Mary A. Risinger

Sites of Regulated Phosphorylation that Control K-Cl Cotransporter Activity

Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3s intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.

AN ERYTHROPOIETIN FUSION PROTEIN COMPRISED OF IDENTICAL REPEATING DOMAINS EXHIBITS ENHANCED BIOLOGICAL PROPERTIES

The hematopoietic growth factor erythropoietin (Epo) initiates its intracellular signaling cascade by binding to and inducing the homodimerization of two identical receptor molecules. We have now constructed and expressed in COS cells a cDNA encoding a fusion protein consisting of two complete human Epo domains linked in tandem by a 17-amino acid flexible peptide. On SDS-polyacrylamide gel electrophoresis, the Epo-Epo fusion protein migrated as a broad band with an average apparent molecular mass of 76 kDa, slightly more than twice the average apparent molecular mass of Epo, 37 kDa. EnzymaticN-deglycosylation resulted in an Epo-Epo species that migrated on SDS-polyacrylamide gel electrophoresis as a narrow band with an average apparent molecular mass of 39 kDa. The specific activity of the Epo-Epo fusion protein in vitro (1,007 IU/μg; 76 IU/pmol) was significantly greater than that of Epo (352 IU/μg; 13 IU/pmol). Moreover, secretion of Epo-Epo by COS cells was 8-fold greater than that of Epo. Subcutaneous administration of a single dose of Epo-Epo to mice resulted in a significant increase in red blood cell production within 7 days. In contrast, administration of an equivalent dose of conventional recombinant Epo was without effect. The pharmacokinetic behavior of Epo-Epo differed significantly from that of Epo. The results suggest that Epo-Epo may have important biological and therapeutic advantages.

A point mutation in the protein 4.2 gene (allele 4.2 Tozeur) associated with hereditary haemolytic anaemia

A recessively transmitted haemolytic anaemia associated with the lack of protein 4 2 was found in a Tunisian kindred. Trace amounts of this protein (72 kD component) became visible using high‐sensitivity Western blots. Band 3 and ankyrin genes were excluded as candidate genes by linkage studies, and nucleotide sequencing of band 3 cytoplasmic domain cDNA revealed no alteration. In contrast, protein 4.2 gene contained in the homozygous state a mutation at position 310: CGA → CAA (Arg → Gln). This mutation defining allele 4.2 Tozeur was co‐inherited with the disease. The mRNA encoding the variant protein was normal in size and approximately normal in amount. Recombinant protein 4.2 Tozeur bound normally to red cell IOVs but disclosed an increased susceptibility to proteolysis in vitro. We infer that the nearly total absence of protein 4.2 in the patients results from imbalance between destruction and synthesis of mutated protein 4.2 prior to its binding to the membrane.

Endocytosis of cell-cell junctions and spontaneous cell disaggregation in a cultured human ovarian adenocarcinoma (COLO 316)

Although cultured COLO 316 human ovarian adenocarcinoma cells are joined by extensive tight junctions and numerous demosomes in confluent monolayers, viable cells may be spontaneously released into the nutrient medium. Intracytoplasmic vesicles containing tight junctional and desmosomal elements were identified in freeze-fracture and thin section preparations of the released cells and some vesicles exhibited structural signs of degradation. Possible mechanisms for tight junctional and desmosomal interiorization and the possible relationship between junctional interiorization and certain malignant behaviors are discussed.

K-Cl Cotransporter Gene Expression during Human and Murine Erythroid Differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1–7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Volume regulation and KCl cotransport in reticulocyte populations of sickle and normal red blood cells

