Abnormal erythrocyte band 4.1 protein in myelodysplastic syndrome with elliptocytosis

Summary A case of myelodysplastic syndrome with haemolytic anaemia and a marked elliptocytosis is reported. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membrane proteins revealed that the patient's band 4.1 was decreased to about 50–70% of that of control and contained abnormal molecule migrating in a faster mobility than normal band 4.1. which was confirmed by immunoblotting. The actin/spectrin ratio of the patient's ghosts diminished to about 70% of that of control ghosts. Flowcytometric analysis showed that the glycophorin C content of the patient's erythrocytes was reduced but maintained the level of about 70% of that of normal, indicating that the glycophorin C-band 4.1 interaction might not be so seriously damaged as to cause elliptocytic shape change. We postulate that the abnormal band 4.1 produced from the abnormal erythroid clone may be the primary molecular defect and result in a dysregulation of spectrinactin interaction to cause erythrocyte shape change and membrane instability.     

A variant of erythrocyte membrane skeletal protein band 4.1 associated with hereditary elliptocytosis

A family comprising three patients (a mother and two children) with mild hereditary elliptocytosis was studied. Each patient had prominent elliptocytosis, reduced red cell deformability, and normal erythrocyte thermal sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the erythrocyte membranes in each patient showed decreased levels of band 4.1 (approximately half of the normal value) and the presence of an additional band migrating below protein band 4.2. This additional band was shown to derive from protein 4.1. Comparative partial proteolytic mapping of protein 4.1 and the additional band revealed a number of common peptides. Enzyme-linked immunoelectrotransfer blots of the patients' erythrocyte membranes using a monoclonal antibody to protein 4.1 revealed that, in addition to protein 4.1, two other bands below protein 4.2 were stained; one of these bands migrated in the same position as the additional band detected in the Coomassie Blue-stained gels. Immunoblotting of the patients' whole cells using the antibody to protein 4.1 revealed that this altered band 4.1 occurred as such in the intact red cell. SDS-PAGE of protein 4.1 purified from one patient showed the presence of two lower molecular weight bands below protein 4.1; the lower band migrated in the same position as the additional band found on SDS-PAGE of the patients' erythrocyte membranes. The patient's purified protein 4.1 displayed a decrease of about 40% in the binding activity with crude spectrin extracted from normal controls. Spectrin-spectrin interactions were normal in the three patients. The additional band present in the patients' red cell membranes probably represents a proteolytic degradation product. This alteration, present both in whole cells and isolated membranes, might affect the intact cells in vivo. We suggest that the patients' erythrocyte membrane instability may be related to the presence of an abnormal protein 4.1 whose modulatory influence on the spectrin-actin interaction in the skeleton is defective.     

Partial deficiency of protein 4.1 in hereditary elliptocytosis

Protein 4.1, an important component of the red cell membrane skeleton, was quantitated relative to protein 3 after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of membranes isolated from red cells of members of 14 kindreds with hereditary elliptocytosis (HE) who reside in South Africa. A partial deficiency of protein 4.1 (mean 30% reduction) was inherited in autosomal dominant fashion in five white kindreds giving a frequency of 0.36 of HE families studied. Immunoblots of membrane proteins separated by SDS-PAGE and probed with a monoclonal antibody to protein 4.1 did not reveal any proteolytic fragments in the 4.1-deficient subjects that could account for the reduction of this protein. These studies draw attention to the relatively high frequency of this condition as a cause of HE in white subjects in this country.     

Hereditary elliptocytosis with protein band 4.1 deficiency in the dog

A dog with persistent elliptocytosis was studied. The dog had membrane protein band 4.1 deficiency, microcytosis, shortened erythrocyte lifespan, increased osmotic sensitivity, and a mild glutathione deficiency. Erythrocyte deformability and membrane stability were adversely effected. The dog's parents had decreased band 4.1, decreased stability, and some elliptocytosis. This disorder in dogs closely resembles human patients with band 4.1 deficiency and should provide a valuable animal model to study the erythrocyte membrane cytoskeleton.     

A molecular study of heterozygous protein 4.1 deficiency in hereditary elliptocytosis

Genomic DNA from five kindreds and two individuals with hereditary elliptocytosis [HE(4.1+)] and a partial deficiency of protein 4.1 [HE(4.1+)] was extracted and probed with a cDNA for protein 4.1. When using a fragment of the cDNA that encompassed the coding region of the gene, two restriction fragment length polymorphisms segregating with protein 4.1 deficiency were found in one kindred when using the enzymes BgIII and PvuII but were not seen in the other HE(4.1+) subjects or in 20 random control individuals. DNA digested with three other enzymes (HindIII, EcoRI, TaqI) produced restriction patterns similar to controls. The unique BgIII and PvuII polymorphisms probably reflect a rearrangement of the coding region of the protein 4.1 gene as the underlying cause of the partial protein 4.1 deficiency in this family. A less likely possibility is that these polymorphisms represent coincidental single base changes unrelated to the primary gene defect.     

