Lactobacillus plantarum 299v Supplementation Improves Vascular Endothelial Function and Reduces Inflammatory Biomarkers in Men With Stable Coronary Artery Disease.

RATIONALE: A strong association has emerged between the gut microbiome and atherosclerotic disease. Our recent data suggest Lactobacillus plantarum 299v (Lp299v) supplementation reduces infarct size in male rats. Limited human data are available on the impact of Lp299v on the vasculature. OBJECTIVE: To determine whether oral Lp299v supplementation improves vascular endothelial function and reduces systemic inflammation in humans with stable coronary artery disease (CAD). METHODS AND RESULTS: Twenty men with stable CAD consumed a drink containing Lp299v (20 billion CFU) once daily for 6 weeks. After a 4-week washout, subjects were given an option of additionally participating in a 10-day study of oral liquid vancomycin (250 mg QID). Vascular endothelial function was measured by brachial artery flow-mediated dilation. Before and after Lp299v, plasma short-chain fatty acids, trimethylamine oxide, and adipokine levels were measured. Additional plasma samples underwent unbiased metabolomic analyses using liquid chromatography/mass spectroscopy. 16S rRNA sequencing was used to determine changes of the stool microbiome. Arterioles from patients with CAD were obtained, and endothelium-dependent vasodilation was measured by video microscopy after intraluminal incubation with plasma from Lp299v study subjects. Lp299v supplementation improved brachial flow-mediated dilation ( P=0.008) without significant changes in plasma cholesterol profiles, fasting glucose, or body mass index. Vancomycin did not impact flow-mediated dilation. Lp299v supplementation decreased circulating levels of IL (interleukin)-8 ( P=0.01), IL-12 ( P=0.02), and leptin ( P=0.0007) but did not significantly change plasma trimethylamine oxide concentrations ( P=0.27). Plasma propionate ( P=0.004) increased, whereas acetate levels decreased ( P=0.03). Post-Lp299v plasma improved endothelium-dependent vasodilation in resistance arteries from patients with CAD ( P=0.02).16S rRNA analysis showed the Lactobacillus genus was enriched in postprobiotic stool samples without other changes. CONCLUSIONS: Lp299v improved vascular endothelial function and decreased systemic inflammation in men with CAD, independent of changes in traditional risk factors and trimethylamine oxide. Circulating gut-derived metabolites likely account for these improvements and merit further study. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01952834.

miR-29 contributes to normal endothelial function and can restore it in cardiometabolic disorders.

We investigated the role of microRNAs (miRNA) in endothelial dysfunction in the setting of cardiometabolic disorders represented by type 2 diabetes mellitus (T2DM). miR-29 was dysregulated in resistance arterioles obtained by biopsy in T2DM patients. Intraluminal delivery of miR-29a-3p or miR-29b-3p mimics restored normal endothelium-dependent vasodilation (EDVD) in T2DM arterioles that otherwise exhibited impaired EDVD Intraluminal delivery of anti-miR-29b-3p in arterioles from non-DM human subjects or rats or targeted mutation of Mir29b-1/a gene in rats led to impaired EDVD and exacerbation of hypertension in the rats. miR-29b-3p mimic increased, while anti-miR-29b-3p or Mir29b-1/a gene mutation decreased, nitric oxide levels in arterioles. The mutation of Mir29b-1/a gene led to preferential differential expression of genes related to nitric oxide including Lypla1. Lypla1 was a direct target of miR-29 and could abrogate the effect of miR-29 in promoting nitric oxide production. Treatment with Lypla1 siRNA improved EDVD in arterioles obtained from T2DM patients or Mir29b-1/a mutant rats or treated with anti-miR-29b-3p. These findings indicate miR-29 is required for normal endothelial function in humans and animal models and has therapeutic potential for cardiometabolic disorders.

Impact of DPP-4 inhibition on acute and chronic endothelial function in humans with type 2 diabetes on background metformin therapy.