The potassium chloride co-transporter (KCC) is a member of the electroneutral cation chloride family of cotransporters found in multiple tissues that are involved in transepithelial ion transport and regulation of intracellular ion content and cell volume. We have shown previously that three of the four KCC genes - KCC1, KCC3, and KCC4 - are expressed in red blood cells (RBC) (Exp. Hem. 33:624, 2005). Functionally, the KCC mediates volume reduction of reticulocytes that establishes the higher cellular hemoglobin concentration (CHC) of mature RBC. KCC activity is higher in reticulocytes and diminishes with age. KCC activity in RBC containing sickle hemoglobin (SS RBC) is elevated compared to normal (AA RBC) in part due to reticulocytosis in SS blood. However, we have demonstrated that SS reticulocytes have abnormal regulation of KCC activity leading to increased CHC upon activation of KCC compared to AA reticulocytes (Blood 104:2954, 2004; Blood 109:1734, 2007). These findings implicate KCC as a factor in the dehydration of SS RBC, which leads to elevated Hb S concentration and enhances Hb S polymerization and hemolysis. Because KCC activity correlates with cell age, standard flux measurements on blood samples with different numbers of reticulocytes or young non-reticulocytes are not comparable. The Advia automated cell counter measures cell volume (MCV) and cellular hemoglobin concentration (CHC) in reticulocytes, an age-defined population of cells, and thus circumvents the problem of variable reticulocyte counts among SS and AA blood samples. In this study, reticulocyte CHC measurements on fresh blood demonstrated a clear difference between AA and SS cells, reflecting in vivo dehydration of SS reticulocytes, although there was significant inter-individual variation, and the CHC distributions of the two groups overlapped. After KCC activation in vitro by cell swelling using the nystatin method, the initial changes in reticulocyte MCV and CHC with time were used to estimate flux rates mediated by KCC, assuming that changes were associated with isotonic KCl movements. After 20-30min a final steady state MCV/CHC (set point) was achieved and maintained, reflecting inactivation of the transporter. CHC set points were 26.5-29g/dl in SS reticulocytes compared to 25-26.5g/dl in AA reticulocytes, reflecting abnormal regulation in SS cells. These results were reproducible in the same individual over time. KCC flux derived from CHC ranged from 5 to 10.3mmolK/kgHb/min in SS reticulocytes, compared to 2.9-7.2mmolK/kgHb/min in AA reticulocytes. Such measures of KCC activity in red cell populations controlled for cell age will facilitate further studies correlating KCC activity with phenotypic or genetic variability in sickle cell disease.

Cytokinesis failure in RhoA-deficient mouse erythroblasts involves actomyosin and midbody dysregulation and triggers p53 activation

RhoA GTPase has been shown in vitro in cell lines and in vivo in nonmammalian organisms to regulate cell division, particularly during cytokinesis and abscission, when 2 daughter cells partition through coordinated actomyosin and microtubule machineries. To investigate the role of this GTPase in the rapidly proliferating mammalian erythroid lineage, we developed a mouse model with erythroid-specific deletion of RhoA. This model was proved embryonic lethal as a result of severe anemia by embryonic day 16.5 (E16.5). The primitive red blood cells were enlarged, poikilocytic, and frequently multinucleated, but were able to sustain life despite experiencing cytokinesis failure. In contrast, definitive erythropoiesis failed and the mice died by E16.5, with profound reduction of maturing erythroblast populations within the fetal liver. RhoA was required to activate myosin-regulatory light chain and localized at the site of the midbody formation in dividing wild-type erythroblasts. Cytokinesis failure caused by RhoA deficiency resulted in p53 activation and p21-transcriptional upregulation with associated cell-cycle arrest, increased DNA damage, and cell death. Our findings demonstrate the role of RhoA as a critical regulator for efficient erythroblast proliferation and the p53 pathway as a powerful quality control mechanism in erythropoiesis.

Genotype-phenotype correlations in hereditary elliptocytosis and hereditary pyropoikilocytosis.

Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are heterogeneous red blood cell (RBC) membrane disorders that result from mutations in the genes encoding α-spectrin (SPTA1), β-spectrin (SPTB), or protein 4.1R (EPB41). The resulting defects alter the horizontal cytoskeletal associations and affect RBC membrane stability and deformability causing shortened RBC survival. The clinical diagnosis of HE and HPP relies on identifying characteristic RBC morphology on peripheral blood smear and specific membrane biomechanical properties using osmotic gradient ektacytometry. However, this phenotypic diagnosis may not be readily available in patients requiring frequent transfusions, and does not predict disease course or severity. Using Next-Generation sequencing, we identified the causative genetic mutations in fifteen patients with clinically suspected HE or HPP and correlated the identified mutations with the clinical phenotype and ektacytometry profile. In addition to identifying three novel mutations, gene sequencing confirmed and, when the RBC morphology was not evaluable, identified the diagnosis. Moreover, genotypic differences justified the phenotypic differences within families with HE/HPP.

