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J A Chasis

Publications and source records attributed to J A Chasis.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of integral membrane protein contributions to the deformability and stability of the human erythrocyte membrane.

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Glycophorin A dimerization and band 3 interaction during erythroid membrane biogenesis: in vivo studies in human glycophorin A transgenic mice.

Band 3 and glycophorin A (GPA) are the 2 most abundant integral proteins in the human erythrocyte membrane. Earlier studies suggested that the 2 proteins may associate not only in the mature erythrocyte membrane, but also during their posttranslational processing and intracellular trafficking. The purpose of this study was to directly examine the GPA-band 3 interaction in vivo and determine the nature of this association during erythroid membrane biogenesis. Transgenic mice were generated expressing the human glycophorin A gene and were used to examine how the induction of human GPA expression affected the levels of murine GPA and band 3 expression in the red cell membrane. Murine GPA expression was reduced in erythrocytes expressing human GPA, whereas the level of band 3 expression remained constant, implying a tight coupling of band 3 and GPA expression in the membrane of mature red cells. In vivo GPA dimerization was not modulated solely by the GPA transmembrane motif, but the distance between this motif and the basic residues on the cytoplasmic side of the transmembrane domain may also be important. In addition, GPA monomers with varying degrees of glycosylation dimerized, providing clear evidence that carbohydrate structures on the extracellular domain do not affect dimerization. The association between the multiple transmembrane-spanning protein, band 3, and the single transmembrane-spanning sialoglycoprotein, GPA, may serve as a model for interactions of other multi-pass and single-pass polypeptides during membrane biogenesis.

Animals↗
PubMed2001

Lutheran blood group glycoprotein and its newly characterized mouse homologue specifically bind alpha5 chain-containing human laminin with high affinity.

Lutheran blood group glycoproteins (Lu gps) are receptors for the extracellular matrix protein, laminin. Studies suggest that Lu gps may contribute to vaso-occlusion in sickle cell disease and it has recently been shown that sickle cells adhere to laminin isoforms containing the alpha5 chain (laminin 10/11). Laminin alpha5 is present in the subendothelium and is also a constituent of bone marrow sinusoids, suggesting a role for the Lu/laminin interaction in erythropoiesis. The objectives of the current study were to define more precisely the molecular interactions of the extracellular and intracellular regions of human Lu and to clone and characterize a mouse homologue. To this end, complementary DNA and genomic clones for the mouse homologue were sequenced and the mouse Lu gene mapped to a region on chromosome 7 with conserved synteny with human 19q13.2. Mouse and human Lu gps are highly conserved (72% identity) at the amino acid sequence level and both mouse and human Lu gps specifically bind laminin 10/11 with high affinity. Furthermore, the first 3, N-terminal, immunoglobulin superfamily domains of human Lu are critical for this interaction. The results indicated that the cytoplasmic domain of BRIC 221-labeled human Lu gp is linked with the spectrin-based skeleton, affording the speculation that this interaction may be critical for signal transduction. These results further support a role for Lu gps in sickle cell disease and indicate the utility of mouse models to explore the function of Lu gp-laminin 10/11 interaction in normal erythropoiesis and in sickle cell disease.

Amino Acid Sequence↗
PubMed2000

Molecular and functional characterization of protein 4.1B, a novel member of the protein 4.1 family with high level, focal expression in brain.

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Amino Acid Sequence↗
PubMed1999

Erythroid cell adhesion molecules Lutheran and LW in health and disease.