Cell culture and animal work indicate that dipeptidyl peptidase-4 (DPP-4) inhibition may exert cardiovascular benefits through favorable effects on the vascular endothelium. Prior human studies evaluating DPP-4 inhibition have shown conflicting results that may in part be related to heterogeneity of background anti-diabetes therapies. No study has evaluated the acute response of the vasculature to DPP-4 inhibition in humans. We recruited 38 patients with type 2 diabetes on stable background metformin therapy for a randomized, double-blind, placebo-controlled crossover trial of DPP-4 inhibition with sitagliptin (100 mg/day). Each treatment period was 8 weeks long separated by 4 weeks of washout. Endothelial function and plasma markers of endothelial activation (intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)) were measured prior to and 2 hours following acute dosing of sitagliptin or placebo, as well as following 8 weeks of intervention with each pill. Thirty subjects completed the study and were included in analyses. Neither acute nor chronic sitagliptin therapy resulted in significant changes in vascular endothelial function. While post-acute sitagliptin ICAM-1 levels were lower than that post-chronic sitagliptin, the ICAM-1 concentration was not significantly different than pre-acute sitagliptin levels or levels measured in relationship to placebo. There were no significant changes in plasma VCAM-1 levels at any time point. Acute and chronic sitagliptin therapies have neutral effects on the vascular endothelium in the setting of metformin background therapy. In conclusion, our findings suggest DPP-4 inhibition has a neutral effect on cardiovascular risk in patients without a history of heart failure or renal insufficiency. TRIAL REGISTRATION: NCT01859793.

Dynamin-related protein 1 mediates low glucose-induced endothelial dysfunction in human arterioles.

Intensive glycemic regulation has resulted in an increased incidence of hypoglycemia. Hypoglycemic burden correlates with adverse cardiovascular complications and contributes acutely and chronically to endothelial dysfunction. Prior data indicate that mitochondrial dysfunction contributes to hypoglycemia-induced endothelial dysfunction, but the mechanisms behind this linkage remain unknown. We attempt to determine whether clinically relevant low-glucose (LG) exposures acutely induce endothelial dysfunction through activation of the mitochondrial fission process. Characterization of mitochondrial morphology was carried out in cultured endothelial cells by using confocal microscopy. Isolated human arterioles were used to explore the effect LG-induced mitochondrial fission has on the formation of detrimental reactive oxygen species (ROS), bioavailability of nitric oxide (NO), and endothelial-dependent vascular relaxation. Fluorescence microscopy was employed to visualize changes in mitochondrial ROS and NO levels and videomicroscopy applied to measure vasodilation response. Pharmacological disruption of the profission protein Drp1 with Mdivi-1 during LG exposure reduced mitochondrial fragmentation among vascular endothelial cells (LG: 0.469; LG+Mdivi-1: 0.276; P = 0.003), prevented formation of vascular ROS (LG: 2.036; LG+Mdivi-1: 1.774; P = 0.005), increased the presence of NO (LG: 1.352; LG+Mdivi-1: 1.502; P = 0.048), and improved vascular dilation response to acetylcholine (LG: 31.6%; LG+Mdivi-1; 78.5% at maximum dose; P < 0.001). Additionally, decreased expression of Drp1 via siRNA knockdown during LG conditions also improved vascular relaxation. Exposure to LG imparts endothelial dysfunction coupled with altered mitochondrial phenotypes among isolated human arterioles. Disruption of Drp1 and subsequent mitochondrial fragmentation events prevents impaired vascular dilation, restores mitochondrial phenotype, and implicates mitochondrial fission as a primary mediator of LG-induced endothelial dysfunction.NEW & NOTEWORTHY Acute low-glucose exposure induces mitochondrial fragmentation in endothelial cells via Drp1 and is associated with impaired endothelial function in human arterioles. Targeting of Drp1 prevents fragmentation, improves vasofunction, and may provide a therapeutic target for improving cardiovascular complications among diabetics.Listen to this article's corresponding podcast @ http://ajpheart.podbean.com/e/mitochondrial-dynamics-impact-endothelial-function/.

