Biomedical subjects

R Winardi

Publications and source records attributed to R Winardi.

7 recordsLinked to original sources

PubMed1995

Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1.

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high-affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding.

Actins↗

PubMed1995

Defining of the minimal domain of protein 4.1 involved in spectrin-actin binding.

The spectrin-actin-binding domain of protein 4.1 is encoded by a 21-amino acid alternative exon and a 59-amino acid constitutive exon. To characterize the minimal domain active for interactions with spectrin and actin, we functionally characterized recombinant 4.1 peptides containing the 21-amino acid cassette plus varying portions of the 59-amino acid cassette (designated 21.10 to 21.59). Peptide 21.43 was shown fully functional in binary interactions with spectrin (by cosedimentation and coimmunoprecipitation experiments) and in ternary complex formation with spectrin and actin (by an in vitro gelation assay). Further truncation produced peptides incapable of binary interactions but fully competent for ternary complex formation (peptides 21.36 and 21.31), shorter peptides with reduced ternary complex activity and altered kinetics (21.26 and 0.59), and inactive peptides (21.20 and 21.10). Binding studies and circular dichroism experiments suggested that residues 37-43 of the constitutive domain were directly involved in spectrin binding. These data indicate that 4.1-spectrin binary interaction requires the 21-amino acid alternative cassette plus the 43 N-terminal residues of the constitutive domain. Moreover, the existence of two possible ternary complex assembly pathways is suggested: one initiated by 4.1-spectrin interactions, and a second by 4.1-actin interactions. The latter may require a putative actin binding motif within the 26 N-terminal residues of the constitutive domain.

Actins↗

PubMed1995

Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.

Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.

Actins↗

PubMed1993

Molecular analysis of glycophorin C deficiency in human erythrocytes.

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.

Base Sequence↗

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↗

PubMed1992

Molecular basis for membrane rigidity of hereditary ovalocytosis. A novel mechanism involving the cytoplasmic domain of band 3.

Hereditary ovalocytic red cells are characterized by a marked increase in membrane rigidity and resistance to invasion by malarial parasites. The underlying molecular defect in ovalocytes remained a mystery until Liu and colleagues (N. Engl. J. Med. 1990. 323:1530-38) made the surprising observation that the ovalocytic phenotype was linked to a structural polymorphism in band 3, the anion transporter. We have now defined the mutation in band 3 gene and established the biophysical sequelae of this mutation. This mutation involves the deletion of amino-acids 400-408 in the boundary between the cytoplasmic and the first transmembrane domains of band 3. The biophysical consequences of this mutation are a marked decrease in lateral mobility of band 3 and an increase in membrane rigidity. Based on these findings, we propose the following model for increased membrane rigidity. The mutation induces a conformational change in the cytoplasmic domain of band 3, leading to its entanglement in the skeletal protein network. This entanglement inhibits the normal unwinding and stretching of the spectrin tetramers necessary for membrane extension, leading to increased rigidity. These findings imply that the cytoplasmic domain of an integral membrane protein can have profound effects on membrane material behavior.

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