart on exon X. Analysis of the PCR products using electrophoresis on polyacrylamide gel showed the presence of 121- and 128-bp bands in all HE subjects, and an additional doublet migrating more slowly than the two bands, which corresponded to the presence of heteroduplexes. The mutation results in a shift of the normal reading frame and leads to a new amino acid sequence at the C-terminus of the mutant beta-Sp chain. A new in-frame stop codon is encountered downstream, leading to premature chain termination. The identification of the molecular defect in Sp beta Tandil provides information regarding the region of the beta-Sp chain that is important for both Sp dimer self-association and an indication of potential sites of phosphorylation of the chain.

scholarly journals Viscoelastic properties of red cell membrane in hereditary elliptocytosis

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 592-595 ◽  
Author(s):  
A Chabanel ◽  
KL Sung ◽  
J Rapiejko ◽  
JT Prchal ◽  
J Palek ◽  
...  
Keyword(s):  
Viscoelastic Properties ◽  
Molecular Defect ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Viscoelastic Parameters ◽  
Rbc Membrane ◽  
Dimensional Network ◽  
Self Association

Abstract The viscoelastic properties of the RBC membrane are in part determined by a submembrane network of proteins consisting of spectrin alpha beta heterodimers (SpD) assembled head-to-head to form spectrin tetramers (SpT) and spectrin oligomers (SpO). SpT, in turn, are connected into a two-dimensional network by the linkage of distal ends of SpT to protein 4.1 and actin. With the micropipette technique, we determined the membrane viscoelastic properties of RBCs from a subset of patients with hereditary elliptocytosis (HE); these RBCs exhibit membrane skeletal instability, defective SpD self-association, and a molecular defect in the alpha I domain of spectrin, which is involved in the SpD-SpD contact (HE SpD alpha-SpD). The elastic modulus and viscosity of the membrane were significantly higher for the HE RBCs than for the control cells. Incubation of normal cells with N-ethyl-maleimide (NEM) produced a similar defective SpD self-association and a significant increase in the viscoelastic parameters of the membrane. The data provide evidence that the mode of assembly of membrane spectrin in the cytoskeletal protein network plays a major role in determining the rheologic behavior of erythrocyte membrane.

scholarly journals Viscoelastic properties of red cell membrane in hereditary elliptocytosis

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 592-595
Author(s):  
A Chabanel ◽  
KL Sung ◽  
J Rapiejko ◽  
JT Prchal ◽  
J Palek ◽  
...  
Keyword(s):  
Viscoelastic Properties ◽  
Molecular Defect ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Protein 4.1 ◽  
Hereditary Elliptocytosis ◽  
Viscoelastic Parameters ◽  
Rbc Membrane ◽  
Dimensional Network ◽  
Self Association

The viscoelastic properties of the RBC membrane are in part determined by a submembrane network of proteins consisting of spectrin alpha beta heterodimers (SpD) assembled head-to-head to form spectrin tetramers (SpT) and spectrin oligomers (SpO). SpT, in turn, are connected into a two-dimensional network by the linkage of distal ends of SpT to protein 4.1 and actin. With the micropipette technique, we determined the membrane viscoelastic properties of RBCs from a subset of patients with hereditary elliptocytosis (HE); these RBCs exhibit membrane skeletal instability, defective SpD self-association, and a molecular defect in the alpha I domain of spectrin, which is involved in the SpD-SpD contact (HE SpD alpha-SpD). The elastic modulus and viscosity of the membrane were significantly higher for the HE RBCs than for the control cells. Incubation of normal cells with N-ethyl-maleimide (NEM) produced a similar defective SpD self-association and a significant increase in the viscoelastic parameters of the membrane. The data provide evidence that the mode of assembly of membrane spectrin in the cytoskeletal protein network plays a major role in determining the rheologic behavior of erythrocyte membrane.

Hereditary Elliptocytosis with a Spectrin Molecular Defect in a White Patient

1984 ◽  
Vol 71 (4) ◽  
pp. 235-240 ◽  
Author(s):  
M.C. Lecomte ◽  
D. Dhermy ◽  
M. Garbarz ◽  
H. Gautero ◽  
O. Bournier ◽  
...  
Keyword(s):  
White Patient ◽  
Molecular Defect ◽  
Hereditary Elliptocytosis
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