Relative importance of step count, intensity, and duration on physical activity's impact on vascular structure and function in previously sedentary older adults.

BACKGROUND: Age-related endothelial dysfunction and vascular stiffening are associated with increased cardiovascular (CV) risk. Many groups have encouraged goals of ≥10 000 steps/day or ≥30 min/day of moderate intensity physical activity (MPA) to reduce age-related CV risk. The impact of MPA on the vasculature of older adults remains unclear. METHODS AND RESULTS: We randomized 114 sedentary older adults ages ≥50 to 12 weeks of either no intervention (group 1), a pedometer-only intervention (group 2), or a pedometer with an interactive website employing strategies to increase the adoption of habitual physical activity (PA, group 3). Endothelial function by brachial flow-mediated dilation (FMD%), vascular stiffness by tonometry, step-count by pedometer, and PA intensity/distribution by accelerometer were measured. Step-count increased in groups 2 (5136±1554 to 9596±3907, P<0.001) and 3 (5474±1512 to 8167±3111, P<0.001) but not in group 1 (4931±1667 to 5410±2410). Both groups 2 and 3 increased MPA ≥30 min/day. Only group 3 increased MPA in continuous bouts of ≥10 minutes (P<0.001) and improved FMD% (P=0.001). Neither achievement of ≥10 000 steps/day nor ≥30 min/day of MPA resulted in improved FMD%. However, achieving ≥20 min/day in MPA bouts resulted in improved FMD%. No changes in vascular stiffness were observed. CONCLUSIONS: MPA reverses age-related endothelial dysfunction, but may require MPA to be performed in bouts of ≥10 minutes duration for ≥20 min/day to be effective. Commonly encouraged PA goals do not guarantee improved endothelial function and may not be as effective in reducing CV risk. CLINICAL TRIAL REGISTRATION URL: Clinicaltrials.gov. UNIQUE IDENTIFIER: NCT-01212978.

Moderate Obesity and Endothelial Dysfunction in Humans: Influence of Gender and Systemic Inflammation.

OBJECTIVE: To determine whether moderate obesity (BMI ≥ 30 kg/m2) is associated with impaired conduit and microvascular endothelial function, and whether men or women are more susceptible to impairment of endothelial function related to moderate obesity. DESIGN AND METHODS: 41 middle aged, non-diabetic moderately obese (BMI 34.7±4.0 kg/m2) and non obese (BMI 24.3±2.6 kg/m2) subjects of both sexes underwent noninvasive studies of endothelial function (brachial reactivity) and measurements of endothelial dependent vasodilation of gluteal subcutaneous arterioles to acetylcholine (Ach). RESULTS: Endothelium dependent vasodilation to Ach was decreased in the moderately obese compared to the non-obese (P<0.001). Stratified analysis based on sex showed impairment of arteriolar endothelial function in women BMI ≥ 30 kg/m2 (P=0.02), but not men. There was no difference between in vivo endothelial function (FMD%, FMD mm) by BMI category. Sex specific analysis showed FMD% was lower in women with BMI ≥ 30 kg/m2 compared to those with BMI < 30 kg/m2 (P=0.02). No differences were seen in men based on BMI category (P=0.18). In women, high sensitivity C-reactive protein (hsCRP) correlated with BMI (ρ=0.68, P=0.006). CONCLUSION: Moderate obesity is associated with impaired resistance arteriolar endothelial function. This is more prominent in women than men and is associated with systemic inflammation.

A conductive pathway generated from fragments of the human red cell anion exchanger AE1.

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO3- exchanger with 12-14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive (36)Cl-influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Delta(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. We found that, whereas AE1Delta(6: 7) has some electroneutral Cl-base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys(539)Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Delta(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3- approximately I- > NO2- > Br- > Cl- > SO4(2-) approximately HCO3- approximately gluconate- approximately aspartate- approximately cyclamate-. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl-HCO3- exchangers, trout AE1 and mammalian SLC26A7.