The Lutheran and LW glycoproteins are blood group-active proteins found at the surface of human red cells. The Lutheran glycoprotein (Lu gp) is a member of the immunoglobulin superfamily (IgSF) that binds the extracellular matrix protein laminin, in particular, laminin isoforms containing the alpha 5 subunit. The LW glycoprotein (LW gp), also an IgSF member, has substantial sequence homology with the family of intercellular adhesion molecules (ICAMs). LW gp binds the integrin very late antigen-4 (VLA-4, alpha 4 beta 1) and alpha V-containing integrins. Studies on the expression of LW and Lu gps during erythropoiesis utilizing in vitro cultures of haemopoietic progenitor cells have shown that LW gp expression precedes that of Lu gp. These observations have led to the suggestion that LW gp on erythroblasts may interact with VLA-4 on macrophages to stabilize erythroblastic islands in normal bone marrow and that Lu gp may facilitate trafficking of more mature erythroid cells to the sinusoidal endothelium where alpha 5-containing laminins are known to be expressed. Levels of Lu gp and LW gp expression on sickle red cells are greater than on normal red cells and sickle red cells adhere to alpha 5-containing laminins. These data suggest that the Lu and LW molecules may contribute to the vaso-occlusive events associated with episodes of acute pain in sickle cell disease.

Anemia, Sickle Cell↗
PubMed1999

Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

3T3 Cells↗
PubMed1999

Protein 4.1R-deficient mice are viable but have erythroid membrane skeleton abnormalities.

A diverse family of protein 4.1R isoforms is encoded by a complex gene on human chromosome 1. Although the prototypical 80-kDa 4.1R in mature erythrocytes is a key component of the erythroid membrane skeleton that regulates erythrocyte morphology and mechanical stability, little is known about 4.1R function in nucleated cells. Using gene knockout technology, we have generated mice with complete deficiency of all 4.1R protein isoforms. These 4.1R-null mice were viable, with moderate hemolytic anemia but no gross abnormalities. Erythrocytes from these mice exhibited abnormal morphology, lowered membrane stability, and reduced expression of other skeletal proteins including spectrin and ankyrin, suggesting that loss of 4. 1R compromises membrane skeleton assembly in erythroid progenitors. Platelet morphology and function were essentially normal, indicating that 4.1R deficiency may have less impact on other hematopoietic lineages. Nonerythroid 4.1R expression patterns, viewed using histochemical staining for lacZ reporter activity incorporated into the targeted gene, revealed focal expression in specific neurons in the brain and in select cells of other major organs, challenging the view that 4.1R expression is widespread among nonerythroid cells. The 4.1R knockout mice represent a valuable animal model for exploring 4.1R function in nonerythroid cells and for determining pathophysiological sequelae to 4.1R deficiency.

Animals↗
PubMed1998

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Alternative Splicing↗
PubMed1997

Erythrocyte membrane vesiculation: model for the molecular mechanism of protein sorting.

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.

Cytoskeleton↗
PubMed1997

Structural protein 4.1 is located in mammalian centrosomes.

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture.

Animals↗
PubMed1997

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

3T3 Cells↗
PubMed1997

Cell shape-dependent regulation of protein 4.1 alternative pre-mRNA splicing in mammary epithelial cells.

Expression of the complex gene encoding multiple isoforms of structural protein 4.1 is regulated by alternative pre-mRNA splicing. During erythropoiesis, developmental stage-specific inclusion of exon 16 generates protein 4.1 isoforms having a fully functional spectrin-actin binding domain. Here we show that human mammary epithelial cells (HMEC), coincident with the dramatic morphological changes induced by altered culture conditions, exhibit a novel pre-mRNA splicing switch involving a new exon (exon 17B, 450 nucleotides) in the COOH-terminal coding region. 4.1 RNA expressed in proliferating HMEC adherent to culture dishes mostly excluded exon 17B, whereas 4.1 transcripts processed in nondividing suspension cultures of HMEC strongly included this exon. This pre-mRNA splicing switch was reversible: cells transferred from poly(2-hydroxyethyl methacrylate) back to plastic resumed cell division and down-regulated exon 17B expression. More detailed studies revealed complex tissue-specific alternative splicing of exon 17B and another new exon 17A (51 nucleotides). These results predict the existence of multiple 4.1 protein isoforms with diverse COOH termini. Moreover, they strongly suggest that regulation of gene expression during differentiation of epithelial cells is mediated not only by transcriptional mechanisms, but also by post-transcriptional processes such as alternative pre-mRNA splicing.