A Model for Protein-Protein Interactions Involved in the Linkage of the Actin Cytoskeleton to Transmembrane Receptors for Extracellular Matrix Proteins

Actin filament bundles in cultured fibroblasts can be seen by electron microscopy to emanate from specialized regions of the cell membrane where the cell adheres tightly to the underlying substratum — the adhesion plaques or focal adhesions (see Chen and Singer, 1982, for discussion). Results from immunofluorescence and protein binding experiments have implicated a number of proteins in the anchorage of the actin filaments (F-actin) to the membrane at these locations (see Burridge, 1986, for a recent review). A schematic model of possible interactions among these proteins based on current information is presented in Figure 1.

Compound heterozygosity for two novel mutations in the erythrocyte protein 4.2 gene causing spherocytosis in a Caucasian patient

The term hereditary spherocytosis (HS) covers a range of genetically and phenotypically variable red blood cell (RBC) cytoskeleton disorders, caused by defects in proteins that link the membrane skeleton to the lipid bilayer. Nonsense and frameshift mutations of ankyrin, band 3, and β-spectrin are often responsible for dominant HS, while homozygosity or compound heterozygosity of defects in ankyrin, α-spectrin, or protein 4.2 cause recessive HS (Eber and Lux 2004, Iolascon and Avvisati 2008). Protein 4.2 is an important component of the RBC cytoskeleton, representing approximately 5% of the membrane protein content (Satchwell, et al 2009). It binds the N-terminal cytoplasmic domain of band 3 and regulates the avidity of the interaction between band 3 and ankyrin within the band 3 macrocomplex (Bruce, et al 2003). Protein 4.2 is encoded by the EPB42 gene, which encompasses approximately 20 kb, containing 13 exons and 12 introns, and maps to 15q15-q21 (Korsgren and Cohen 1991). Recessive HS caused by protein 4.2 deficiency accounts for less than 5% of all HS cases and is common in Japan but rare in other populations. Eleven mutations have been reported so far in the literature, summarized by Satchwell, et al (2009). Here we report a child of Northern European descent diagnosed with HS due to protein 4.2 deficiency. Molecular analysis demonstrated two novel mutations of EPB42 gene in a compound heterozygous state. A 16-month-old Caucasian girl was referred to our clinic for severe anaemia. Past medical history revealed a 3-day postnatal hospitalization for hyperbilirubinaemia requiring phototherapy. Family history was negative for anaemia, gallstones, or splenectomy. Nutritional history revealed that her diet contained excessive cow-milk intake (24 oz daily). Her complete blood cont revealed haemoglobin of 41 g/l, mean cell volume 62.6 fl and an absolute reticulocyte count (ARC) of 251 × 109/l (normal range 44-111 × 109/l). Her smear showed microcytic, hypochromic anaemia with polychromasia and significant anisocytosis. There were occasional ovalocytes and teardrops, and occasional macrocytes. No significant spherocytosis was noted. Further evaluation at that time revealed evidence of iron deficiency anaemia (decreased ferritin at 3 μg/l with elevated total iron-binding capacity [TIBC] 100.4 μmol/l) but also indicated an underlying haemolytic process with significantly elevated aspartate transaminase (200 u/l) and lactate dehydrogenase (1861 u/l) levels, although bilirubin levels were normal (12 μmol/l, all unconjugated). Direct Coombs test was negative; haemoglobin electrophoresis, glucose-6-phosphate dehydrogenase and pyruvate kinase screen were within normal limits. The patient was transfused to a haemoglobin concentration of 80 g/l and started on iron replacement therapy at 6 mg/kg elemental iron daily. After approximately 10 weeks of iron therapy her ferritin and TIBC normalized. The patient achieved and maintained an almost normal haemoglobin level (110-118 g/l) but ARC remained elevated at 160-240 × 109/L. A peripheral blood smear at 6 months after presentation