Recessive distal renal tubular acidosis in Sarawak caused by AE1 mutations.

Mutations of the AE1 (SLC4A1, Anion-Exchanger 1) gene that codes for band 3, the renal and red cell anion exchanger, are responsible for many cases of familial distal renal tubular acidosis (dRTA). In Southeast Asia this disease is usually recessive, caused either by homozygosity of a single AE1 mutation or by compound heterozygosity of two different AE1 mutations. We describe two unrelated boys in Sarawak with dRTA associated with compound heterozygosity of AE1 mutations. Both had Southeast Asian ovalocytosis (SAO), a morphological abnormality of red cells caused by a deletion of band 3 residues 400-408. In addition, one boy had a DNA sequence abnormality of band 3 residue (G701D), which has been reported from elsewhere in Southeast Asia. The other boy had the novel sequence abnormality of band 3 (Q759H) and profound hemolytic anemia.

Protein-4.2 association with band 3 (AE1, SLCA4) in Xenopus oocytes: effects of three natural protein-4.2 mutations associated with hemolytic anemia.

We have investigated the effects of coexpression of protein 4.2 and three protein-4.2 variants with band 3 in the Xenopus oocyte expression system. Normal protein 4.2 increased band-3-specific chloride transport in the oocytes. Protein 4.2 also coimmunoprecipitated with band 3 and colocalized with band 3 at the oocyte plasma membrane. The increase in band-3-mediated chloride transport and coimmunoprecipitation of protein 4.2 required the presence of the N-terminal cytoplasmic domain of band 3. Protein 4.2 also localized to the oocyte plasma membrane in the absence of band 3. The protein-4.2 variants 4.2 Tozeur (R310Q) and 4.2 Komatsu (D175Y) had impaired ability to bind to band 3 and these variants did not localize to the oocyte plasma membrane when expressed on their own or when coexpressed with band 3. Unexpectedly, 4.2 Nippon (A142T) behaved similarly to normal protein 4.2. In the absence of a crystal structure of protein 4.2, we propose a homology model of protein 4.2 based on the structure of the sequence-related protein transglutaminase. Using our results in oocytes and this homology model we speculate how these mutations affect protein 4.2 and result in hereditary spherocytosis.

The disruption of the third extracellular loop of the red cell anion exchanger AE1 does not affect electroneutral Cl-/HCO3- exchange activity.

The red cell anion exchanger (band 3; AE1) is a multispanning membrane protein that traverses the bilayer up to 14 times and mediates the stilbene-disulfonate-sensitive, electroneutral exchange of chloride and bicarbonate. Previous studies showed that the integrity of the third extracellular loop (EC3) of the protein was not essential for stilbene-disulfonate-sensitive chloride uptake. Here we demonstrate that the chloride uptake mediated by assemblies separated at EC3 represents the physiological electroneutral Cl(-)/HCO(3)(-) activity associated with intact AE1 protein. This provides further evidence that the 1:5 and 6:14 regions of the protein form discrete folding domains and confirms that the third extracellular loop does not play a pivotal role in AE1 transport function.

Regions of human kidney anion exchanger 1 (kAE1) required for basolateral targeting of kAE1 in polarised kidney cells: mis-targeting explains dominant renal tubular acidosis (dRTA).