Alternative Splicing↗
PubMed1996

Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid development.

Expression of multiple protein 4.1 isoforms in erythroid progenitors and in a variety of nonerythroid tissues results from alternative pre-mRNA splicing. In 4.1 pre-mRNA, several translation initiation sites are present; synthesis of isoforms larger than 80 kD occurs when an upstream 5' AUG is spliced in, whereas the 80-kD mature erythroid isoform is produced when the upstream AUG is spliced out and translation is initiated at the downstream AUG. During erythropoiesis, this splicing switch is developmentally regulated. We studied this developmental switch in hereditary elliptocytosis 4.1Alg, in which a DNA rearrangement involving the exon containing the downstream AUG results in loss of coding capacity for the 80-kD 4.1, leading to mature red blood cells deficient in 4.1 with decreased membrane mechanical stability. Analysis of erythroblast RNA by reverse transcriptase-polymerase chain reaction showed that, although it retained the upstream AUG, its coding region was approximately 2.2 kb, compared with approximately 2.5 kb of normal 4.1 mRNA, because of the deletion of exons, including the one that codes for the downstream AUG. Immunofluorescent microscopy and Western blot analysis documented protein 4.1 expression in HE 4.1Alg erythroblasts. These studies emphasize the crucial role of differentiation-regulated RNA splicing because, within the same erythroid tissue, the HE 4.1Alg phenotype did not appear until after the differentiation-associated splicing event.

Base Sequence↗
PubMed1994

Cooperative action between band 3 and glycophorin A in human erythrocytes: immobilization of band 3 induced by antibodies to glycophorin A.

The ability of transmembrane receptor proteins to change their association with the cytoskeleton in response to ligand binding seems to be a key mechanism of signal transduction across membranes. To investigate the molecular features of this mechanism we have used the red cell membrane as a model system to study signal transduction through the integral protein, glycophorin A. In these studies the lateral mobility of integral proteins was measured in situ by fluorescence recovery after photobleaching, and membrane rigidity was characterized by micropipette aspiration technique. We found that binding either a monoclonal antibody or its monovalent Fab to the exoplasmic domain of glycophorin A in normal red cells immobilized the receptor and rigidified the membrane. Further, immobilization and rigidification did not occur when antibodies were bound to Miltenberger V cells containing a mutant form of glycophorin A lacking the cytoplasmic domain. These results imply that the site of the immobilization/rigidification lies within the membrane skeletal structure, not in exofacial receptor crosslinking, and requires the extended cytoplasmic domain of normal glycophorin A. In addition, we found that glycophorin A immobilization and membrane skeletal rigidification were accompanied by immobilization of band 3 receptors. This unexpected result indicates a cooperative coupling between liganded glycophorin A, band 3, and the membrane skeleton. We speculate that cooperation of this type may represent a general mechanism for cytoskeletal linkage and transformation initiated by receptors with short cytoplasmic sequences, such as integrins.

Animals↗
PubMed1993

Initial assessment of the beneficial effect of partial splenectomy in hereditary spherocytosis.

Clinical manifestations of hereditary spherocytosis (HS), the most common red blood cell (RBC) membrane disorder, can be abrogated or markedly reduced by splenectomy. However, concerns regarding risks from overwhelming infections after splenectomy have restricted its use, especially in children. This study was designed to determine if partial splenectomy can decrease the hemolytic rate while maintaining phagocytic function of the spleen. Partial splenectomy was performed in 11 children (age 2 to 13) with HS. The effect on hemolytic rate was assessed by comparing the presurgical and postsurgical values for hemoglobin, reticulocyte number, and RBC life span. The residual splenic phagocytic function was assessed using technetium 99m scans and by enumerating the percentage of pitted RBCs in circulation. There were no complications from the surgical procedure in any of the 11 individuals. Following partial splenectomy, hemoglobin increased on the average by 3 g/dL, reticulocyte count decreased by 300 x 10(6)/L, and RBC life span was substantially prolonged. Normal technetium uptake was noted in the splenic remnant and the percentage of pitted RBCs was in the normal range. Partial splenectomy is effective in decreasing the hemolytic rate while maintaining residual splenic phagocytic function of the spleen in HS. We conclude that the use of this procedure as treatment for RBC membrane disorders warrants consideration, especially in infants under 5 years of age who need frequent transfusions.

