Heterozygosity of CDAN II (HEMPAS) gene may be detected by the analysis of erythrocyte membrane glycoconjugates from healthy carriers.
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BACKGROUND AND OBJECTIVES: Congenital dyserythropoietic anemia (CDA) type I, II, and III, is associated with abnormalities of erythrocyte membrane glycoconjugates that are most pronounced in type II CDA or hereditary erythroblastic multinuclearity with a positive acidified-serum test (HEMPAS). The abnormalities consist in hypoglycosylation of polylactoaminoglycans linked to proteins (as in band 3 glycoprotein) and ceramides (known under the name of polyglycosylceramides) as well as in accumulation of some oligoglycosylceramides: lactotriaosylceramide, neolactotetraosylceramide, and sometimes globotetraosylceramide. Glycophorin A is partially unglycosylated with respect to O-linked glycans. Types I and II of the disease are inherited in an autosomal recessive fashion. The aim of the present study was to investigate a possibility that heterozygosity with respect to CDAN2 gene in healthy carriers could be detected by analysis of erythrocyte membrane glycoconjugates. DESIGN AND METHODS: We examined a family which consisted of heterozygous parents and their two sons, one of whom was afflicted with CDA II (proband) while the other was healthy. In all family members the glycosylation status of band 3 glycoprotein, polyglycosylceramides and glycophorin A was evaluated from their carbohydrate molar composition. In addition we determined erythrocyte membrane contents of oligo- and polyglycosylceramides, and agglutinability of erythrocytes by anti-i antibody. RESULTS: We found that the heterozygous parents showed, but about 50% less pronounced, most of the typical abnormalities of erythrocyte membrane glycoconjugates that were present in the proband. These abnormalities included: hypoglycosylation of band 3, accumulation and hypoglycosylation of polyglycosylceramides, and accumulation of lactotriaosylceramide. The level of neolactotetraosylceramide in the erythrocyte membranes of the parents was, however, normal. Globotetraosylceramide content was elevated in erythrocytes from the proband and, surprisingly, even more so in the parents. Glycophorin A in the proband was only slightly abnormal. Erythrocytes from both the parents and the proband expressed increased agglutinability with anti-i antibody. All glycoconjugates examined were normal in erythrocytes from the healthy son. INTERPRETATION AND CONCLUSIONS: Individuals heterozygous with respect to CDAN2 gene can be identified through determination of the carbohydrate molar composition of band 3 and polyglycosylceramides as well as by an elevated erythrocyte content of polyglycosylceramides. In the parents these abnormalities show dosage effects. Determination of the carbohydrate molar composition of glycophorin A and of oligoglycosylceramides seems to be less promising. These findings indicate that the analysis of erythrocyte membrane glycoconjugates may be a valuable addition to the repertoire of methods used in studies on the genetics of CDA. (+info)
Anion exchanger isoform 2 operates in parallel with Na(+)/H(+) exchanger isoform 1 during regulatory volume decrease of human cervical cancer cells.
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Intracellular pH (pH(i)) homeostasis was investigated in human cervical cancer SiHa cells undergoing regulatory volume decrease (RVD) to determine which transport systems were involved. Using isoform-specific primers, mRNA transcripts of Na(+)/H(+) exchanger isoform 1 (NHE1) and isoform 3 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) and the results confirmed by Western immunoblotting. From anion exchanger isoforms 1-3 (AE1-3), only the mRNA transcript of AE2 was identified by RT-PCR and the identity was confirmed by digestion with a specific restriction endonuclease. SiHa cells loaded with the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and resuspended in isotonic media showed a stable pH(i). In contrast, a gradual internal acidification took place following resuspension in hypotonic media. The NHE inhibitors, HOE694 (10 microM) and amiloride (1 mM), showed a similar potency in enhancing the rate and extent of the hypotonicity-induced internal acidification. The absence of extracellular Na(+) also substantially enhanced the acidification during RVD. These results suggest that internal acidification during RVD is mainly compensated by the operation of NHE1. Extracellular Cl(-) was critically necessary for the pH(i) acidification during RVD. The hypotonicity-induced acidification was significantly attenuated by 100 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, a concentration inhibiting more than 90% AE activity. This indicates that AE2 mediates a net Cl(-) influx with compensating HCO(3)(-) efflux during RVD. We conclude that AE2 operates in parallel with NHE1 to regulate pH(i) during RVD of human cervical cancer cells. (+info)
Impaired trafficking of distal renal tubular acidosis mutants of the human kidney anion exchanger kAE1.
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Distal renal tubular acidosis (dRTA) is an inherited disease characterized by the failure of the kidneys to appropriately acidify urine and is associated with mutations in the anion exchanger (AE)1 gene. The effect of the R589H dRTA mutation on the expression of the human erythroid AE1 and the truncated kidney form (kAE1) was examined in transfected human embryonic kidney 293 cells. AE1, AE1 R589H, and kAE1 were present at the cell surface, whereas kAE1 R589H was located primarily intracellularly as shown by immunofluorescence, cell surface biotinylation, N-glycosylation, and anion transport assays. Coexpression of kAE1 R589H reduced the cell surface expression of kAE1 and AE1 by a dominant-negative effect, due to heterodimer formation. The mutant AE1 and kAE1 bound to an inhibitor affinity resin, suggesting that they were not grossly misfolded. Other mutations at R589 also prevented the formation of the cell surface form of kAE1, indicating that this conserved arginine residue is important for proper trafficking. The R589H dRTA mutation creates a severe trafficking defect in kAE1 but not in erythroid AE1. (+info)
The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV.