Distal renal tubular acidosis (dRTA) is characterised by defective acid secretion by kidney alpha-intercalated cells. Some dominantly inherited forms of dRTA result from anion exchanger 1 (AE1) mutations. We have developed a stably transfected cell model for the expression of human kidney AE1 (kAE1) and mutant kAE1 proteins in MDCKI cells. Normal kAE1 was delivered to the plasma membrane of non-polarised cells and to the basolateral membrane of polarised cells. The AE1 N-glycan was processed to a complex form. Surprisingly, expression of kAE1 increased the permeability of the paracellular barrier of polarised MDCKI monolayers. All dominant dRTA mutations examined altered the targeting of kAE1 in MDCKI cells. The mutant proteins kAE1(R589H), kAE1(S613F) and kAE1(R901Stop) were retained in the ER in non-polarised cells, but the kAE1(R901Stop) protein was also present in late endosomes/lysosomes. The complex N-glycan of kAE1(R901Stop) was larger than that of normal kAE1. In polarised cells, the mutant kAE1(R901Stop) was mis-targeted to the apical membrane, while the kAE1(R589H) and kAE1(S613F) mutants did not reach the cell surface. These results demonstrate that dominant dRTA mutations cause aberrant targeting of kAE1 in polarised kidney cells and provide an explanation for the origin of dominant dRTA. Our data also demonstrate that the 11 C-terminal residues of kAE1 contain a tyrosine-dependent basolateral targeting signal that is not recognised by mu 1B-containing AP-1 adaptor complexes. In the absence of the N-terminus of kAE1, the C-terminus was not sufficient to localise kAE1 to the basolateral membrane. These results suggest that a determinant within the kAE1 N-terminus co-operates with the C-terminus for kAE1 basolateral localisation.

Altered structure and anion transport properties of band 3 (AE1, SLC4A1) in human red cells lacking glycophorin A.

We have studied the properties of band 3 in different glycophorin A (GPA)-deficient red cells. These red cells lack either both GPA and glycophorin B (GPB) (M(k)M(k) cells) or GPA (En(a-) cells) or contain a hybrid of GPA and GPB (MiV cells). Sulfate transport was reduced in all three red cell types to approximately 60% of that in normal control red cells as a result of an increased apparent K(m) for sulfate. Transport of the monovalent anions iodide and chloride was also reduced. The reduced iodide transport resulted from a reduction in the V(max) for iodide transport. The anion transport site was investigated by measuring iodide fluorescence quenching of eosin-5-maleimide (EMA)-labeled band 3. The GPA-deficient cells had a normal K(d) for iodide binding, in agreement with the unchanged K(m) found in transport studies. However, the apparent diffusion quenching constant (K(q)) was increased, and the fluorescence polarization of band 3-bound EMA decreased in the variant cells, suggesting increased flexibility of the protein in the region of the EMA-binding site. This increased flexibility is probably associated with the decrease in V(max) observed for iodide transport. Our results suggest that band 3 in the red cell can take up two different structures: one with high anion transport activity when GPA is present and one with lower anion transport activity when GPA is absent.

Erythrocyte detergent-resistant membrane proteins: their characterization and selective uptake during malarial infection.

Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.

Distinct regions of human glycophorin A enhance human red cell anion exchanger (band 3; AE1) transport function and surface trafficking.

Human red cell glycophorin A (GPA) enhances the expression of band 3 anion transport activity at the cell surface of Xenopus oocytes. This effect of GPA could occur in two ways, enhancement of band 3 anion transport function or enhancement of band 3 trafficking to the cell surface. We have examined the GPA effect using GPA mutants. We compared the sequences of GPA and its homolog glycophorin B (GPB; which does not facilitate band 3 cell-surface activity or trafficking) to identify candidate regions of GPA for study. We constructed several GPA or GPB mutants, including naturally occurring GPA/GPB hybrid molecules and insertion, deletion, and substitution mutants. We analyzed the effects of the mutant proteins on band 3-specific chloride transport and surface presentation using co-expression in Xenopus oocytes. We find that the C-terminal cytoplasmic tail of GPA enhances trafficking of band 3 to the cell surface, whereas the extracellular residues 68-70 increase the specific anion transport activity of band 3. In addition, examination of the oligomerization of GPA mutants showed that single amino acid substitutions N-terminal to the transmembrane domain greatly reduce SDS-stable GPA dimer formation, implying that regions outside the transmembrane domain of GPA are important for GPA dimer formation.

A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane.