Molecular pathology of inherited erythrocyte membrane disorders: hereditary spherocytosis and elliptocytosis.

Hereditary spherocytosis and elliptocytosis are common genetic defects of the red blood cell membrane skeleton. In recent years rapid advances have been made in the knowledge of the protein structure and assembly of the cytoskeleton. Thanks to the wide use of protein analysis methods several alterations have been discovered in functionally important domains of the different cytoskeletal proteins in these diseases. The cloning of cDNA for the majority of the cytoskeletal proteins allows us to begin elucidating some of these defects at the DNA level. This paper will review the effects of recent advances upon: cytoskeleton structure and assembly; molecular pathology of spherocytosis, elliptocytosis and pyropoikilocytosis.     

Gerbich reactivity in 4.1(—) hereditary elliptocytosis and protein 4.1 level in blood group Gerbich deficiency

The membrane polypeptide composition and the blood group Gerbich phenotype of red cells from 4.1 (-) hereditary elliptocytic patients and from Gerbich-negative donors, who display two unrelated genetic abnormalities, were compared. In homozygous 4.1 (-) hereditary elliptocytosis where the primary defect was presumably the absence of the membrane skeletal protein 4.1, there was approximatively a 70% reduction in the minor sialoglycoproteins beta and gamma. This was associated with a severe reduction of blood group Gerbich reactivity as determined with both murine monoclonal and human anti-Gerbich antibodies. In the heterozygous state in the presence of one haploid set of protein 4.1 gene there was only a modest decrease in glycoproteins beta and gamma and the Gerbich serological reactivity was within normal limits. In homozygous Gerbich-negative red cells which lack glycoproteins beta and gamma but do not display elliptocytic red cells, the levels of protein 4.1 was repeatedly found within or just below the lowest values of normal controls. In the heterozygous Gerbich-negative conditions, glycoproteins beta and gamma were present in reduced amounts but the blood group Gerbich reactivity fell within normal limits since the anti-Gerbich reagents used were unable to detect a dosage effect. The amount of protein 4.1 was normal. These results add further support to the view that protein 4.1 and the sialoglycoproteins beta and gamma are physically linked in vivo which in some way serve to maintain red cell shape and integrity. Of interest was the finding that absence of protein 4.1 had a greater influence on the level of membrane glycoproteins beta and gamma than did the absence of beta and gamma glycoproteins on band 4.1.     

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.     

Autoantibody against erythrocyte protein 4.1 in a patient with autoimmune hemolytic anemia

We observed the presence of a new autoantibody, anti-erythrocyte protein 4.1, in a patient with autoimmune hemolytic anemia (AIHA). Western blotting analysis revealed that IgG from the patient's plasma reacted with erythrocyte protein 4.1. However, among other patients with hemolytic diseases (six having AIHA and three each having either hereditary spherocytosis, elliptocytosis, or lead poisoning) as well as among control subjects, no antibody activity to protein 4.1 was observed. In addition to the anti-protein 4.1 antibody, two different kinds of anti-erythrocyte antibodies were detected by conventional serological studies in this patient. One of them was an anti-Ena-like antibody in the eluate from the patient's erythrocytes, while another was the anti-S-specific antibody in the plasma. An elution study and an absorption study using S antigen-positive erythrocytes demonstrated that the anti-protein 4.1 antibody differed from both the anti-Ena-like antibody and the anti-S antibody. Familial analysis of the patient revealed the same antibody in her brother, who did not have hemolytic anemia. These results demonstrate that anti-protein 4.1 antibody is considered to be included in the spectrum of anti-cytoskeleton autoantibodies, which have been observed in patients having increased cell lysis as well as in healthy subjects.     

Analyses of HLA linkage in white families with multiple cases of seropositive rheumatoid arthritis

Families with multiple cases of seropositive rheumatoid arthritis (RA), identified through 2 probands with the disease, were analyzed for genetic linkage between an autosomal susceptibility gene for RA and the HLA loci. These analyses were carried out over a wide range of penetrances (0.1-0.4), with prevalence for the disease fixed at 0.01 or 0.04. In some models, a sporadic frequency of 20% was assumed. Close linkage to the HLA loci was ruled out for all models where the gene was autosomal dominant, except for those with low penetrance and with prevalence set at 0.04. The models in which the gene was autosomal recessive produced results similar to those in models where it was autosomal dominant when prevalence was set at 0.01, and close linkage was ruled out, except at very low penetrances. With prevalence set at 0.04, the autosomal recessive gene model produced log odds scores which were all negative (-2.6 to -0.3). Therefore, the evidence in these families does not support close linkage with HLA, even though a significant DR4 RA population association exists. The data suggest that non-major histocompatibility complex (MHC) genes may contribute to the development of RA and, therefore, disease susceptibility may involve the genetic interaction of one or more MHC genes and non-MHC genes, in addition to environmental agents.     

Deamidation of human erythrocyte protein 4.1: possible role in aging.