Adolescent↗
PubMed1993

Differentiation-associated switches in protein 4.1 expression. Synthesis of multiple structural isoforms during normal human erythropoiesis.

Erythroid differentiation is accompanied by dramatic alterations in morphology and membrane mechanical properties resulting, in large part, from reorganization of the membrane skeletal protein network. The 80-kD protein 4.1 is an important organizational component of this membrane skeleton. Recently, it has been recognized that multiple structural isoforms of 4.1 are encoded by a single gene via alternative pre-mRNA splicing, and that an upstream ATG can be spliced in and used for translation of high molecular weight 4.1. We are exploring the hypothesis that differentiation-associated switches in protein 4.1 structure play an important role in membrane reorganization. To study changes in 4.1 gene expression during normal human differentiation, we analyzed 4.1 protein and mRNA structure at various developmental stages. Using immunofluorescence microscopy, we observed high molecular weight 4.1 isoforms in preproerythroblasts producing punctate, predominantly cytoplasmic staining with a perinuclear area of intense fluorescence, while mature red cells expressed very little high molecular weight 4.1. Isoforms containing an alternatively expressed 102-nucleotide exon near the COOH terminus were abundant in both preproerythroblasts and mature cells but produced a punctate distribution of fluorescence over the entire preproerythroblast and intense membrane-associated fluorescence in the erythrocyte. Characterization of RNA by polymerase chain reaction and nuclease protection assays revealed a differentiation-associated switch in pre-mRNA splicing in the spectrin-actin binding domain. Since this domain plays a critical role in regulating membrane material properties, we speculate that this switch may be crucial to reorganization of the skeletal network during erythropoiesis. We conclude that 4.1 isoforms are differentially expressed and differentially localized during erythropoiesis, and that this isoform family is likely to have diverse functions during terminal differentiation.

Alternative Splicing↗
PubMed1993

An isoform-specific mutation in the protein 4.1 gene results in hereditary elliptocytosis and complete deficiency of protein 4.1 in erythrocytes but not in nonerythroid cells.

Multiple protein 4.1 isoforms are expressed in a variety of tissues through complex alternative pre-mRNA splicing events, one function of which is to regulate use of two alternative translation initiation signals. Late erythroid cells express mainly the downstream initiation site for synthesis of prototypical 80-kD isoforms; nonerythroid cells in addition use an upstream site to encode higher molecular mass isoform(s). In this study, we examined the effects of a 5' gene rearrangement in a family with hereditary elliptocytosis and complete deficiency of erythrocyte 4.1 protein on 4.1 isoform expression in erythroid vs. nonerythroid cells. Patient 4.1 mRNAs from reticulocytes, fibroblasts, and B lymphocytes were amplified by reverse transcriptase/polymerase chain reaction techniques and shown to exhibit a 318-nucleotide deletion that encompasses the downstream AUG, but leaves intact the upstream AUG. Immunoblot analysis revealed a total deficiency of 4.1 in patient red cells and a selective deficiency of 80-kD isoform(s) but not high molecular weight 4.1 in patient nonerythroid cells. Thus, the 4.1 gene mutation in this family produces an isoform-specific deficiency that is manifested clinically in tissue-specific fashion, such that red cells are affected but other cell types are unaffected because of tissue-specific differences in RNA splicing and translation initiation.

B-Lymphocytes↗