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Cytosolic carbonic anhydrase II (CAII) and the cytoplasmic C-terminal tails of chloride/bicarbonate anion exchange (AE) proteins associate to form a bicarbonate transport metabolon, which maximizes the bicarbonate transport rate. To determine whether cell surface-anchored carbonic anhydrase IV (CAIV) interacts with AE proteins to accelerate the bicarbonate transport rate, AE1-mediated bicarbonate transport was monitored in transfected HEK293 cells. Expression of the inactive CAII V143Y mutant blocked the interaction between endogenous cytosolic CAII and AE1, AE2, and AE3 and inhibited their transport activity (53 +/- 3, 49 +/- 10, and 35 +/- 1% inhibition, respectively). However, in the presence of V143Y CAII, expression of CAIV restored full functional activity to AE1, AE2, and AE3 (AE1, 101 +/- 3; AE2, 85 +/- 5; AE3, 108 +/- 1%). In Triton X-100 extracts of transfected HEK293 cells, resolved by sucrose gradient ultracentrifugation, CAIV recruitment to the position of AE1 suggested a physical interaction between CAIV and AE1. Gel overlay assays showed a specific interaction between CAIV and AE1, AE2, and AE3. Glutathione S-transferase pull-down assays revealed that the interaction between CAIV and AE1 occurs on the large fourth extracellular loop of AE1. We conclude that AE1 and CAIV interact on extracellular loop 4 of AE1, forming the extracellular component of a bicarbonate transport metabolon, which accelerates the rate of AE-mediated bicarbonate transport. (+info)
Secretory-defect distal renal tubular acidosis is associated with transporter defect in H(+)-ATPase and anion exchanger-1.
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Recent progress in molecular physiology has permitted us to understand pathophysiology of various channelopathies at a molecular level. The secretion of H(+) from alpha-intercalated cells is mediated by apical plasma membrane H(+)-ATPase and basolateral plasma membrane anion exchanger-1 (AE1). Studies have demonstrated the lack of H(+)-ATPase immunostaining in the intercalated cells in a few patients with distal renal tubular acidosis (dRTA). Mutations in H(+)-ATPase and AE1 gene have recently been reported to cause dRTA. This study extends the investigation of the role of transporter defect in dRTA by using immunohistochemical methods. Eleven patients with hyperchloremic metabolic acidosis were diagnosed functionally to have secretory-defect dRTA: urine pH >5.5 during acidemia, normokalemia or hypokalemia, and urine-to-blood pCO(2) <25 mmHg during bicarbonaturia. Renal biopsy tissue was obtained from each patient, and immunohistochemistry was carried out using antibodies to H(+)-ATPase and AE1. For comparison, renal tissues from the patients who had no evidences of distal acidification defect by functional studies were used: four with glomerulopathy or tubulointerstitial nephritis (disease controls) and three from nephrectomized kidneys for renal cell carcinoma (normal controls). The H(+)-ATPase immunoreactivity in alpha-intercalated cells was almost absent in all of the 11 patients with secretory-defect dRTA. In addition, 7 of 11 patients with secretory-defect dRTA were accompanied by negative AE1 immunoreactivity. In both disease controls and normal controls, the immunoreactivity of H(+)-ATPase and AE1 was strong in alpha-intercalated cells. In conclusion, significant defect in acid-base transporters is the major cause of secretory-defect dRTA. (+info)
Band 3 is an anchor protein and a target for SHP-2 tyrosine phosphatase in human erythrocytes.
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Tyr phosphorylation of the multifunctional transmembrane protein band 3 has been implicated in several erythrocyte functions and disorders. We previously demonstrated that pervanadate treatment of human erythrocytes induces band-3 Tyr phosphorylation, which is catalyzed by the sequential action of tyrosine kinase Syk and tyrosine kinase(s) belonging to the Src family. In this study, we show that Tyr phosphorylation of band 3, elicited by pervanadate, N-ethylmaleimide, or diamide, greatly increases band-3 interaction with the tyrosine phosphatase SHP-2 in parallel with the translocation of SHP-2 to erythrocyte membranes. These events seem to be mediated by Src-like catalyzed phosphorylation of band 3 because both SHP-2 translocation to cellular membranes and its interaction with Tyr-phosphorylated protein are greatly counteracted by PP2, a specific inhibitor of Src kinases. Binding-competition experiments demonstrate that SHP-2 recruitment to band 3 occurs via its SH2 domain(s). In particular, our data support the view that SHP-2 docks specifically with P-Y359 of band 3. Experiments performed with intact erythrocytes in the presence of the SHP-2 inhibitor calpeptin suggest that, once recruited to Tyr-phosphorylated band 3, the tyrosine phosphatase dephosphorylates the protein. P-Y8, 21, and 904 are the residues affected by SHP-2, as judged by (32)P-peptide mapping of band 3 digested with trypsin. These results indicate that in treated erythrocytes, recruitment of cytosolic SHP-2 to band 3 is a prerequisite for the subsequent dephosphorylation of the transmembrane protein. (+info)
Band 3 mutations, distal renal tubular acidosis, and Southeast Asian ovalocytosis.
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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. (+info)
Band-3 protein function in human erythrocytes: effect of oxygenation-deoxygenation.
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Sulfate transport by band-3 protein in adult human erythrocytes was shown to be modulated by oxygen pressure. In particular, a higher transport activity was measured under high oxygen pressure than at low one (0.0242+/-0.0073 vs. 0.0074+/-0.0010 min(-1)). Other factors, such as magnesium ions and orthovanadate, which can indirectly affect the binding properties of the cytoplasmic domain of band 3 (cdb3), influence significantly the anion exchanger activity. No effect of oxygen pressure on sulfate transport was found in chicken erythrocytes, which may be related to their lacking the cdb3 binding site. These findings are fully consistent with a molecular mechanism where the oxygen-linked transition of hemoglobin (T-->R) could play a key role in the regulation of anion exchanger activity. (+info)