The human erythrocyte membrane protein 4.1 exists in two major electrophoretic forms: 4.1a (80 Kd) and 4.1b (78 Kd). Mass spectrometry and amino acid analysis of the proteolytic peptides derived from carboxyl-terminal regions of these proteins indicate that they differ by deamidation of two aspargine residues at positions 478 and 502. Electrophoretic analysis of carboxyl-terminal peptides has shown that the mobility difference between the two polypeptides is due to the deamidation of Asn502 and not that of Asn478. This observation was confirmed by converting a congener of the protein 4.1b to 4.1a by site-directed mutagenesis of Asn502 to Asp. These results unambiguously demonstrate that deamidation of Asn502 is responsible for conversion of protein 4.1b to 4.1a. Since the conversion of protein 4.1b to 4.1a, under physiological conditions, occurs in a time-dependent manner, our study clearly shows that deamidation is an excellent marker for red blood cell aging.     

Red cell membrane alteration involving protein 4.1 and protein 3 in a case of recessively inherited haemolytic anaemia

We present 2 siblings with a severe congenital haemolytic anaemia. Red cells displayed a variety of abnormal shapes, including leptocytes, schizocytes and elliptocytes. Repeatedly, skeletal protein 4.1 appeared reduced by 30%. The 4.1a/4.1b ratio was normal despite the haemolytic state. No change could be detected in spectrin, nor in sialoglycoproteins. Band 3 was denser, narrower and displaced downward. The parents, who are consanguineous, were devoid of any obvious biochemical abnormality; however, their red cells were not normal. These 2 cases with reduced protein 4.1 clearly depart from 4.1 (-) hereditary elliptocytosis. The possibility of an altered binding of protein 4.1 to some other membrane component is discussed.     

Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis

Mice with disruptions of the red blood cell (RBC) cytoskeleton provide severe hemolytic anemia models in which to study multiorgan thrombosis and infarction. The incidence of cerebral infarction ranges from 70% to 100% in mice with alpha-spectrin deficiency. To determine whether mutant RBCs abnormally bind adhesive vascular components, we measured adhesion of mouse and human RBCs to immobilized human thrombospondin (TSP) and laminin (LM) under controlled flow conditions. Mutant RBCs had at least 10-fold higher adhesion to TSP compared with normal RBCs (P <.006). Mutant relative to unaffected RBC adhesion to LM was significantly (P <.01) increased as well. Treatment of RBCs with the anionic polysaccharide dextran sulfate inhibited mutant RBC adhesion to TSP (P <.001). Treatment of RBCs with antibodies to CD47 or the CD47-binding TSP peptide 4N1K did not inhibit TSP adhesion of RBCs. Previously, we have shown that infarcts in alpha-spectrin-deficient sph/sph mice become histologically evident beginning at 6 weeks of age. TSP adhesion of RBCs from 3- to 4- and 6- to 8-week-old sph/sph mice was significantly higher than RBCs from adult mice (> 12 weeks old; P <.005). While the mechanism of infarction in these mice is unknown, we speculate that changes in RBC adhesive characteristics contribute to this pathology.     

Association of inherited dysfibrinogenaemia and protein C deficiency in two unrelated families

Summary An inherited association of dysfibrinogenaemia and protein C deficiency was found in three members of the same family. The propositus was a 48-year-old man who suffered from severe and rapidly complicated atherosclerosis of the aorta and lower limbs arteries, which perhaps suggests that the association of these two molecular abnormalities may have enhanced the thrombotic process. The abnormal fibrinogen had a reduced ability to bind thrombin which may be thrombogenic. We found the same inherited association of dysfibrinogenaemia and protein C deficiency in a patient with venous thrombosis. The functional abnormality of the fibrinogen, which could have been responsible for thrombosis, was delayed proteolysis by plasmin. Not only fibrinogen, but also fibrin clots were resistant to plasmic degradation. These observations raise two questions: (1) Is the association of a protein C deficiency with a dysfibrinogenaemia fortuitous or the result of a common mechanism? (2) Is there a link between an increased thrombotic tendency and either both of the defects of haemostasis that we have found, or only one of them?     

Protein 4.1 deficiency and deletion of chromosome 20q are associated with acquired elliptocytosis in myelodysplastic syndrome

We report a case of myelodysplastic syndrome (MDS), associated with prominent elliptocytosis. A 66-year-old male presented with peripheral pancytopenia, and was diagnosed with MDS [refractory anaemia (RA)]. Apart from marked elliptocytosis, dyshaematopoietic features were not evident in his peripheral blood or hypercellular bone marrow. After 18 months, he had progressed to RA with excess blasts in transformation. Analysis of red blood cell membrane proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a reduced quantity of protein 4.1 (30% of control). Deletion of chromosome 20q was identified by conventional cytogenetic analysis and fluorescence in situ hybridization. Marked elliptocytosis, persistent for more than 17 months, decreased strikingly after chemotherapy with idarubicin and Ara-C. These findings suggest that acquired elliptocytosis occurred as an unusual morphological feature of MDS, associated with abnormalities of protein 4.1 and chromosome 20q.