revealed rare spherocytes (Figure 1A). Her mean cell haemoglobin concentration was increased (355-367 g/l) indicating the possibility of spherocytosis. Osmotic fragility was mildly increased (Figure 1B). Ektacytometry revealed a slightly decreased maximal deformability index (DImax) and increased Omin (osmolality at which 50% of the cells haemolyse), indicative of a spherocytic membrane disorder (Figure 1C). RBC membrane protein gel electrophoresis demonstrated absence of protein 4.2, confirmed by immunoblotting, thereby establishing the diagnosis of HS (Figure 1D and E). Figure 1 A. Blood smear (obtained when patient at baseline health status) showing a slight poikilocytosis and anisocytosis and a few spherocytes (arrowheads). B. Osmotic fragility was borderline increased. C. Ektacytometry indicated the possibility of mild spherocytosis ... CD47 has been shown to be concomitantly decreased with 4.2 deficiency (Bruce, et al 2002), while an increase in the cell adhesion molecule CD44 has been noted (van den Akker, et al 2010). Indeed, immunoblotting demonstrated decreased CD47 and increased CD44 in the patients RBC membrane (Figure 1E). Flow cytometry revealed a 2.5- to 3-fold decrease in CD47 surface expression on the patients RBCs as compared to normal control RBCs (Figure 1F). Informed consent was obtained from the family under an Institutional Review Board approved protocol for EPB42 gene analysis. DNA sequencing revealed that the child was compound heterozygous for two novel mutations in the EBP42 gene. The paternal copy of EBP42 carried a C→T nucleotide change in exon 6, resulting in a non-conservative substitution of the hydrophilic threonine-307 by the hydrophobic isoleucine (T307I), which we propose to name 4.2Cincinnati (Figure 2A). The region 306-CTVLRCLG-313 has been highly conserved evolutionarily (Toye, et al 2005). SIFT software (sift.jcvi.org) analysis (Ng and Henikoff 2003) revealed that 10 of the 15 amino acid residues from 306-320 can tolerate no substitutions, including residues 307, 310, and 317. In fact, four of the known mutations in protein 4.2 occur in this area: missense mutations 4.2Tozeur (R310Q) and 4.2Shiga (R317C), as well as nonsense mutations 4.2Nancy (frameshift 317/term 319) and 4.2Notame (del exon 6/frameshift 308). It is likely that protein 4.2Cincinnati behaves similarly to protein 4.2Tozeur, which is more susceptible to proteolysis, because it is not incorporated normally within the band 3 macrocomplex due to alteration of the domain containing the band 3-binding hairpin (Hayette, et al 1995, Satchwell, et al 2009). The fact that CD47 is decreased in our patients RBCs confirms prior observations that protein 4.2 acts as a linker between band 3 and CD47 (Bruce, et al 2002). The maternal copy of EBP42 carried a deletion of 32 nucleotides within exon 10, resulting in a frame shift starting at codon L505T. The resulting protein, which we named 4.2Ohio, terminates at amino acid 517 instead of 691, with 13 amino acids modified at the C-terminus (Figure 2B). Figure 2 The 13 exons of EPB42 in genomic DNA extracted from whole blood were amplified individually by polymerase chain reaction and sequenced bidirectionally using ByDye v1.1 chemistry on an ABI 3737xl DNA Analyzer. A. DNA sequencing of exon 6 revealed a heterozygote ... While mutations in protein 4.2 are much more common in Japan, we present here a Caucasian patient who is compound heterozygous for two previously undescribed mutations in EBP42, resulting in protein 4.2 absence accompanied by decreased CD47 and increased CD44 in the membrane. Protein 4.2 deficiency results in HS inherited through an autosomal recessive pattern, with a mildly abnormal smear and modest decreases in osmotic resistance and deformability. In addition to the functional assays (osmotic fragility and ektacytometry) traditionally used to diagnose erythrocyte cytoskeleton disorders, membrane protein analysis, flow cytometry, and gene sequencing can be employed to offer a definitive diagnosis of such atypical cases.