We have studied the membrane proteins of band 3 anion exchanger (AE1)-deficient mouse and human red blood cells. It has been shown previously that proteins of the band 3 complex are reduced or absent in these cells. In this study we show that proteins of the Rh complex are also greatly reduced (Rh-associated glycoprotein, Rh polypeptides, CD47, glycophorin B) or absent (LW). These observations suggest that the Rh complex is associated with the band 3 complex in healthy RBCs. Mouse band 3(-/-) RBCs differed from the human band 3-deficient RBCs in that they retained CD47. Aquaporin 1 was reduced, and its glycosylation was altered in mouse and human band 3-deficient RBCs. Proteins of the glycophorin C complex, and other proteins with independent cytoskeletal interactions, were present in normal or increased amounts. To obtain direct evidence for the association of the band 3 and the Rh protein complexes in the RBC, we examined whether Rh complex proteins were coimmunoprecipitated with band 3 from membranes. RhAG and Rh were found to be efficiently coimmunoprecipitated with band 3 from deoxycholate-solubilized membranes. Results suggest that band 3 forms the core of a macrocomplex of integral and peripheral RBC membrane proteins. The presence of these proteins in a single structural macrocomplex makes it likely that they have linked functional or regulatory roles. We speculate that this macrocomplex may function as an integrated CO(2)/O(2) gas exchange unit (metabolon) in the erythrocyte.

The N-terminal region of the transmembrane domain of human erythrocyte band 3. Residues critical for membrane insertion and transport activity.

We studied the role of the N-terminal region of the transmembrane domain of the human erythrocyte anion exchanger (band 3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which gives rise to nonfunctional band 3, and also on the portion of the protein N-terminal to the transmembrane domain (amino acids 361-396). We examined the effects of mutations in these regions on endoplasmic reticulum insertion (using cell-free translation), chloride transport, and cell-surface movement in Xenopus oocytes. We found that the hydrophobic length of TM1 was critical for membrane insertion and that formation of a transport-active structure also depended on the presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that a polar residue was located 2 or 3 amino acids on the C-terminal side of Asp-399. Finally, deletion of the cytoplasmic surface sequence G(381)LVRD abolished chloride transport, but not surface expression, indicating that this sequence makes an essential structural contribution to the anion transport site of band 3.

An N-terminal GFP tag does not alter the functional expression to the plasma membrane of red cell and kidney anion exchanger (AE1) in mammalian cells.

Two isoforms of the band 3 anion exchanger are expressed in mammalian cells, a 911 residue protein (B3) in red cells, and a truncated protein (KB3) in the alpha-intercalated cells of the kidney. Mutants of both isoforms are known to be associated with human disease, and mistargeting of the mutated proteins has been suggested as the mechanism of pathogenesis in several cases but has been difficult to prove. The present study demonstrates the feasibility of using confocal microscopy for investigating the targeting of homozygous and heterozygous B3 and KB3 mutants. K562 erythroleukemia cells offer several advantages for the stable expression of B3, but have not previously been used for its visualization. A wide range of cell attachment factors, growth conditions, fixation reagents and primary antibodies were investigated to enable imaging of B3 and endogenous GPA by immunofluorescence confocal microscopy in stable K562/B3 clones. B3 co-localized with GPA at the cell surface and also in an intracellular compartment. Functional cell surface expression of KB3 in stable K562 clones was also obtained. Importantly, both B3 and KB3 could be expressed as stable fusion proteins tagged with green fluorescent protein (GFP) in K562 cells, and it was demonstrated that N-terminal GFP-tagging does not affect the targeting or chloride transport properties of B3 or KB3. The use of GFP as well as double-labelling methods developed for immunostaining will be invaluable for investigating the interactions of band 3 with itself and other proteins during its trafficking in erythroid and kidney cells. This will help elucidate how band 3 mutations can cause human diseases such as hereditary spherocytosis and distal renal tubular acidosis.

Absence of CD47 in protein 4.2-deficient hereditary spherocytosis in man: an interaction between the Rh complex and the band 3 complex.

We present data on a patient of South Asian origin with recessive hereditary spherocytosis (HS) due to absence of protein 4.2 [4.2 (-) HS]. Protein 4.2 cDNA sequence analysis showed the presence of a novel 41-bp frameshift deletion that predicts a truncated peptide designated protein 4.2 Hammersmith. Quantitative reverse transcription-polymerase chain reaction indicated that the mutant mRNA was unstable. Sequencing of protein 4.2 genomic DNA revealed that the deletion stems from aberrant splicing. The proband was homozygous for a G>T substitution at position 1747 (cDNA numbering) that activates a cryptic acceptor splice site within exon 11 of the protein 4.2 gene (EPB42). The proband's mother was found to be heterozygous for this substitution. Unlike protein 4.2 null mice, the proband's red cells showed no evidence for abnormal cation permeability. Quantitation of red cell membrane proteins was carried out by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and flow cytometric measurement. CD47, a protein associated with the Rh complex, was markedly reduced to about 1% (in the proband) and 65% (in the mother) that found in healthy controls. The Rh-associated glycoprotein migrated with a higher than normal apparent molecular weight on SDS-PAGE. There was no obvious reduction in Rh polypeptides. These observations indicate that protein 4.2 and CD47 interact in the human red cell membrane. They provide further evidence for an association between the band 3 complex (band 3, ankyrin, protein 4.2, glycophorin A) and the Rh complex (Rh-associated glycoprotein, Rh polypeptides, glycophorin B, CD47, LW) and define a point of attachment between the Rh complex and the red cell cytoskeleton.

Band 3 mutations, distal renal tubular acidosis, and Southeast Asian ovalocytosis.

Familial distal renal tubular acidosis (dRTA) and Southeast Asian ovalocytosis (SAO) may coexist in the same patient. Both can originate in mutations of the anion-exchanger 1 gene (AE1), which codes for band 3, the bicarbonate/chloride exchanger in both the red cell membrane and the basolateral membrane of the collecting tubule alpha-intercalated cell. Dominant dRTA is usually due to a mutation of the AE1 gene, which does not alter red cell morphology. SAO is caused by an AE1 mutation that leads to a nine amino acid deletion of red cell band 3, but by itself does not cause dRTA. Recent gene studies have shown that AE1 mutations are responsible for autosomal recessive dRTA in several countries in Southeast Asia; these patients may be homozygous for the mutation or be compound heterozygotes of two different AE1 mutations, one of which is usually the SAO mutation.

Molecular basis and functional consequences of the dominant effects of the mutant band 3 on the structure of normal band 3 in Southeast Asian ovalocytosis.

Southeast Asian ovalocytosis (SAO) human red cell membranes contain similar proportions of normal band 3 and a mutant band 3 with a nine amino acid deletion (band 3 SAO). We employed specific chemical modification and proteolytic cleavage to probe the structures of band 3 in normal and SAO membranes. When the membranes were modified specifically at lysine residues with N-hydroxysulfosuccinimide-SS-biotin, band 3 Lys-851 was not modified in normal membranes but quantitatively modified in SAO membranes. Normal and SAO membranes showed different patterns of band 3 proteolytic cleavage. Notably, many sites cleaved in normal membranes were not cleaved in SAO membranes, despite the presence of normal band 3 in these membranes. The mutant band 3 changes the structure of essentially all the normal band 3 present in the SAO membranes, and these changes extend throughout the normal band 3 molecules. The results also imply that band 3 in SAO membranes is present as hetero-tetramers or higher hetero-oligomers. The dominant structural effects of band 3 SAO on the other band 3 allele have important consequences on the functional and hematological properties of human red cells heterozygous for band 3 SAO. Analysis of the altered profile of biotinylation and protease cleavage sites suggests the location of exposed surfaces in the band 3 membrane domain and identifies likely interacting regions within the molecule. Our approach provides a sensitive method for studying structural changes in polytopic membrane proteins.